Characterization of Radical S-adenosyl-L-methionine

Epimerase, NeoNSamender Randhawa1, John Zhang1, Yuan Xia1, Cindy Hunt1, Daniel P. Dowling1

1Department of Chemistry, University of Massachusetts Boston, Boston, MAFunding 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. A subset of

radical SAM enzymes, such as 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 grow NeoN protein crystals and obtain its

crystallographic information in order to understand the underlying biochemical

basis for its catalytic activity. We hypothesize that NeoN could be used as a

biocatalyst tool in the future for developing specific epimerases that could

produce novel compounds.

Methods and Development

Conclusions

Introduction

Future Work

Radical S-adenosyl-L-methionine (SAM) enzymes (RS enzymes)

carryout a variety of biological functions, such as synthesis of cofactors, and

antibiotics1. Till date 113,6334 RS function units have been characterized both

biochemically and structurally. 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 fold2 (Fig.1a). The presence of a [4Fe- 4S] cluster bound to a

conserved cysteine triad (CxxxCxxC) 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

synthase3) binds proximal to SAM, poised for catalysis (Fig. 1b). 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). We are interested in how NeoN is

capable of specifically catalyzing the epimerization of one site of neomycin C,

and future studies of this system may lead to the development of

bioengineered epimerases.

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.

• Cloning of NeoN genome, expressing, and crystallizing the protein.

• 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.

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 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 5.

Figure 4. RS enzyme

sequence similarity.

Sequence analysis can be

used to assign function to

proteins. A similarity cut

off e-value of 10-28 has

separated the RS enzyme

sequences into clusters

that largely group by

reaction type.

• 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. Shisler, Krista. et al. Curr. Opin. Struct. Biol. (2012) 22, 701-710.

3. Berkovitch, F. et al. Science (2004) 303, 76–79.

4. SFLD - Superfamily List." SFLD - Superfamily List. Web. 26 Apr. 2015.

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 5. Protein X-ray crystallography methods scheme. We will be cloning

the gene for NeoN from Streptomyces fradiae into an expression vector

containing a hexa-histidine affinity tag for protein purification. Purified protein

will be concentrated and used for crystallization experiments.

a

Figure 3. NeoN epi-

merization of Neomycin C

to Neomycin B. (a) 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. (b) The first

cluster is made up of C26,

C30, and C33. The second

cluster involves C247,

C249 C271, and C274.

NeoN

C C C C 416C C C

b

b

Figure 1. Structure of BioB. (a) Overall

TIM barrel structure is colored in red (α

helices) and yellow (β strands). (b) 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 1R30.

a b