1. Characterization of Radical S-adenosyl-L-methionine
Epimerase, NeoN
Samender Randhawa1, John Zhang1, Daniel P. Dowling1
1Department of Chemistry, University of Massachusetts Boston, Boston, MA
Funding for this research was provided by the Oracle Education Foundation grant to the CSM
Abstract
A vast number of enzymes are being characterized that belong to a
superfamily known as radical S-adenosyl-L-methionine (SAM) enzymes, whose
members contain a [4Fe–4S] cluster ligated by three cysteine residues. Radical SAM
enzymes generally initiate catalysis by reductively cleaving SAM, yielding a
molecule of L-methionine and the 5′-deoxyadenosyl radical species. This radical
species can then be utilized in Nature to initiate a numerous amount of radical
chemistry through substrate hydrogen atom abstraction. A number of radical SAM
enzymes, including the Neomycin C epimerase (NeoN), contain additional iron–
sulfur clusters that are required for the reactions they catalyze.
The goal of this work is to understand the underlying biochemical basis for
NeoN catalytic activity. To accomplish this, the technique of X-ray crystallography
will be utilized to obtain atomic resolution information for this system. We are
interested in how NeoN is capable of specifically catalyzing the epimerization of
one, stereospecific site of neomycin C, which contains multiple possible sites that
could be acted upon. Future studies of this system may lead to the development of
bioengineered epimerases, and we hypothesize that NeoN could be used as a
biocatalyst tool in the future for developing specific epimerases that could produce
novel compounds. Here, initial experiments to clone the neoN gene from
Streptomyces fradiae into an expression platform are presented.
Introduction
Radical S-adenosyl-L-methionine (SAM) enzymes (RS enzymes) carryout a
variety of biological functions, such as synthesis of cofactors, and antibiotics1. To
date 113,744 RS functional units have been characterized both biochemically and
structurally2. The majority of the RS enzymes adopt a full or partial triosephosphate
isomerase barrel (TIM) fold, with one of the first reported RS enzyme structures,
that of biotin synthase (BioB), adopting a full TIM barrel fold3 (Fig.1). The presence
of a [4Fe- 4S] cluster bound to a conserved cysteine triad is common to all RS
enzymes, and the cluster is positioned at the carboxy terminus of the protein barrel.
SAM binds to the cluster within the barrel, and the substrate (dethiobiotin for biotin
synthase4) binds proximal to SAM, poised for catalysis. The RS enzymes use a
common mechanism: the generation of a primary carbon- centered radical
intermediate, the 5′ deoxyadenosyl radical (dAdo●) (Fig.2).
NeoN is a recently characterized epimerase that plays a vital role in the last
biosynthetic step of neomycin B, an aminoglycoside antibiotic produced by
Streptomyces fradiae1. NeoN is a RS enzyme that selectively epimerizes the C-5'''
carbon of Neomycin C1 (Fig.3). The auxiliary clusters in SAM enzymes, including
NeoN, may have multiple uses, such as accepting or donating electrons during the
course of the reaction, or they may be anchoring points for substrates to bind. Their
utility expands the radical SAM enzyme functionality, enabling radical SAM
enzymes to perform added chemical reactions. We are interested in how NeoN is
capable of specifically catalyzing the epimerization of one, stereospecific site of
neomycin C, which contains multiple possible sites that could be acted upon.
Aims:
• To obtain atomic resolution information in order to characterize and understand
how NeoN functions.
• To identify important residues within the active site that play a role in catalytic
epimerization.
• To characterize substrate recognition in order to bind other non-substrate
molecules for specific epimerization reactions.
Future Work
• Cloning of NeoN genome, expressing, and crystallizing the protein using a
different strain of Streptomyces fradiae.
• Gibson assembly method of molecular cloning will be preferred over colony
PCR for correct expression of the gene.
• Ensuring that the expressed crystallized protein is soluble and functionally
active.
• Analyzing the active site residues to explore incorporating non-substrate
molecules for possible epimerization by the enzyme.
Methods, Results, and Development
X-ray crystallography is a tool used to identify the atomic and molecular
structure of a crystal which is a highly ordered structure. The crystalline arrangement of
atoms causes the incident X-rays to diffract into many specific directions. The
diffraction pattern can then be analyzed using computational programs to obtain an
electron density map, which reveals the protein’s structure and the binding of cofactors
and ligands. The overall scheme of crystallizing NeoN is shown in Figure 6.
The main idea behind crystallizing a protein is that most proteins are soluble at
physiological conditions, but as the concentration of solutes rises, the protein becomes
less soluble, leading it to crystallize or precipitate. The goal of crystallization is to
produce a well-ordered crystal lattice that is able to provide a diffraction pattern on
exposure to X-rays.
The amplification of neoN gene using the ATCC 19760 strain of Streptomyces
fradiae resulted in the production of two fairly close bands in the 900bp region (Fig.4).
Multiple annealing temperatures were tested to understand this causation (Fig.5). Due
to the difficulties in obtaining a positive clone using the standard ligation protocol, a
different method called the Gibson Assembly was used with the pET28a vector (Fig.7).
The amplification of neoN did yield a single band in the 900bp region, but the band
intensity was not high to be conclusive as an effective method for neoN gene
amplification. Therefore, the results of the Gibson assembly, unfortunately, were not
successful.
Conclusions
• The primer design plays an important role in the complete expression of the
NeoN protein and its further optimization will yield better results in the future.
• Epimerization of Neomycin C to Neomycin B is accomplished by NeoN through
its two [4Fe-4S] clusters at C-5′′′.
• A thorough understanding of the NeoN structure for Neomycin B synthesis will
open up the possibility of modifying the enzyme to recognize different types of
substrates, making NeoN a tractable tool as a biocatalyst.
References:
1. Fumitaka Kudo. et al. JACS (2014) 136, 13909-13915.
2. SFFLD - Superfamily List." SFLD - Superfamily List. Web. 26 Apr. 2015
3. Shisler, Krista. et al. Curr. Opin. Struct. Biol. (2012) 22, 701-710.
4. Berkovitch, F. et al. Science (2004) 303, 76–79.
Figure 2. Reductive cleavage of SAM.
RS enzymes use [4Fe-4S] clusters to bind
to SAM and transfer an electron to the
sulfonium of SAM, producing methionine
and dAdo● through homolytic cleavage of
S-5′C bond in SAM. The dAdo● then
abstracts an H-atom from the substrate to
initiate a radical-mediated transformation.
Figure 6. Protein X-ray
crystallography methods scheme.
The neoN gene was cloned from
Streptomyces fradiae (ATCC 19760)
into an expression vector containing N
terminal 6xHis tag (Hexa-histidine).
pET28 vector was selected for ligation
reactions with the neoN DNA as it
contains a selectable marker for
kanamycin resistance which is useful
for isolating cells with the correctly
inserted plasmid. Additionally, a
thrombin cleavage site is also a feature
of this vector whose role is crucial
during protein purification.
Figure 3. NeoN epimerization of
Neomycin C to Neomycin B. NeoN is a
putative RS enzyme which is encoded in
the neomycin gene cluster. This gene
cluster is structurally related to
aminoglycoside biosynthetic gene clusters
such as lividomycin B and paromomycin1.
Figure 1. Structure of BioB. Overall
TIM barrel structure is colored in red
(α helices) and yellow (β strands).
The cofactors and substrates are
arranged vertically as follows: [4Fe-
4S] cluster on the bottom, SAM,
dethiobiotin, and the [2Fe-2S] cluster.
Carbon atoms are colored cyan for
substrates. PDB accession code 1R30.
Figure 5. Colony PCR gel analysis. The
annealing temperature was varied to 62℃,
68℃, and 72℃ to account for the presence of
magnesium in the buffer which raises the
melting point of the primer as well as the
unwanted hairpin structures. A ten-fold dilution
(represented by ‘D’ next to temperature) was
also performed to examine any differences in
the amount of intensities obtained for neoN.
The agarose gel was analyzed and it was noted
that there was no impact of varying the
annealing temperature from the ideal
temperature of 55℃.
62 62D 68 68D 72 72D 1kb ladder
A1 B1
A2 B2
Figure 4. Colony PCR gel analysis. The
presence of neoN gene was confirmed in
Streptomyces fradiae cell line (ATCC 19760).
The PCR was performed at the ideal annealing
temperature of 55℃. Interestingly, there were
two bands present closely and this region of
gel was cut and the DNA was extracted from
it. The bands present closely in the 900bp
region indicated the possible amplification of
neoN in the colony PCR samples.
Figure 7. Gibson Assembly PCR gel analysis. (A) The Gibson assembly method of
cloning the neoN gene resulted in the presence of one band in the 900bp region when the
agarose gel was analyzed. However, the low intensity of the band suggested that the amount
of DNA was not high. (B) Further analysis of the gel with GelQuant software indicated that
the amount of DNA amplification was less than 5%. For the band in lane 6, the percentage
of neoN DNA in the sample against the background was 1362275/ 36190215=0.0376 or
3.76%
A B