Protein metabolism and nitrogen fixation and metabolism

1. Biochemistry
and
Metabolism
Dr. Kirpa Ram (Assistant Professor, Botany)
Ph. +91-9468393474, Mail- dr.kirparamjangra@gmail.com
Protein Metabolism And Nitrogen fixation and N & S Metabolism

2. Protein Metabolism
And
Nitrogen fixation and N & S Metabolism
Biochemistry by Dr. Kirpa Ram

3. Part - I
Protein Metabolism
Biochemistry by Dr. Kirpa Ram

4. Protein
• Proteins are complex, organic compounds composed of
many amino acids linked together through peptide
bonds and cross-linked between chains by sulfhydryl
bonds, hydrogen bonds and van der Waals forces.
• Proteins are complex biomolecules that are made up
of smaller units known as amino acids.
Or
• Proteins are organic nitrogenous compounds in which
a large number of amino acids are joined together by
peptide linkages to form long polypeptide chains.
• Peptide-linkage (-CONH-) is formed between amino
group (-NH2) abd carboxylic group (-COOH) of another
amino acid eliminating one molecule of water.
• That end of the polypeptide chain where the -COOH group of the amino
acid is not involved in peptide linkage is called as C-terminal end.
• The other end of the polypeptide chain with amino acid having free -NH2
group is called as N-terminal end.
Protein is a macronutrient that is essential to building muscle mass

5. Classification of Proteins
Proteins are classified based on the following THREE criterions:

6. (I). Classification of Proteins based on the Structure of Proteins
(B). Globular Proteins
1. Globular proteins are spherical
or globular in shape.
2. The polypeptide chain is tightly
folded into spherical shapes.
3. Tertiary structure is the most
important functional structure
in globular proteins.
4. Physically they are soft than
fibrous proteins.
5. They are readily soluble in
water.
6. Most of the proteins in the cells
belong to the category of
globular proteins.
Functions: Form enzymes,
antibodies and some hormones.
Example: Insulin, Haemoglobin,
DNA Polymerase and RNA
Polymerase
(A). Fibrous Proteins
1. They are linear (long fibrous) in
shape.
2. Secondary structure is the most
important functional structure of
fibrous proteins.
3. Usually, these proteins do not have
tertiary structures.
4. Physically fibrous proteins are very
tough and strong.
5. They are insoluble in the water.
6. Long parallel polypeptide chains
cross linked at regular intervals.
Fibrous proteins form long fibres or
sheaths.
Functions: perform the structural
functions in the cells.
Examples: Collagen, Myosin, Silk and
Keratin.
(C). Intermediate Proteins
i. Their structure is
intermediate to linear and
globular structures.
ii. They are short and more or
less linear shaped proteins
iii. Unlike fibrous proteins, they
are soluble in water.
Function: blood clotting
proteins
Example: Fibrinogen

7. II). Classification of Proteins based on Composition
(A). Simple Proteins
i. Simple proteins composed of
ONLY amino acids.
ii. Proteins may be fibrous or
globular.
iii. They possess relatively simple
structural organization.
Example: Collagen, Myosin,
Insulin, Keratin
(B). Conjugated Proteins
i. Conjugated proteins are complex proteins.
ii. They contain one or more non-amino acid components.
iii. Here the protein part is tightly or loosely bound to one or more non-protein
part(s).
iv. The non-protein parts of these proteins are called prosthetic groups.
v. The prosthetic group may be metal ions, carbohydrates, lipids, phosphoric acids,
nucleic acids and FAD.
vi. The prosthetic group is essential for the biological functions of these proteins.
vii. Conjugated proteins are usually globular in shape and are soluble in water.
viii.Most of the enzymes are conjugated proteins.
Based on the nature of prosthetic
groups, the conjugated proteins are
further classified as follows:
a) Phosphoprotein: Prosthetic group is
phosphoric acid, Example:- Casein of milk,
Vitellin of egg yolk.
b) Glycoproteins: Prosthetic group is
carbohydrates, Example:- Most of the
membrane proteins, Mucin (component of
saliva).
c) Nucleoprotein: Prosthetic group is nucleic acid, Example:- proteins in
chromosomes, structural proteins of ribosome.
d) Chromoproteins: Prosthetic group is pigment or chrome, Example:-
Haemoglobin, Phytochrome and Cytochrome.
e) Lipoproteins: Prosthetic group is Lipids, Example:- Membrane proteins
f) Flavoproteins: Prosthetic group is FAD (Flavin Adenine Dinucleotide),
Example:- Proteins of Electron Transport System (ETS).
g) Metalloproteins: Prosthetic group is Metal ions, Example:- Nitrate
Reductase

8. (III). Classification of Protein based on Functions
(A). Structural Proteins:
Form the component of the connective tissue, bone,
tendons, cartilage, skin, feathers, nail, hairs and
horn. Most of them are fibrous proteins and are
insoluble in water. They are usually inert to
biochemical reactions. They maintain the native
form and position of the organs..
Example:
Collagen: It is found in connective tissue such as
tendons, cartilage, a matrix of bones and cornea of
the eye. Leather is almost pure collagen.
Elastin: It is found in ligaments. It is capable of
stretching in two dimensions.
Keratin: It constitutes almost the entire dry weight
of hair, wool, feathers, nails, claws, quills, scales,
horns, hooves, tortoise shell and much of the outer
layer of skin.
Fibroin: It is the major component of silk fibres
and spider webs.
Resilin: The wing hinges of some insects are made
of resilin, which has nearly perfect elastic
properties.
(B). Enzymes:
They are the biological catalysts. Most of them are globular
conjugated proteins. Example: DNA Polymerase, Nitrogenase,
Lipase
(C). Hormones:They include the proteinaceous hormones in the cells.
Example: Insulin, Glucagon, ACH
(D). Respiratory Pigments
They are coloured proteins. All of them are conjugated proteins
and they contain pigments (chrome) as their prosthetic group.
Example: Haemoglobin, Myoglobin
(E). Transport Proteins
They form channels in the plasma membrane. They also form one
of the components of blood and lymph in animals. Example:
Serum albumin
(F). Contractile proteins
They are the force generators of muscles. Example: Actin,
Myosin
(G). Storage Proteins
They act as the store of metal ions and amino acids in the cells.
Found in seeds, egg and milk. Example: Ferritin which stores
iron, Casein, Ovalbumin, Gluten of Wheat
(F). Toxins They are toxic proteins.Example: Snake venom

9. Protein Structure
Proteins are a polymeric chain of amino acid
residues. The structure of a protein is mainly composed of
long chains of amino acids. The structure and position of
amino acids give particular properties to the proteins.
Amino acids are made up of an amino functional group (-
NH2) and a carboxyl group (-COOH).
1. Primary – The primary structure of a protein is the linear polypeptide chain
formed by the amino acids in a particular sequence.
2. Secondary – The secondary structure of a protein is formed by hydrogen
bonding in the polypeptide chain. These bonds cause the chain to fold and coil
in two different conformations known as the α-helix or β-pleated sheets.
3. Tertiary – The tertiary structure is the final 3-dimensional shape acquired by
the polypeptide chains under the attractive and repulsive forces of the different
R-groups of each amino acid. This is a coiled structure that is very necessary
for protein functions.
4. Quaternary – This structure is exhibited only by those proteins which have
multiple polypeptide chains combined to form a large complex. The individual
chains are then called subunits.

10. Primary Structure of Protein
All amino acids have the alpha carbon bonded to a hydrogen
atom, carboxyl group, and an amino group.
The "R" group varies among amino acids and determines
the differences between these protein monomers.
The amino acid sequence of a protein is determined by the
information found in the cellular genetic code.
Primary Structure describes the unique order in which amino acids are linked together to
form a protein. Proteins are constructed from a set of 20 amino acids.
Generally, amino acids have the following structural properties:
A carbon (the alpha carbon) bonded to the four groups below:
1. A hydrogen atom (H)
2. A Carboxyl group (-COOH)
3. An Amino group (-NH2)
4. A "variable" group or "R" group

11. Secondary Structure of Protein
• The overall three-dimensional structure of a
polypeptide is called its tertiary structure. The
tertiary structure is primarily due to interactions
between the R groups of the amino acids that make
up the protein.
• Secondary Structure refers to the coiling or folding
of a polypeptide chain that gives the protein its 3-D
shape. There are two types of secondary structures
observed in proteins.
• One type is the alpha (α) helix structure. This
structure resembles a coiled spring and is secured by
hydrogen bonding in the polypeptide chain.
• The second type of secondary structure in proteins is
the beta (β) pleated sheet. This structure appears to
be folded or pleated and is held together by hydrogen
bonding between polypeptide units of the folded
chain that lie adjacent to one another.

12. Tertiary Structure of Protein
• Tertiary Structure refers to the comprehensive 3-D
structure of the polypeptide chain of a protein. There
are several types of bonds and forces that hold a
protein in its tertiary structure.
• Hydrophobic interactions greatly contribute to the
folding and shaping of a protein. The "R" group of the
amino acid is either hydrophobic or hydrophilic.
• Hydrogen bonding in the polypeptide chain and
between amino acid "R" groups helps to stabilize
protein structure by holding the protein in the shape
established by the hydrophobic interactions.
• Protein folding, ionic bonding can occur between the
positively and negatively charged "R" groups that
come in close contact with one another.
• Folding can also result in covalent bonding between
the "R" groups of cysteine amino acids. This type of
bonding forms what is called a disulfide bridge.

13. Quaternary Structure of Protein
• Quaternary Structure refers to the structure of a
protein macromolecule formed by interactions
between multiple polypeptide chains. Each
polypeptide chain is referred to as a subunit.
• Proteins with quaternary structure may consist of
more than one of the same type of protein subunit.
They may also be composed of different subunits.
• Hemoglobin is an example of a protein with
quaternary structure. Hemoglobin, found in
the blood, is an iron-containing protein that binds
oxygen molecules. It contains four subunits: two
alpha subunits and two beta subunits.

14. How to Determine Protein Structure or confirmation of protein structure?
• The three-dimensional shape of a protein is determined by
its primary structure.
• Protein structure characterization and conformation
done on the basis of:
1. Amino Acid Sequence
• Amino Acid sequence normally involving high-resolution
accurate-mass spectrometry (Orbitrap, QToF) coupled
with U(H)PLC technology and Amino Acid Analysis
methods. Proteins or peptides are normally digested with
various enzymes in order to produce suitable peptide
fragments for LC-MS/MS.
2. Peptide Mapping
• Intertek conduct selective fragmentation of the selected
protein into discrete peptides by enzyme or chemical
digestion followed by high-resolution mass spectrometry
(Orbitrap, QToF) analysis. Peptide map methods can then
be validated as UPLC-UV (MS) and used routinely for
batch release or stability studies.

15. 3. Terminal Amino Acid Sequence
• Confirmation of the amino-terminal (N-terminal) and carboxy-terminal (C-terminal) amino acids is
performed by MALDI-MS and high resolution mass spectrometry (Orbitrap, QToF) for product
identification and to establish homogeneity, where understanding the type and extent of modifications at
either termini, is a fundamental aspect of product quality control
4. Disulphide Bridges and Sulfhydryl group
• Where cysteine residues are present in the molecule, our scientists perform a qualitative / semi-
quantitative assessment of the position and extent of expected and mismatched disulphide bridges by
high resolution mass spectrometry (Orbitrap, QToF) and colorimetric tests for free sulfhydryl groups.
5. Post Translational Modifications
• Our post translational modification analysis experts apply a strategic approach to PTM analysis during
early development phases to help you to establish product acceptance criteria and as part of structural
characterization studies and comparability programs, stability studies or release testing.
6. Higher Order Structure
• The higher-order structure is examined using far and near-UV circular dichroism (CD), nuclear
magnetic resonance (NMR), infrared spectroscopy (FTIR), intrinsic fluorescence studies or ultraviolet-
visible (UV-vis, second derivative) spectroscopy. Our biophysical characterisation suite of technologies
also includes protein aggregation studies through dynamic light scattering, SEC with multi-angle laser
light scattering (MALS), sedimentation velocity analytical ultracentrifugation (SV-AUC) and
Differential Scanning Calorimetry (DSC).

16. Properties of Proteins
1. Denaturation: Partial or complete unfolding of the native (natural) conformation of
the polypeptide chain is known as denaturation. This is caused by heat, acids,
alkalies, alcohol, acetone, urea, beta- mercaptoethanol.
2. Coagulation: When proteins are denatured by heat, they form insoluble aggregates
known as coagulum. All the proteins are not heat coagulable, only a few like the
albumins, globulins are heat coagulable
3. Isoelectric pH (pH1): The pH at which a protein has equal number of positive and
negative charges is known as isoelectric pH. When subjected to an electric field the
proteins do not move either towards anode or cathode, hence this property is used to
isolate proteins. The proteins become least soluble at pHI and get precipitated. The
pHI of casein is 4.5 and at this pH the casein in milk curdles producing the curd.
4. Molecular Weights of Proteins: The average molecular weight of an amino acid is
taken to be 110. The total number of amino acids in a protein multiplied by 110 gives
the approximate molecular weight of that protein. Different proteins have different
amino acid composition and hence their molecular weights differ. The molecular
weights of proteins range from 5000 to 109 Daltons. Experimentally the molecular
weight can be determined by methods like gel filtration, PAGE, ultra-centrifugation
or viscosity measurements.

17. Ramachandran Plot for protein structure
• In a polypeptide the main chain N-Calpha and Calpha-C
bonds relatively are free to rotate. These rotations are
represented by the torsion angles phi and psi,
respectively.
• G N Ramachandran used computer models of small
polypeptides to systematically vary phi and psi with the
objective of finding stable conformations. For each
conformation, the structure was examined for close
contacts between atoms. Atoms were treated as hard
spheres with dimensions corresponding to their van der
Waals radii. Therefore, phi and psi angles which cause
spheres to collide correspond to sterically disallowed
conformations of the polypeptide backbone.
• The Ramachandran Plot originally developed in 1963
by G. N. Ramachandran, C. Ramakrishnan, and V.
Sasisekharan, is a way to visualize energetically allowed
regions for backbone dihedral angles ψ against φ
of amino acid residues in protein structure.

18. 1. The white areas correspond to conformations where atoms
in the polypeptide come closer than the sum of their van
der Waals radi.
2. The yellow areas show the allowed regions if slightly
shorter van der Waals radi are used in the calculation, ie
the atoms are allowed to come a little closer together. This
brings out an additional region which corresponds to the
left-handed alpha-helix.
3. L-amino acids cannot form extended regions of left-handed
helix but occassionally individual residues adopt this
conformation The 3(10) helix occurs close to the upper
right of the alpha-helical region and is on the edge of
allowed region indicating lower stability.
4. Disallowed regions generally involve steric hindrance
between the side chain C-beta methylene group and main
chain atoms.
5. Glycine has no side chain and therefore can adopt phi and
psi angles in all four quadrants of the Ramachandran plot.
Hence it frequently occurs in turn regions of proteins
where any other residue would be sterically hindered

20. Concept of Domains, motifs, and folds in protein structure
Proteins are frequently described as consisting of several structural units. These units include
domains, motifs, and folds. Despite the fact that there are about 100,000 different proteins
expressed in eukaryotic systems, there are many fewer different domains, structural motifs
and folds.
1. Structural domain
A structural domain is an element of the protein's
overall structure that is self-stabilizing and often
folds independently of the rest of the protein chain.
Many domains are not unique to the protein products
of one gene or one gene family but instead appear in
a variety of proteins. Domains often are named and
singled out because they figure prominently in the
biological function of the protein they belong to; for
example, the "calcium-binding domain of
calmodulin". Because they are independently stable,
domains can be "swapped" by genetic engineering
between one protein and another to make chimera
proteins.

21. Structural and sequence motif
The structural and sequence motifs refer to short segments of
protein three-dimensional structure or amino acid sequence that
were found in a large number of different proteins.
Supersecondary structure
The supersecondary structure refers to a specific combination of
secondary structure elements, such as β-α-β units or a helix-turn-
helix motif. Some of them may be also referred to as structural
motifs.
Protein fold
A protein fold refers to the general protein architecture, like a
helix bundle, β-barrel, Rossmann fold or different "folds"
provided in the Structural Classification of Proteins database. A
related concept is protein topology that refers to the arrangement
of contacts within the protein.

22. Superdomain
A superdomain consists of two or more
nominally unrelated structural domains that are
inherited as a single unit and occur in different
proteins.
An example is provided by the protein tyrosine
phosphatase domain and C2 domain pair in
PTEN, several tensin proteins, auxilin and
proteins in plants and fungi. The PTP-C2
superdomain evidently came into existence
prior to the divergence of fungi, plants and
animals is therefore likely to be about 1.5
billion years old.

23. Concept of Amino Acids
Amino acids are the building blocks of proteins.
• Among the thousands of amino acids available in
nature, proteins contain only 20 different kinds of
amino acids, all of them are L-alpha-amino acids.
• The same 20 standard amino acids make proteins in all
the living cells, may it either be a virus, bacteria, yeast,
plant or human cell.
• These 20 amino acids combine in different sequences
and numbers to form various kinds of proteins.
• The general formulae for an amino acid can be written
as ‘R-CH-NH2—COOH’. Depending upon the ‘R’
group present in the amino acid it is named
accordingly.

24. Classification of Amino Acids
I. Depending upon the Charge:
Amino acids can be broadly classified into three major groups:
(1) Neutral
(2) Acidic and
(3) Basic.
1. Neutral amino acids: Those amino acids that do not contain any charge on the ‘R’ group.
They are further classified into the following categories:
a) Aliphatic: Those amino acids whose ‘R’ group contains a chain of carbon atoms—Gly,
Ala, Ser, Thr, Val, Leu, lie, Asn, Gin.
b) Aromatic: Those amino acids whose ‘R’ group has a benzene ring—Phe, Tyr, Trp.
c) Heterocyclic: The “R” group has a heterocylic ring, i.e., any of the ring structures which
contain different atoms—Pro, His.
d) Sulphur containing: Those amino acids which contain a sulphur atom-Cys, Met.
2. Acidic amino acids: Those amino acids that contain a negative charge or an acidic group-Asp,
Glu.
3. Basic amino acids: Those amino acids that contain a positive charge or a basic group-Arg, Lys
and His.

25. II. Depending upon the Solubility in Water:
The amino acids can also be grouped into two different categories, depending upon their solubility in water. They
are-
1. Hydrophobic amino acids: Amino acids insoluble in water are known as hydrophobic amino acids. They are—Ala, Val,
Leu, lie, Pro, Met, Phe, Trp.
2. Hydrophilic amino acids: Amino acids soluble in water are known as hydrophilic amino acids. They are—Gly, Ser, Thr,
Cys, Tyr, Asp, Asn, Glu, Gin, Lys, Arg, His.
III. Depending upon their Nutritional Requirements: The amino acids are classified into two groups.
1. Essential amino acids:
Are those which cannot be synthesized by the human body and hence they should be taken through the diet. There are 10
essential amino acids (Arginine*, Histidine, Isoleucine, Leucine, Lysine, Methionine*, Phenylalanine*,
Threonine, Tryptophan, Valine). Among these amino acids, arginine and histidine are known as semi-essential
amino acids.
2. Non-essential amino acids:
These acids are those that can be synthesized in the human body and are not required in the diet. These include gly, ala,
ser, pro, tyr, cys, asp, asn, glu, gin.
Non-essential Alanine, Asparagine, Aspartate, Cysteine, Glutamate, Glutamine, Glycine, Proline, Serine,
Tyrosine
Essential Arginine*, Histidine, Isoleucine, Leucine, Lysine, Methionine*, Phenylalanine*,
Threonine, Tryptophan, Valine

26. Biosynthesis of Amino Acids:
Plants and bacteria can form all 22
amino acids from amphibolic
intermediates.
But humans and other animals cannot
synthesize some of these.
Therefore, these are supplied by the diet
and are termed nutritionally essential
amino acids.
The remainders are synthesized in the
body. Therefore, these are termed
nutritionally nonessential amino acids.
Overview of Amino Acid Biosynthesis
I. Line A (blue line), glycolysis
II. Line B (green line) catabolism of aspartate
III. Line C (pink line) catabolism of threonine
IV. Line D (orange line), pyruvate station
V. Line E (purple line) TCA cycle
VI. Line F (gray line) fructose-6-phosphate
VII. Line G (red line) the anabolism of leucine
VIII. Finally, line H (dark blue) anabolism of
histidine

27. I. Line A (blue line), for example, represents the glycolysis which leads to the formation of
pyruvate, which is a precursor of alanine.
II.Line B (green line) shows the catabolism of aspartate resulting in lysine, threonine and
isoleucine.
III.Line C (pink line) displays the catabolism of threonine which products are converted
into glycine and serine. Serine can in turn be catabolised to yield cysteine,
IV.Line D (orange line), and used, via the pyruvate station, for the biosynthesis of valine
and alanine.
V. Line E (purple line) shows the products formed after the TCA cycle: aspartate and
glutamate, which can be catabolised leading to arginine, proline and glutamine.
VI.Line F (gray line) represents the aromatic biosynthesis pathway from fructose-6-
phosphate.
VII.Line G (red line) shows the anabolism of leucine from a common precursor with
valine, 2-oxoisovalerate.
VIII.Finally, line H (dark blue) represents the anabolism of histidine from fructose-6-
phosphate.

28. Synthesis of Simple Amino acid

29. Catabolism of Amino Acids
List of amino acids converted to carbohydrate and fat or both:
Glycogen
(Glycogenic L-amino acids)
Fat
(“Ketogenig” L-amino acid)
From Acetyl_CoA
Both Glycogen and Fat
(Glycogenic and Ketogenig”
L-amino acid
Alanine Ala A Leucine Leu L Isoleucine Ile I
Arginine Arg R Lysine Lys K
Aspartate Asp D Phenylalanine Phe F
Cysteine Cys C Tyrosine Tyr Y
Glutamate Glu E Tryptophan Trp W
Glycine Gly G
Histidine His H
Hydroxyproline Hyp
Proline Pro P
Methionine Met M
Serine Ser S
Threonine Thr T
Valine Val V

30. Breakdown of glutamine by glutaminase is a source of ammonium ion in the
cell. The other product is glutamate. Glutamate, of course, can be converted by a
transamination reaction to alpha-ketoglutarate, which can be oxidized in the
citric acid cycle.

31. Proteomics
The entire protein component of a given organism is called ‘proteome’, the
term coined by Wasinger in 1995.
The term “proteomics” was first used by Marc Wilkins in 1996 to denote the
“PROTein complement of a genOME”
A proteome is a quantitatively expressed protein of a genome that provides
information on the gene products that are translated, amount of products
and any post translational modifications.

32. Proteome is a defines the complete set of proteins expressed during a cell’s
entire lifetime.
• Proteomics is the study of the proteome; it uses technologies ranging from
genetic analysis to mass spectrometry.
• Proteomics assesses activities, modifications, localization, and interactions of
proteins in complexes.
• Proteome indicates the total proteins expressed by a genome in a cell or tissue.
• Biomarkers detection might allow identification of patients who would benefit
from further evaluation.
• With the development of proteomic techniques, proteome analysis provides a
fast, non-invasive diagnostic tool for patients with various diseases.
• The advent of highly sensitive proteomic technologies can identify proteins
associated with development of diseases well before any clinically identifiable
alteration.
• MS has a high resolving power and identifies proteins with more accuracy.

33. 1. The behavior of gene products is difficult or impossible to predict from gene sequence.
2. Even if a gene is transcribed, its expression may be regulated at the level of translation.
3. Genome sequence tells us about the sequence of proteins but there are many post
translational modification taking place in cells. Genomics fails to explain these
modifications.
4. Proteomic is the field that bridge the gap between genome sequencing and cellular
behavior.
Why Proteomics?

34. Types of Proteomics
1. Structural Proteomics:- The ultimate aim
of this proteomics is to build a body of
structural information that will help predict
the probable structure and potential function
for almost any protein from knowledge of its
coding sequence.
2. Functional proteomics:- It refers to the use
of proteomics techniques to analyze the
characteristics of molecular protein-
networks involved in a living cell.
3. Expression proteomics:- It refers to the
quantitative study of protein expression
between sample differing by some variable.

35. Structural Proteomics
1. Structural proteomics helps to understand three dimensional shape
and structural complexities of functional proteins.
2. It determine either by amino acid sequence in protein or from a gene
this process is known as homology modeling.
3. It identify all the protein present in complex system or protein-protein
interaction.
4. Mass spectroscopy is used for structure determination.

36. Functional proteomics
Functional proteomics is the analysis of protein function
at larger scale.
1. The characterization of protein-protein interactions
are useful to determine the protein functions
2. It also explains the way proteins assemble in bigger
complexes.
3. Technologies such as affinity purification, mass
spectrometry, and the yeast two-hybrid system are
particularly useful in interaction proteomics
Functional Proteomics

37. Expression proteomics
Expression proteomics includes the analysis of protein expression at larger
scale.
• It helps identify main proteins in a particular sample, and those proteins
differentially expressed in related samples—such as diseased vs. healthy
tissue.
• Major Techniques in Expression Proteomics Expression proteomics
1. 1D- and 2D-SDS PAGE
2. MALDI-TOF
3. Liquid chromatography
4. MS-MS
5. SELDI-MS
6. Protein microarray

38. Methods of Proteome Analysis
1. Resolution and identification of proteins are possible
by 2D-PAGE (Polyacrylamide Gel Electrophoresis) and
Mass Spectrometry; comparative 2-D gel approach or
protein chip approach helps to identify the proteins in
up or down regulated system.
2. Matrix assisted laser desorption ionization time-
of-flight mass spectrometry (MALDI-TOF MS).
The hybrid electrospray ionization (ESI) method of
quadrupole TOF-MS with its increased mass
accuracy is becoming increasingly established.

39. Tools and technology of proteomics
Tools Techniques Involved In Separations
1. 2-DGE (two-dimensional gel electrophoresis)
2. MudPIT (multidimensional protein
identification technology)
3. Protein chips
4. 2-Hybrid systems
5. ICAT (isotope-coded affinity tagging)
6. ABPs (activity-based probe)

40. Technology of Proteomics
The measurement of real gene expression, the proteins should be analyzed.
Before the identification and measurement of the activity, all the proteins in
a proteome for any instant should be separated from each other.
• A Typical Proteomics Experiment (e.g. Protein Expression Profiling)
can be Divided into the following Categories:
1. Separation and isolation of protein
2. The acquisition of protein structural information for protein
identification and characterization
3. Database utilization.

41. (i) Protein Separation and Isolation
An essential component of proteomics is the protein
electrophoresis, the most effective way to resolve a
complex mixture of proteins.
• In one dimensional gel electrophoresis (1-DE),
proteins are resolved on the basis of their molecular
masses. Proteins are stable enough during 1-DE due
to their solubility in sodium dodycyl sulphate (SDS).
Proteins with molecular mass of 10-300 kDa can be
easily separated through 1-DE.
• As a two dimensional gel electrophoresis (2-DE) can
resolve thousands of proteins, that have gone under
some post-translational modifications as well as
protein expression of any two samples can be
compared quantitatively and qualitatively.

42. (ii) Acquisition of Protein Structures: Protein Identification
A. Edman Sequencing (ES)
• One of the earliest methods used for
protein identification was micro
sequencing by Edman chemistry to
obtain N-terminal amino acid
sequences. This technique was
introduced by Edman in 1949.
• In Edman sequencing, N-terminal of
a protein is sequenced to determine
its true start site.
• Edman sequencing is more
applicable sequencing method for the
identification of proteins separated
by SDS-Polyacrylamide gel
electrophoresis.

43. B. Mass Spectrometry (MS)
• The most significant breakthrough in proteomics has been the mass
spectrometric identification of gel-separated proteins.
• Due to its high sensitivity levels, identification of proteins in protein
complexes/mixtures and high throughput, this technique has been proved far
better than ES.
•In mass spectrometry,
proteins are digested into
peptides in the gel itself
by suitable protease such
as trypsin, because
proteins, as such, are
difficult to elute out from
the gels

44. There are Two Main Approaches to Mass Spectrometric Protein
Identification
1. Electrospray ionization: In Electrospray ionization (ESI) involves the
fragmentation of individual peptides followed by direct ionization through
electrospray in a tandem mass spectrometer. In ESI, a liquid sample flows from a
microcapillary tube into the orifice of the mass spectrometer, where a potential
difference between the capillary and the inlet to the mass spectrometer results in
the generation of a fine mist of charged droplets. It has the ability to resolve
peptides in a mixture, isolate one species at a time and dissociate it into amino or
carboxy-terminal containing fragments designated ‘b’ and ‘y’, respectively.
2. Peptide mass mapping: In peptide mass mapping approach (Henzel et al. 1993)
the mass spectrum of the eluted peptide mixture is acquired, which result in a
peptide mass fingerprint of the protein being studied. The mass spectrum is
obtained by a relatively simple ‘mass spectrometric method-matrix assisted laser
desorption/ ionization’ (MALDI).

45. Steps involved in mass spectrometry

46. Mass spectrometry
Mass spectrometry (MS) is an analytical
technique that ionizes chemical species and sorts
the ions based on their mass-to-charge ratio.
Basic principle
1. Mass spectroscopy is the most accurate method for determining the molecular mass of
the compound and its elemental composition.
2. In this technique, molecules are bombarded with a beam of energetic electrons.
3. The molecules are ionised and broken up into many fragments, some of which are
positive ions.
4. Each kind of ion has a particular ratio of mass to charge, i.e. m/e ratio(value). For
most ions, the charge is one and thus, m/e ratio is simply the molecular mass of the
ion.
In simpler terms, a mass spectrum measures the masses within a sample. Mass
spectrometry is used in many different fields and is applied to pure samples as
well as complex mixtures.

47. MALDI-TOF Mass Spectrophotometry Instrumentation

48. 1. Ionization
• The atom is ionised by knocking one or more
electrons off to give a positive ion. (Mass
spectrometers always work with positive ions).
• The particles in the sample (atoms or
molecules) are bombarded with a stream of
electrons to knock one or more electrons out of
the sample particles to make positive ions.
• Most of the positive ions formed will carry a
charge of +1.
• These positive ions are persuaded out into the
rest of the machine by the ion repeller which is
another metal plate carrying a slight positive
charge
Steps involve in Mass Spectrophotometry

49. 2. Acceleration
The ions are accelerated so that they
all have the same kinetic energy.
The positive ions are repelled away
from the positive ionisation chamber
and pass through three slits with
voltage in the decreasing order.
The middle slit carries some
intermediate voltage and the final at
‘0’ volts.
All the ions are accelerated into a
finely focused beam

50. 3. Deflection
• The ions are then deflected by a magnetic field
according to their masses. The lighter they are, the
more they are deflected.
• The amount of deflection also depends on the
number of positive charges on the ion -The more
the ion is charged, the more it gets deflected.
• Different ions are deflected by the magnetic field
by different amounts.
• The amount of deflection depends on: The mass
of the ion: Lighter ions are deflected more than
heavier ones.
• The charge on the ion: Ions with 2 (or more)
positive charges are deflected more than ones with
only 1 positive charge.

51. 4. Detection
• The beam of ions passing through the
machine is detected electrically.
• When an ion hits the metal box, its
charge is neutralised by an electron
jumping from the metal on to the ion.
• That leaves a space amongst the
electrons in the metal, and the electrons
in the wire shuffle along to fill it.
• A flow of electrons in the wire is
detected as an electric current which can
be amplified and recorded. The more
ions arriving, the greater the current.
Only ion stream B makes it right through the machine to the ion
detector. The other ions collide with the walls where they will pick up
electrons and be neutralised. They get removed from the mass
spectrometer by the vacuum pump.

52. Practical Applications of Proteomics
1. Post-Translational Modifications:
• Proteomics studies involve certain unique features as the ability to analyze
post- translational modifications of proteins. These modifications can be
phosphorylation, glycosylation and sulphation as well as some other
modifications involved in the maintenance of the structure of a protein.
2. Protein-Protein Interactions:
• The major attribution of proteomics towards the development of protein
interactions map of a cell is of immense value to understand the biology of a
cell. The knowledge about the time of expression of a particular protein, its
level of expression, and, finally, its interaction with another protein to form
an intermediate for the performance of a specific biological function is
currently available.

53. 3. Protein Expression Profiling:
• The largest application of proteomics continues to be protein expression
profiling. The expression levels of a protein sample could be measured by
2-DE or other novel technique such as isotope coded affinity tag (ICAT).
• Biochemical genomics approach Method, which uses parallel biochemical
analysis of a proteome comprised of pools of purified proteins in order to
identify proteins and the corresponding ORFs responsible for a biochemical
activity.
• Functional protein microarrays, in which individually purified proteins are
separately spotted on a surface such as a glass slide and then analyzed for
activity.
• These approach has huge potential for rapid high-throughput analysis of
proteomes and other large collections of proteins, and promises to transform
the field of biochemical analysis.

54. 4. Molecular Medicine:
• Proteomic technologies will play an important role in drug discovery,
diagnostics and molecular medicine because of the link between
genes, proteins and disease.
• This aims to discover the proteins with medical relevance to identify a
potential target for pharmaceutical development, a marker(s) for
disease diagnosis or staging, and risk assessment—both for medical
and environmental studies.

55. Protein–Protein Interaction Analysis
Protein–protein interactions (PPIs) are the physical contacts of high
specificity established between two or more protein molecules as a result of
biochemical events steered by interactions that include electrostatic forces,
hydrogen bonding and the hydrophobic effect.
Many are physical contacts with molecular associations between chains that
occur in a cell or in a living organism in a specific biomolecular context.

56. Need to study Protein-Protein Interaction
1. Protein sequence and structure—used to discover motifs that predict
protein function
2. Evolutionary history and conserved sequences—identifies key
regulatory residues
3. Expression profile—reveals cell-type specificity and how expression is
regulated
4. Post-translational modifications—phosphorylation, acylation,
glycosylation and ubiquitination suggest localization, activation and/or
function
5. Interactions with other proteins—function may be extrapolated by
knowing the function of binding partners
6. Intracellular localization—may allude to the function of the protein

57. Types of protein–protein interactions
Protein interactions are fundamentally two type (Both types of interactions
can be either strong or weak)
1. Stable
2. Transient
• Stable interactions are those associated with proteins that are purified as
multisubunit complexes, and the subunits of these complexes can be
identical or different.
• Hemoglobin and core RNA polymerase are examples of multi-subunit
interactions that form stable complexes.
• Transient interactions are expected to control the majority of cellular
processes.
• As the name implies, transient interactions are temporary in nature and
typically require a set of conditions that promote the interaction, such as
phosphorylation, conformational changes or localization to discrete areas of
the cell.

58. Biological effects of protein–protein interactions
The result of two or more proteins that interact with a specific functional objective can
be demonstrated in several different ways. The measurable effects of protein
interactions have been outlined as follows:
1. Alter the kinetic properties of enzymes, which may be the result of subtle changes
in substrate binding or allosteric effects
2. Allow for substrate channeling by moving a substrate between domains or
subunits, resulting ultimately in an intended end product
3. Create a new binding site, typically for small effector molecules
4. Inactivate or destroy a protein
5. Change the specificity of a protein for its substrate through the interaction with
different binding partners, e.g., demonstrate a new function that neither protein can
exhibit alone
6. Serve a regulatory role in either an upstream or a downstream event

59. Common methods to analyze the various
types of protein interactions
Method Protein–protein interactions type
Co-immunoprecipitation (co-IP) Stable or strong
Pull-down assay Stable or strong
Crosslinking protein interaction analysis Transient or weak
Label transfer protein interaction analysis Transient or weak
Far–western blot analysis Moderately stable

60. Co-immunoprecipitation (co-IP)
• Co-immunoprecipitation (co-IP) is a popular technique for protein interaction
discovery. Co-IP is conducted in essentially the same manner as
an immunoprecipitation (IP) of a single protein, except that the target protein
precipitated by the antibody, also called the "bait", is used to co-precipitate a
binding partner/protein complex, or "prey", from a lysate. Essentially, the
interacting protein is bound to the target antigen, which is bound by the
antibody that is immobilized to the support.
Immuno-precipitated proteins and their
binding partners are commonly detected
by sodium dodecyl sulfate–
polyacrylamide gel electrophoresis (SDS-
PAGE) and western blot analysis.

61. Crosslinking protein interaction analysis
Most protein–protein interactions are transient, occurring only briefly as part of a single
cascade or other metabolic function within cells. Crosslinking interacting can be used to
analyze the protein–protein interaction while maintaining the original interacting
complex.
Homobifunctional, amine-
reactive crosslinkers can
be added to cells to
crosslink potentially
interacting proteins
together, which can then
be analyzed after lysis by
western blotting.

62. Far–western blot analysis
• Just as pull-down assays differ from co-IP in the detection of protein–protein
interactions by using tagged proteins instead of antibodies, so is far–western
blot analysis different from western blot analysis, as protein–protein
interactions are detected by incubating electrophoresed proteins with a
purified, tagged bait protein instead of a target protein-specific antibody,
respectively. The term "far" was adopted to emphasize this distinction.

63. Label transfer protein interaction analysis
Label transfer involves crosslinking
interacting molecules (i.e., bait and prey
proteins) with a labeled crosslinking agent
and then cleaving the linkage between the
bait and prey so that the label remains
attached to the prey.
This method is particularly valuable
because of its ability to identify proteins
that interact weakly or transiently with the
protein of interest.
New non-isotopic reagents and methods
continue to make this technique more
accessible and simple to perform by any
researcher.

64. Pull-down assays
Pull-down assays are similar in methodology to co-immunoprecipitation
because of the use of beaded support to purify interacting proteins.
The difference between these two
approaches, though, is that while
co-IP uses antibodies to capture
protein complexes, pull-down
assays use a "bait" protein to purify
any proteins in a lysate that bind to
the bait.
Pull-down assays are ideal for
studying strong or stable
interactions or those for which no
antibody is available for co-
immunoprecipitation.

65. Part-II
Nitrogen fixation and N & S
Metabolism

66. Nitrogen fixation
• Nitrogen fixation, any natural or industrial process that causes free nitrogen (N2),
which is a relatively inert gas plentiful in air, to combine chemically with other
elements to form more-reactive nitrogen compounds such as ammonia, nitrates, or
nitrites.
• Nitrogen fixation is carried out naturally in soil by microorganisms termed
diazotrophs that include bacteria such as Azotobacter and archaea.
• All biological nitrogen fixation is effected by enzymes called nitrogenases. These
enzymes contain iron, often with a second metal, usually molybdenum but
sometimes vanadium.
The phenomenon of conversion of free nitrogen (molecular and elemental) into
nitrogenous compounds (to make it available to the plants for absorption) is called
nitrogen fixation.
• This incorporation of mineral nutrients into organic substances such as pigments,
enzyme cofactors, lipids, nucleic acids, and amino acids is termed nutrient
assimilation.

67. Nitrogen's functions in plants
1. Nitrogen is an essential element of all the amino acids in plant structures
which are the building blocks of plant proteins, important in the growth and
development of vital plant tissues and cells like the cell membranes and
chlorophyll.
2. Nitrogen is a component of nucleic acid that forms DNA a genetic material
significant in the transfer of certain crop traits and characteristics that aid in
plant survival. It also helps hold the genetic code in the plant nucleus.
3. Chlorophyll being an organelle essential for carbohydrate formation by
photosynthesis and a substance that gives the plant their green color,
nitrogen is a component in it that aids in enhancing these features.
4. Nitrogen is essential in plant processes such as photosynthesis. Thus, plants
with sufficient nitrogen will experience high rates of photosynthesis and
typically exhibit vigorous plant growth and development.

68. Types of nitrogen fixation
1. Physical Nitrogen Fixation:
A. Natural Nitrogen Fixation
Natural processes fix about 190×1012 g yr–1 of nitrogen through the following
processes
Lightning: Lightning is responsible for about 8% of the nitrogen fixed as nitric acid
(HNO3). This nitric acid subsequently falls to Earth with rain.
Photochemical reactions: Approximately 2% of the nitrogen fixed derives from
photochemical reactions between gaseous nitric oxide and ozone (O3).
B. Industrial Nitrogen Fixation: Ammonia is produced industrially by direct
combination of nitrogen with hydrogen (obtained from water) at high
temperature and pressure. Later, it is converted into various kinds of fertilizers,
such as urea etc.
1. Physical Nitrogen Fixation
2. Biological nitrogen fixation

69. 2. Biological nitrogen fixation:
The remaining 90% results from biological nitrogen fixation, in which bacteria
or blue-green algae (cyanobacteria) fix N2 into ammonium (NH4
+).
The conversion of atmospheric nitrogen into the nitrogenous compounds
through the agency of living organisms is called biological nitrogen
fixation.
Or
The phenomenon of reduction of inert gaseous di-nitrogen (N2) into
ammonia (NH3) through the agency of some microorganisms so that it can
be made available to the plants is called as biological nitrogen fixation or
diazotrophy.
• The process is carried out by two main types of microorganism: those which
live in close symbiotic association with other plants and those which are “free
living” or non-symbiotic.
Biological nitrogen fixation (BNF) is the process whereby atmospheric
nitrogen is reduced to ammonia in the presence of nitrogenize.

70. Microorganism as Nitrogen Fixers
Among the earth’s organisms, only some prokaryotes like bacteria and cyanobacteria
can fix atmosphere nitrogen. They are called nitrogen fixers or diazotrophs. They fix
about 95% of the total global nitrogen fixed annually (-200 million matric tones) by
natural process.
Diazotrophs may be asymbiotic (free living) or symbiotic
i) Free Living Nitrogen Fixing Bacteria:
• Azotobacter, Beijerinckia (bothaerobic) and
Clostridium (anaerobic) are saprophytic
bacteria that perform nitrogen fixation.
• Desulphovibrio is chemotrophic nitrogen
fixing bacterium. Rhodopseudomonas,
Rhodospirillum and Chromatium are
nitrogen fixing photoautotrophic bacteria.
• These bacteria add up to 10-25 kg, of
nitrogen/ha/annum.
(ii) Free living Nitrogen Fixing Cyanobacteria:
• Many free living blue-green algae (cyanobacteria)
perform nitrogen fixation, e.g., Anabaena, Nustoc,
Aulosira, Cylmdrospermum, Trichodesmium.
• These are also important ecologically as they live in
water-logged sods where denitrifing bacteria can be
active.
• Aulosira fertilissima is the most active nitrogen fixer
in Rice fields, while Cylindrospermum is active in
sugarcane and maize fields. They add 20-30 kg
Nitrogen/ha/annum.

71. Contrasting habitats of free-living and
symbiotic nitrogen fixation.
(a) FLNF is carried out by a diverse array of N
fixers living in a community, while
symbiotic N fixation is performed only by a
few bacteria (e.g., rhizobia and Frankia)
living in a population.
(b) FLNF is supported by dissolved organic
carbon (DOC) in the soil, a variable and
complex C source, while symbiotic N fixers
receive a constant supply of simple C
compounds (i.e., succinate) directly from
the host plant.
(c) Oxygen concentration in the rhizosphere is
highly variable and driven by soil structure
and texture and respiration by microbes and
roots. Conversely, symbiotic N fixers are
supplied oxygen at low concentrations by
their host plant.
(d) Nutrients necessary to support FLNF (e.g., P, Fe, Mo, and V) must be acquired by the diazotroph. However, these
nutrients are delivered to symbiotic N fixers by the host plant.
(e) Diazotrophs in the rhizosphere can access N from soil and FLNF, while all symbiotically fixed N is delivered to the
plant.

72. (iii) Symbiotic Nitrogen Fixing Cyanobacteria:
Anabaena and Nostoc species are common symbionts in lichens, Anthoceros, Azolla and
cycad roots. Azolla pinnata (a water fern) has Anabaena azollae in its fronds. It is often
inoculated to Rice fields for nitrogen fixation.
(iv) Symbiotic Nitrogen Fixing Bacteria:
Rhizobium is aerobic, gram negative nitrogen
fixing bacterial symbionts of Papilionaceous
roots. Sesbania rostrata has Rhizobium in
root nodules and Aerorhizobium in stem
nodules. Frankia is symbiont in root nodules
of many non-leguminous plants like
Casuarina and Alnus.
Xanthomonas and Mycobacterium occur as
symbiont in the leaves of some members of
the families Rubiaceae and Myrsinaceae
(e.g., Ardisia).

73. A. Symbiotic Nitrogen Fixation
Both Rhizobium sp. and Frankia are free living in soil, but only as symbionts,
can fix atmospheric di-nitrogen.
The symbiotic nitrogen fixation can be discussed under following steps
(i) Nodule formation
(ii)Mechanism of nitrogen fixation
(i) Nodule formation
It involves multiple interactions between free-living
soil Rizobium and roots of the host plant. The
important stages involved in nodule formation are as
follows-Host Specificity: A variety of microorganisms
exist in the rhizosphere (i.e. immediate vicinity of
roots) of host roots.

74. I. The roots of young leguminous plants secrete
a group of chemical attractants like flavonoids
and betaines. In response to these chemical
attractants specific rhizobial Tells migrate
towards the root hairs and produce nod
(nodulation) factors.
II. The nod factors found on bacterial surface
bind to the lectin proteins present on the
surface of root hairs. This lectinnod factor
interaction induces growth and curling of root
hairs around Rhizobia.
III.At these regions wall degrades in response to
node-factors and Rhizobia enter the root hair
invagination of plasma membrane called
infection thread.

75. IV. The infection thread filled with
dividing Rhizobia elongate
through the root hair and later
branched to reach different cortical
cells.
V. The Rhizobia are released into the
cortical cells either single or in
groups enclosed by a membrane.
VI.The Rhizobia stop dividing, loose
cell wall and become nitrogen fixing
cells as led bacteroids.
VII.The membrane surrounding the
bacteroids is called peribacteroid
membrane. The infected cortical
cells divide to form nodule

76. The infection process during nodule organogenesis.
A. Rhizobia bind to an emerging root hair in
response to chemical attractants sent by the
plant.
B. In response to factors produced by the bacteria,
the root hair exhibits abnormal curling growth,
and rhizobia cells proliferate within the coils.
C. Localized degradation of the roothair wall leads
to infection and formation of the infection
thread from Golgi secretory vesicles of root
cells.
D. The infection thread reaches the end of the cell,
and its membrane fuses with the plasma
membrane of the root hair cell.
E. Rhizobia are released into the apoplast and
penetrate the compound middle lamella to the
subepidermal cell plasma membrane, leading to
the initiation of a new infection thread, which
forms an open channel with the first.
F. The infection thread extends and branches until
it reaches target cells, where vesicles composed
of plant membrane that enclose bacterial cells
are released into the cytosol.

77. (ii) Mechanism of nitrogen fixation
The nodule serves as site for N2 fixation.
• It contains all the necessary bio-chemicals such as the enzyme complex
called nitrogenase and leghaemoglobin (leguminous haemoglobin).
• The nitrogenase has 2 components i.e. Mo-Fe protein
(molybdoferredoxin) and Fe-protein (azoferredoxin).
• The nitrogenase catalyzes the conversion of atmosphere di-nitrogen (N2)
to 2NH3. The ammonia is the first stable product of nitrogen fixation.
Mg2-
N2 + 8e- + 8H+ + 16 ATP 2NH3 + H2 + 16 ADP + 16 Pi
Nitrogenase

78. The nitrogenase is extremely sensitive to oxygen. To protect these enzymes, nodule
contains an oxygen scavenger called leghaemoglobin (Lb), which is a reddish-pink
pigment located outside the peribacteroid membrane or located in between bacteroids.
1. During nitrogen fixation, the free di-
nitrogen first bound to MoFe protein
and stepwise formed to form
ammonia (NH3) which is protonated
and form NH4
+.
2. In this process ferredoxin serves as an
electron donor to Fe-protein
(nitrogenase reductase) which in turn
hydrolyzes ATP and reduce MoFe
protein, the MoFe protein in Turn
reduce the substrate N2.
3. The electrons and ATP are provided
by photosynthesis and respiration of
the host cells.

79. Signals for Symbiosis
Nod Factors Produced by Bacteria Act as Signals for Symbiosis
Nod factors are lipochitin oligosaccharide signal molecules, all of which have a chitin β-1→4-
linked Nacetyl-D-glucosamine backbone and a fatty acyl chain on the C-2 position of the
nonreducing sugar.
Three of the nod genes (nodA, nodB, and nodC) encode enzymes (NodA, NodB, and NodC, respectively) that
are required for synthesizing this basic structure:
1. NodA is an N-acyltransferase that catalyzes the addition of a fatty acyl chain.
2. NodB is a chitin-oligosaccharide deacetylase that removes the acetyl group from the terminal nonreducing
sugar.
3. NodC is a chitin-oligosaccharide synthase that links N-acetyl-D-glucosamine monomers.
Host-specific nod genes that vary among rhizobial species are involved in the modification of the fatty acyl
chain or the addition of groups important in determining host specificity:
• NodE and NodF determine the length and degree of saturation of the fatty acyl chain; those of Rhizobium
leguminosarum bv. viciae and R. meliloti result in the synthesis of an 18:4 and a 16:2 fatty acyl group,
respectively.
• Other enzymes, such as NodL, influence the host specificity of Nod factors through the addition of specific
substitutions at the reducing or nonreducing sugar moieties of the chitin backbone.

80. • Rhizobia in the surrounding soil
secrete NodD, which is a protein that
recognizes the flavonoids secreted by
the plants.
• Interaction with the flavonoids
activates NodD, then NodD returns to
the rhizobium to induce the
transcription of nodABC genes.
• nodABC proteins modify the Nod
factor in response to the flavonoids
released by the plant.
• The Nod factors interact with the
plant and cause root hair curling and
root hair deformation. This leads to
the formation of an infection thread
into the root hair.
• Nod factors cause cortical cell division in the primordium of the plant and form a nodule.
• The rhizobia move through the plant root hair via the infection thread to the nodule within the plant.

81. Pairing Mechanisms of HOST and PATHOGEN
• A particular legume host responds to a specific Nod factor. The legume
receptors for Nod factors appear to be special lectins (sugar-binding proteins)
produced in the root hairs.
• Nod factors activate these
lectins, increasing their
hydrolysis of
phosphoanhydride bonds of
nucleoside di and triphosphates.
• This lectin activation directs
particular rhizobia to
appropriate hosts and facilitates
attachment of the rhizobia to the
cell walls of a root hair.

82. Nitrogen assimilation
Nitrogen assimilation is the formation of
organic nitrogen compounds like amino acids
from inorganic nitrogen compounds present
in the environment.
• Organisms like plants, fungi and certain
bacteria that cannot fix nitrogen gas (N2)
depend on the ability to assimilate nitrate or
ammonia for their needs.
• Other organisms, like animals, depend
entirely on organic nitrogen from their food.
1. NITRATE ASSIMILATION
2. AMMONIUM ASSIMILATION

83. NITRATE ASSIMILATION
A. Conversion of Nitrate to Nitrite:
• Plants assimilate most of the nitrate absorbed by their roots into organic
nitrogen compounds. The first step of this process is the reduction of nitrate to
nitrite in the cytosol (Oaks 1994).
• The enzyme nitrate reductase catalyzes this reaction:
NO3
– + NAD(P)H + H + + 2 e– → NO2– + NAD(P)+ + H2O
Nitrate reductase dimer, illustrating the three binding
domains whose polypeptide sequences are similar in
eukaryotes: molybdenum complex (MoCo), heme,
and FAD. The NADH binds at the FADbinding
region of each subunit and initiates a two-electron
transfer from the carboxyl (C) terminus, through
each of the electron transfer components, to the
amino (N) terminus. Nitrate is reduced at the
molybdenum complex near the amino terminus.

84. B. Conversion of Nitrite to ammonia:
• Nitrite (NO2
–) is a highly reactive, potentially toxic ion. Plant cells
immediately transport the nitrite generated by nitrate reduction from the
cytosol into chloroplasts in leaves and plastids in roots.
• In these organelles, the enzyme nitrite reductase reduces nitrite to ammonium
according to the following overall reaction:
NO2
– + 6 Fdred + 8 H+ + 6e–→ NH4
+ + 6 Fdox + 2 H2O
Photosynthetic electron flow, via
ferredoxin, to the reduction of nitrite
by nitrite reductase. The enzyme
contains two prosthetic groups,
Fe4S4 and heme, which participate in
the reduction of nitrite to
ammonium.

85. AMMONIUM ASSIMILATION
• Plant cells avoid ammonium toxicity by rapidly converting the ammonium
generated from nitrate assimilation or photorespiration into amino acids.
• The primary pathway for this conversion involves the sequential actions of
glutamine synthetase and glutamate synthase.
Conversion of Ammonium to Amino Acids Requires Two Enzymes
A. Glutamine synthetase (GS) combines ammonium with glutamate to form glutamine.
Glutamate + NH4
+ + ATP → glutamine + ADP + Pi
GS-GOGAT pathway

86. B. Elevated plastid levels of glutamine stimulate the activity of glutamate synthase (also known as
glutamine:2-oxoglutarate aminotransferase, or GOGAT). This enzyme transfers the amide group
of glutamine to 2-oxoglutarate, yielding two molecules of glutamate. Plants contain two types of
GOGAT: One accepts electrons from NADH; the other accepts electrons from ferredoxin (Fd):
Glutamine + 2-oxoglutarate + NADH + H+ → 2 glutamate + NAD+
Glutamine + 2-oxoglutarate + Fdred → 2 glutamate + Fdox
GS-GOGAT pathway

87. 2. Ammonium Can Be Assimilated via an Alternative Pathway
Glutamate dehydrogenase (GDH) catalyzes a reversible reaction that
synthesizes or deaminates glutamate:
2-Oxoglutarate + NH4
+ + NAD(P)H ↔ Glutamate + H2O + NAD(P)+
GDH pathway

88. Sulfate uptake, transport and assimilation
• Sulphur, which is also spelt sulfur, is a chemical element with symbol S and
atomic number of 16. It is a non-metal and is readily available at room
temperature as a bright yellow crystalline solid.
• Sulfur is an essential element for growth and physiological functioning of
plants. However, its content strongly varies between plant species and it
ranges from 0.1 to 6% of the plants' dry weight.
Sources of Sulphur
Sulphur can be found from the following sources:
I. Extraction from beneath the earth crust - this is
the most important source.
II. From natural gas - this is the second most
important source.
III.From other processes - example, as a by-product
of the purification of crude coal gas and the
refining of petroleum.

89. Physiological Functions of Sulfur:
• Sulfur compounds are also of great importance for food quality and for the
production of phyto-pharmaceutics. Sulfur deficiency will result in the loss
of plant production, fitness and resistance to environmental stress and pests.
1. Sulfur is present primarily in the cell protein in the form of cysteine and methio-
nine.
2. The cysteine is important in protein structure and in enzymic activity.
3. Methionine is the principal methyl group donor in the body. The activated form
of methionine, S-adenosylmethionine, is the precursor in the synthesis of large
number of methylated compounds which are involved in intermediary metabolism
and detoxification mechanism.
4. Sulfur is a constituent of coenzyme A and lipoic acid which are utilized for the syn-
thesis of acetyl-COA and S-acetyl lipoate, respectively.
5. Sulfur is a component of other organic compounds, such as heparin, glutathione,
thiamine, biotin, ergothioneine, taurocholic acid, sulfocyanides, indoxyl sulfate,
chondroitin sulfate, insulin, penicillin, anterior pituitary hormones and melanin.

90. Sulphur Uptake and Assimilation
Conversion of inorganic sulphur compounds such as SO4
2-
into sulfur-containing organic compounds such as cysteine
by plants is called as sulfur or sulfate assimilation.
Root plastids contain all sulfate reduction enzymes, but the
reduction of sulfate to sulfide and its subsequent
incorporation into cysteine predominantly takes place in the
shoot, in the chloroplasts.
The organic sulfur is present in the protein fraction (up to
70% of total sulfur), as cysteine and methionine (two amino
acids) residues.
Plants contain a large variety of other organic sulfur compounds, as thiols (glutathione),
sulfolipids and secondary sulfur compounds (alliins, glucosinolates, phytochelatins), which
play an important role in physiology and protection against environmental stress and pests.
Sulfate Is the Absorbed Form of Sulfur in Plants
Most of the sulfur in higher-plant cells derives from sulfate (SO4
2–) absorbed via an H+–SO4
2
symporter from the soil solution. Sulfate in the soil comes predominantly from the weathering
of parent rock material.

91. Sulfate Assimilation Requires the Reduction of Sulfate to Cysteine
The first step in the synthesis of sulfur-containing organic compounds
is the reduction of sulfate to the amino acid cysteine.
1. Activation begins with the reaction between sulfate and ATP to
form 5′-adenylylsulfate (which is sometimes referred to as
adenosine-5′-phosphosulfate and thus is abbreviated APS) and
pyrophosphate (PPi).
SO4
2– + Mg-ATP →APS + Ppi
2. The reduction of APS is a multistep process that occurs
exclusively in the plastids. APS reductase transfers two electrons
apparently from reduced glutathione (GSH) to produce sulfite
(SO3
2–):
APS + 2 GSH → SO3
2– + 2H+ + GSSG + AMP
3. where GSSG stands for oxidized glutathione. (The SH in GSH and
the SS in GSSG stand for S-H and S—S bonds, respectively.)
Second, sulfite reductase transfers six electrons from ferredoxin
(Fdred) to produce sulfide (S2–):
SO3
2– + 6 Fdred → S2– + 6 Fdox

92. 5. The resultant sulfide then reacts with O-acetylserine
(OAS) to form cysteine and acetate. The O-acetylserine
that reacts with S2– is formed in a reaction catalyzed by
serine acetyltransferase:
Serine + acetyl-CoA→ OAS + CoA
6. The reaction that produces cysteine and acetate is
catalyzed by OAS thiol-lyase:
OAS + S2– → cysteine + acetate
7. The sulfation of APS, localized in the cytosol, is the
alternative pathway. First, APS kinase catalyzes a
reaction of APS with ATP to form 3′-phosphoadenosine-
5′-phosphosulfate (PAPS).
APS + ATP → PAPS + ADP
Sulfotransferases then may transfer the sulfate group from PAPS to
various compounds, including choline, brassinosteroids, flavonol, gallic
acid glucoside, glucosinolates, peptides, and polysaccharides.

93. • The enzyme ATP sulfurylase cleaves
pyrophosphate from ATP and replaces it
with sulfate.
• Sulfide is produced from APS through
reactions involving reduction by
glutathione and ferredoxin.
• The sulfide or thiosulfide reacts with O-
acetylserine to form cysteine. Fd,
ferredoxin; GSH, glutathione, reduced;
GSSG, glutathione, oxidized.
Structure and pathways of compounds involved in sulfur assimilation
Sulfate Assimilation Occurs
Mostly in Leaves
The reduction of sulfate to cysteine
changes the oxidation number of
sulfur from +6 to –4, thus entailing
the transfer of 10 electrons.
Glutathione, ferredoxin, NAD(P)H,
or Oacetylserine may serve as
electron donors at various steps of
the pathway.

94. Methionine Is Synthesized from Cysteine
Methionine, the other sulfur-containing amino acid found in proteins, is
synthesized in plastids from cysteine. After cysteine and methionine are
synthesized, sulfur can be incorporated into proteins and a variety of other
compounds, such as acetyl-CoA and S-adenosylmethionine.
The latter compound is important in the synthesis of ethylene and in
reactions involving the transfer of methyl groups, as in lignin synthesis.
In Chloroplast
i. Synthesis of methionine from cysteine also occurs in chloroplasts in leaves.
ii. The sulfur-containing organic compounds produced after sulfur (sulfate) assimilation are exported to other
parts of the plant, such as root and shoot apices and fruits, through phloem chiefly as glutathione.
iii. It is believed that glutathione may also coordinate absorption of sulfate by roots and its assimilation by shoot.
In Plastid
i. In the first step, cysteine reacts with O-phosphohomoserine in the
presence of the enzyme cystathionine γ-synthase to form
cystathionine. Inorganic phosphate (Pi) is released in the reaction.
ii. In the next step, the enzyme cystathionine β-Iyase splits
cystathionine into homocysteine, NH3 and pyruvate.
iii. Finally, homocysteine is methylated by the enzyme methionine
synthase to form methionine.