MOLECULAR EVOLUTION-DIRECTED APPROACH FOR DESIGNING OF -METHYLASPARTATE MUTASE FROM THE SEQUENCES OF...

International Journal of Chemical Modeling ISSN 1941-3955

Volume 3, Number 3 © 2011 Nova Science Publishers, Inc.

MOLECULAR EVOLUTION-DIRECTED APPROACH

FOR DESIGNING OF -METHYLASPARTATE MUTASE

FROM THE SEQUENCES OF HALOARCHAEA

P. Chellapandi1,*

and J. Balachandramohan2

1Department of Bioinformatics, Centre for Excellence in School of Life Sciences,

Bharathidasan University, Tiruchirappalli-620024, Tamil Nadu, India 2Department of Bioinformatics, School of Chemical and Biotechnology, SASTRA

University, Thanjavur-613402, Tamil Nadu, India

Abstract

Methylaspartate mutase is one of the cobalt containing enzymes, which is mainly involved in

vitamin B12 biosynthesis and also in C5 dibasic acid metabolism of both prokaryotes and

eukaryotes. It is widely distributed in archaea, particularly haloarchaean Haloarcula

marismortui. Thus, the present work was aimed to use conserved regions of this enzyme

sequence at substrate- and metal-binding sites, and active cleft for designing enzyme

constructs so as to understand the possible rearrangement reactions. The sequences of -

methylaspartate mutase were searched from haloarchaean and then opted regions were chosen

to model 3D-structure by ModWeb server. As it was in the native enzyme sequence,

conserved domain of the same enzyme was simply modeled to bring new substrate specificity

and catalytic rearrangement reactions. The top-five energy conformer of models were selected

and allowed to interact with different substrates by using molecular dynamics simulation and

molecular docking methods, respectively. From the results obtained from this study, we

proposed that this enzyme construct has more stable at high salt concentration, and has a

strong binding affinity with theo-3-hydroxy-L-aspartate, and has also preferably interacted

with other substrates. Since, this approach can significantly imply more reliability of

designing -methylaspartate mutase construct. Perhaps, the designed enzyme would catalyze

the rearrangement of glutamate radical to methylaspartate radical in biotransformation

reactions.

Keywords: Molecular docking; Molecular dynamics; β-Methylaspartate mutase;

Haloarchaea; Molecular evolution; Enzyme design.

* E-mail address: [email protected]. Phone: +91-431-2407071, Fax: +91-431-2407045 (Corresponding

author)

P. Chellapandi and J. Balachandramohan 298

1. Introduction

Cobalt acts as an essential metal ion found associated with a number of enzymes in both

prokaryotes and eukaryotes [1-3]. It is also found in isobacterioclorins and porphyrins

isolated from sulfate reducing bacteria [4]. Methionine aminopeptidase, prolidase, nitrile

hydratase, glucose isomerase, methylmalonyl-CoA carboxytransferase, aldehyde

decarbonylase, lysine 2, 3 aminomutase, -methylaspartate mutase, and bromoperoxidase are

some examples of cobalt-containing enzymes [5]. Among these enzymes, -methylaspartate

mutase (EC 5.4.99.1) has a potential importance in industrial application include vitamin B12

biosynthesis [1] and some chemical rearrangement reactions in biotransformation [5]. It is

also participated in C5-branched dibasic acid metabolism of organism [6]. It belongs to the

family of isomerases, specifically those intramolecular transferases transferring other groups.

The systematic name of this enzyme class is L-threo-3-methylaspartate carboxy-

aminomethylmutase. Other names in common use include glutamate mutase, glutamic

mutase, glutamic isomerase, glutamic acid mutase, glutamic acid isomerase, methylaspartic

acid mutase, -methylaspartate-glutamate mutase, and glutamate isomerase. The first step in

glutamate breakdown is a conversion to β-methylaspartate by glutamate mutase (isomerase),

and the second step is the deamination of β-methylaspartate to mesaconate by β-

methylaspartase. The substrates and products of the reversible mutase reaction are identified

as L-glutamate and L-threo-β-methylaspartate, respectively [1, 6]. A rearrangement reaction

is initiated with extracting hydrogen from the protein-bound substrate by a 5'-desoxyadenosyl

radical, which is generated by the homolytic cleavage of the organometallic bond of the

cofactor B12 [7, 8]. Several glutamate derivatives and β-methylaspartate could not serve as

substrates [6].

Many organisms have been reported to produce β-methylaspartate mutase. However, only

few of haloarchaean have to be investigated for its enzyme activity, one of which is

Haloarcula marismortui. This is a halophilic red archaeon (from the Halobacteriaceae family)

found in Dead Sea (a high saline and low oxygen solubility) and in high light intensity

environment. Like other halophilic archaebacteria, H. marismortui thrives in extreme

environment due to several adaptations in protein structure, metabolic strategies and

physiologic responses [9, 10]. Therefore, much more attempts have been focused on the

biochemical function of β-methylaspartate mutase subunit S of this organism and its

appropriate application in biotransformation reactions [6].

The evolutionary conservation in sequence as well as structure would make contribution

in enzyme catalysis. Such conserved amino acid residues are accounted to consider of

designing enzymes on which metalloenzymes have been the main concern due to extensive

role in biocatalytic activity and enzyme stability. Thus, the present work was focused on how

evolutionary conservation of β-methylaspartate mutase subunit S sequences of haloarchaean

at metal- and substrate-binding sites has to be contributed for designing enzyme constructs

with broad-substrate specificities. This study has also focused the following tasks; i) to search

occurrence of this enzyme within archaea, ii) to identify conserved domain architecture

enabling to catalyze metal-dependent biochemical reaction, iii) to develop protein 3D-

structure models wherein catalytic domain, substrate- and metal-binding sites are embossed,

iv) to find out the lowest energy conformers of enzyme constructs, and v) to calculate binding

energies of enzyme-substrate complexes that are formed.

Molecular Evolution-Directed Approach… 299

2. Materials and Methods

2.1. Evolutionary Conservation Analysis

Complete sequences of archaeal β-methylaspartate mutase subunit S were retrieved from

GenPept of NCBI. Conserved domains architecture of these sequences were searched from

NCBI-CDD (Conserved Domain Database) [11] by using conserved domain (CD) search tool

[12] with expected value threshold 10 and a low complexity filter. Metal- and substrate-

binding sites of these sequences were compared with availed PDB structures. The parameter

was set as 10 expected threshold, 3 word size, 11 gap existence cost, 1 gap extension cost,

Blossum 62 scoring matrix, conditional compositional score alignment, and automatically

adjust the parameter for short input sequences. The query sequences were compared to a

position-specific score matrix prepared from the underlying conserved domain alignment.

The selected sequences were clustered together with complete deletion of gaps and correction

in multiple substitutions by ClustalX 2.0 software [13]. All aligned sequences were iterated at

each alignment step, and manually inspected to delete the low scoring sequences. Neighbor

joining (NJ) algorithm was used to search homogeneous patterns among all lineages, and then

an unrooted phylogenetic tree was built by using MEGA 4.0 software [14] with 1000

bootstraps values [15].

2.2. Molecular Modeling

PSI-BLAST tool [16] with a default parameter was carried out to obtain a suitable PDB

template (homolog) for 3D-structure modeling from query sequences. ModWeb is an automatic

comparative protein modeling server that was used to build 3D-structures from these sequences

using comparative modeling by satisfaction of spatial restraints as implemented in Modeller [17,

18]. The resulting models were evaluated using several model assessment schemes and the best

scoring models selected. ProFunc server was used to predict the corresponding function and

active sites of each model [19]. Every model was compared with crystallographic protein

structures whose catalytic domains are similar to metal- and substrate-binding sites. Atomic

coordinates of entire structures which are not covering the position of metal- and substrate-

binding, and also active site residues have been removed.

2.3. Molecular Dynamics Simulation

Structural conformers of these models were generated by standard dynamic simulation

cascade module in Discovery Studio software using CHARMM force field and steepest

descent as well as adopted basis Newton-Raphson algorithms. The

terms conformation and conformer are used to represent different geometrical configurations

of atoms in a molecule. In Discovery Studio, these terms can also apply to subsets of atoms of

a molecule, such as a single amino acid residue. Conformers are typically created for

molecules that have a small number of alternate locations for a small subset of the atoms.

Conformers are represented by conformer atoms and conformer bonds, which can be

manipulated similarly to, and independently of, other atoms and bonds. There are no


constraints on the number of conformers, although there are usually between 2 and 4 for the

residues where conformers are defined. Distance constraint of the model was between N-

terminal to C-terminal and dihedral restraint was started from C to Cα (Ф) of first amino acid

residue and Cα to N (ψ) of second amino acid residue until the last amino acid residue in a

molecular dynamic ensemble. Molecular dynamic simulation was executed for 2.24 hrs, and

after that 30 conforms of the same model were generated based on the relationship of

conformation index and total energy. The lowest energy conformer of model was selected for

further docking studies with respective substrates. Conformers belonging to the same model

were grouped under a conformer model, which was listed under the Conformer Model tab in

Discovery Studio. Generalized Born with a simple Switching implicit solvent model was used

with spherical cut-off (electrostatic), 80 implicit dielectric constant, 1 dielectric constant, 0

salt concentration and non-polar surface area for conformational analysis.

2.4. Molecular Docking

AutoDock software 4.0 was used for finding inhibition constants, free energy of binding,

intermolecular and internal energies of selected low energy conformers with substrates by using

Genetic algorithm [20] and AMBER force field parameters set. SIMCOM software of KEGG

(http://www.genome.jp/tools/simcomp) was used to retrieve related strutures for substrate in

MOL2 files and then converted to PDB format. In docking process, enzyme construct was fixed

as regid whereas substrate was set as flexible. Active site amino acid residues of enzyme was

selected in an enegy grid and prefered to dock with a flexible susbstrate. To find out the lowest

binding energy, all ligands (substrates) were allowed to interat with correponding enzyme. The

molecular mechanics-based and empirical terms were multiplied by coefficients that are

determined by linear regression analysis of complexes with known 3D-structures and known

binding free energies. Minimization of docked structures was performed with smart energy

minimization algorithm to refine the orientation of the substrate in the receptor site. After

docking, this construct was solvated with explicit periodic boundary solvation model. Solvation

parameter was set as 20 radius of sphere, 7.0 minimum distance from boundary, orthorhombic

cell shape, false counter ion, 0.145 salt concentration, 3145150 random speed, sodium type

cation and chloride type anion. Accurate treatment of solvation effects, at the level of individual

water molecules, is essential for enzymatic activity and formation of enzyme-substrate complex

in water. Several strategies exist for explicit water conformational sampling, but none of those

proposed thus far are suitable for the simultaneous sequence/conformational sampling necessary

for modeling of evolutionary dynamics. A semi-explicit water sampling algorithm implemented

in AutoDock that combines continuum and explicit solvation (an explicit/implicit water

interface), creating and annihilating waters as needed based on a grand canonical ensemble

Monte Carlo simulation method. AutoDock scoring system, binding energy and RMSD (root

mean square deviation) were used to check the quality of docking models.

3. Results

While searching the sequences of -methylaspartate mutase small subunit S from

archaea, many sequences were deposited only for haloarchaea (Haloarcula marismortui


ATCC 43049, H. salinarum R1, H. mukohataei DSM 12286 and Natrialba magadii ATCC

43099). No crystallographic data availed in PDB for structural information of this enzyme.

PSI-BLAST search results showed that overall structural identity of this enzyme was ranged

from 45 to 51% in which a sequence with accession Q5V467 (construct) was more opted for

designing enzyme. It has the shortest amino acid length and embossed metal-binding as well

as catalytic domains. Other protein models were neglected due to low modeling scores and

diverged positions of the active residues, and substrate-binding sites in conserved domains

(Table 1 and 2). The amino acid position of 8-139 was modeled with a template 1ccwA and

48% identity and 1.62 MPQS obtained. Amino acid residues Ala34, Gly35 and Phe36 were

predicted in the metal binding position (15-130) as nest sites of this enzyme, and showed

conservation score of 2.626. At the amino acid position 15-130, metal binding sites of

construct was found as B12 glm_B12_BD (B12 binding domain of glutamate mutase) of B12

binding like superfamily with e-value of 3e-46 that can contribute to harbor its catalytic

function on methylaspartate.

Table 1. Homology modeling for predicting 3D-structure of -methylaspartate mutase

Accession AA Template Identity Position MPQS Z-Dope

Q9HN21 149 1be1A 45 7-138 1.35 0.81

YP_001690048 149 1be1A 45 7-138 1.35 0.81

NP_280920 149 1be1A 45 7-138 1.35 0.81

Q5V467 151 1ccwA 48 8-139 1.62 -1.71

Q5V3F0 148 1be1A 51 2-132 1.46 0.22

YP_135658 148 1be1A 51 2-132 1.46 0.22

All of the protein models had modeling score of 1.00.

Table 2. Data mining for searching metal binding and active site similarity regions

of -methylaspartate mutase models (PSSM-ID: 30210)

Accession Template Metal

binding site Active site Score

Q9HN21 1be1A 15-126 Val20, Gly21, Ile22, Thr23 3.724

YP_001690048 1be1A 15-126 Val20, Gly21, Ile22, Thr23 3.724

NP_280920 1be1A 15-126 Val20, Gly21, Ile22, Thr23 3.724

Q5V467 1ccwA 15-130 Ala34, Gly35, Phe36 2.626

Q5V3F0 1be1A 10-125 Val16, Gly17, Ile18, Thr19 4.713

YP_135658 1be1A 10-125 Val16, Gly17, Ile18, Thr19 4.713

Phylogenetic analysis of this study showed that β-methylaspartate mutase subunit S

sequences of haloarchaea were formed a separate clade that revealed its evolutionary

resemblance within closely related species (Fig. 1). Later on, it was formed a cluster with

halophilic bacteria such as Oceanospirillum sp. MED92 and Salinispora arwnicola CNS-205.

The sequences of β-methylaspartate mutase subunit S of proteobacteria was also showed

phylogenetic correspondence to haloarchaean.


Figure 1. NJ Phylogenetic tree of -methylaspartate mutase sequences obtained from archaea.

Figure 2. Total energy versus conformational index relationship of searching the lowest energy

conformers of -methylaspartate mutase construct using energy minimization and dynamic simulation.

As shown in Fig. 2, the total energy and conformational index liaison of searching the

lowest energy conformers of -methylaspartate mutase construct during energy minimization

and dynamic simulation. Based on the conformation energies, the top-five lowest energy

conformers have been selected. Molecular dynamics studies as shown in Table 3 pointed out

that the total energy of top-five conformers was not changed significantly. vander Waals

energies of the conformers were ranged from -681.94 to 705.81 kcal/mol where as

electrostatic energies were ranged from -3125.96 to 3215.91 kcal/mol. It indicated that the

structure of this enzyme construct is stabilized better by electrostatic interaction than vander

Halobacterium salinarum

Halobacterium salinarum R1

Halobacterium sp. NRC-1

Halobacterium salinarum R1

Halomicrobium mukohataei DSM 12286

Halomicrobium mukohataei DSM 12286

Haloarcula marismortui (Model )

Natrialba magadii ATCC 43099

Haloarcula marismortui

Haloarcula marismortui ATCC 43049

Oceanospirillum sp. MED92

Salinispora arenicola CNS-205

Pelotomaculum thermopropionicum SI

Dictyoglomus turgidum DSM 6724

Desulfotomaculum reducens MI-1

Carboxydothermus hydrogenoformans Z-2901

Alkaliphilus metalliredigens QYMF

Escherichia coli O157:H7 str. EC4024


Waals interaction. The best conformational energy of construct was -3778 kcal/mol (252

kcal.mol torsion energy) at 309 K.

Table 3. Molecular dynamic simulation for searching the lowest energy conformers

of -methylaspartate mutase constructs.

Conformer Total

energy

Vander

Waals energy

Electrostatic

energy

Torsion

energy

Temperature

K

1 -3778.66 -685.90 -3244.20 252.19 309.35

2 -3777.66 -681.21 -3125.96 261.29 304.65

3 -3777.14 -697.24 -3188.22 259.58 304.39

4 -3777.11 -705.81 -3215.35 253.65 301.70

5 -3777.08 -681.94 -3215.91 250.13 301.16

All of the molecular energies are expressed as kcal/mole.

Figure 3. Representative docking model of threo-3-hydroxy-L-aspartate into -methylaspartate mutase

construct (Q5V467) is represented interaction site view of enzyme-substrate complex. After docking,

this model was solvated with explicit periodic boundary solvation model. The yellow dot lines denote

the hydrogen bonds. All the amino acid residues which involved in molecular interaction are shown in

line drawing and colored by residue types in which hydrogen is colored white, carbon green, oxygen

red, nitrogen blue, and sulfur orange. Ligands are shown in stick in which carbon is colored tints,

hydrogen gray, nitrogen blue, and sulfur orange. All the interaction distances are represented as RMSD

and expressed as Angstrom (Å). The binding energy as well as intermolecular forces acting on this

docking model is represented in Table 4.


Table 4. Molecular docking for predicting binding energy of -methylaspartate mutase

constructs and substrates interactions

Substrates

Binding

energy

(kcal/mol)

Inhibition

constant

(Ki mM)

Intermolecular

energy

(kcal/mol)

Internal

energy

(kcal/mol)

L-threo-3-Methylaspartate -4.16 0.89 -3.65 -3.13

L-threo-3-Methylmalate -2.65 11.46 -3.51 -3.39

threo-3-Hydroxy-L-aspartate -5.47 0.10 -4.48 -3.79

4-Methyl-L-glutamate -5.02 0.21 -5.01 -4.47

L-Tartaric acid -2.26 22.02 -3.07 -2.64

meso-2,3-Dimercaptosuccinic

acid

-2.07 30.25 -3.35 -3.12

Figure 4. 3D-structural representation (Rasmol view) of designed -methylaspartate mutase construct.

In molecular docking studies, the substrates include L-threo-3-methylaspartate, L-threo-

3-methylmalate, threo-3-hydroxy-L-aspartate, 4-methyl-L-glutamate, L-tartaric acid and

meso-2, 3-dimercaptosuccinic acid have docked into enzyme construct (Fig. 3). Upon

considering strong molecular interactions, enzyme construct-threo-3-hydroxy-L-aspartate

complex was selected as the best docking model of this study. Atom N1 of threo-3-hydroxy-

L-aspartate has made H-bonding to OH group of Tyr92 residue in enzyme construct with

interaction distance of 2.66 Å whereas atoms of OXT (2.83 Å) and O1 (2.92Å) formed H-

bands with the side chain residue Arg118. As shown in Fig. 4, 71 H-bands donors, 4 helixes,

10 turns and 2 strands were found in this construct. Catalytically, it was more suited for threo-

3-hydroxy-L-aspartate rather than the native substrate L- threo-3-methylaspartate due a strong

binding affinity was predicted for first one.


4. Discussion

Most of the members of B12 binding-like superfamily are attracted different cobalamide

derivates [21]. This clan comprises of several enzymes, such as glutamate mutase, methionine

synthase and methylmalonyl-CoA mutase enzymes [5]. Cobalamin has undergone a

conformational change on binding the protein. Dimethylbenzimidazole group can coordinate

the cobalt in the free cofactor, moved away from the corrin, which is replaced by a histidine

contributed by protein. The sequence motif Asp-X-His-X-X-Gly is contained this histidine

ligand that is conserved in many cobalamin-binding proteins. Not all members of this family

have such conserved binding motif [3]. Hence, the metal- and substrate-binding sites and

catalytic domain of this enzyme construct showed more evolutionary conservation, and

resemblance with closely related species. It suggests the occurrence of possible molecular

interactions with different substrates at cobalamin-binding regions by this construct.

Comparing the large subunits of glutamate mutases and related enzymes have showed the

highest degree of similarity for GlmB and NikV proteins of Streptomyces tendae [2].

Similarly, haloarchaean‘s enzyme sequences have showed phylogenetic correspondence to

halophilic bacteria and proteobacteria, reflecting that the functional divergence of this enzyme

may be resulted among prokaryotes. It suggested that its enzyme function can be evolved

from proteobacteria, and halotolerant capability of it should be acquired as such through

subsequent evolutionary process within haloarchaea. It is agreed earlier work done on

halotolerant malate dehydrogenase and such ability, stability and activity developed as the

results of growing H. marismortui in the Dead Sea [22]. Crystallographic studies proved that

the surface of halophilic proteins is enriched by acidic amino acids, which maintains stability

of them in high salt concentration [3, 9]. Anion-binding sites in halophilic enzymes are also

stabilized in the environment containing salt [22]. It is clearly known that -methylaspartate

mutase is one of the candidates in malate dehydrogenase family. Since the sequence used in

this study was obtained from H. marismortui and the surface of this enzyme has enriched

with acidic amino acids, and anion-binding sites, this enzyme construct would be stable even

at high salt concentration. Phylogenetic analysis of this study also supported that conserved

regions of this enzyme was strongly correlated its relationship with haloarchaean family.

In glutamate mutase catalyzed reaction, hydrogen on carbon-4 of glutamate is

interchanged with the glycyl group on carbon-3 to give methylaspartate. Glutamate mutase

catalyzes an unusual rearrangement reaction that involves radical intermediates [21]. The

adenosyl radical generated by B12 can be used to remove the migrating hydrogen from the

substrate. It is a step common to all B12 isomerases in many organisms. The substrate radical

can be rearranged to form a product radical, in this case methylaspartyl radical. Then, the

hydrogen is replaced from the coenzyme to give methylaspartate and regenerate the adenosyl

radical, which may be 'stored' by reforming the cobalt-carbon bond. In essence, the

introduction of the unpaired electron onto the substrate serves to activate it towards chemical

reactions that would not otherwise be feasible [7, 8, 21]. The results obtained from molecular

docking studies proposed that the rearrangement of glutamyl radical to methylaspartyl radical

can be occurred by fragmentation of the glutamyl radical. This rearrangement reaction gives

acrylate and a glycyl radical as intermediates, followed by recombination of the glycyl radical

with the other end of the acrylate double bond to yield the methylaspartyl radical.


Chemical models suggests the formation of a substituted cyclopropyl methylene radical

as an intermediate in the rearrangement of 2-methyleneglutarate to (R)-3-methylitaconate

catalyzed by the related 2-methyleneglutarate mutase. Such a mechanism is unlikely in the

glutamate mutase reaction due to the lack of a double bond at the migrating carbon. Indeed,

chemical modeling showed that glutamate derivatives only rearranged if the amino acid was

converted to a ketimine suggesting the participation of an electrophilic center in the reaction

[1]. The side chains of Val20, Gly21, IIe22 and Thr23 residues in this construct are the

primary covalent attachment sites to a substrate L-threo-3-methylaspartate, but the binding

energy and other molecular energies of enzyme-substrate complex are not significantly stable.

However, a molecular interaction and binding affinity attributed by the side chains of Tyr92

or Arg118 residues in this construct with threo-3-hydroxy-L-aspartate are stable.

Accordingly, (a strong enzyme-substrate complex and a low energy conformation of model),

the amino acids residues as it is in native form can be served as catalytic regions, and thereby

it would catalyze to convert threo-3-hydroxy-L-aspartate into glutamate.

Conclusion

The enzymes manganese superoxide dismutase by molecular mechanics calculations

employing the CAChe system [23] and rational design using DEZYMER algorithm [24],

nuclease and protease by modification of protein scaffold [25], deoxyribose-phosphate

aldolase by the recapitulation of active sites of native enzymes [26], isochorismate pyruvate

lyase by quantum mechanics/molecular mechanics [27], and chorismate mutase by computing

empirical valence bonds [28] have already been designed by computational approaches.

However, those approaches have been employed complex mathematical derivations and

altered protein scaffold or amino acids as compared to the evolution-directed approach.

Unfortunately the resulting enzyme constructs have been significantly less effective than the

corresponding natural enzymes [29] and the reasons for these limited successes are not

completely clear [28]. Herein, we have not done any alternation in native amino acids

position or replacing residues. It is directly based on evolutionary conservation of substrate-

and metal-binding domains of the enzyme. This hypothesis is that if any enzyme has similar

or identical conserved domain in its catalytic region, it can bring similar catalytic activity and

broad substrate specificity. Generally, most of the haloarchaean enzymes are highly

conserved within this group and are unique as compared to bacterial enzymes. The enzyme

sequences of this study are obtained from Haloarchaea. Consequently, this approach is

strongly supported for designing enzyme construct with more biological significance that may

be accorded with experimental work. In this perspective, few experimental success have been

made for proteinase K invariant by machine learning algorithms [30], -glycosidases by

amino acid replacements [31], and L-aminoacylase by alternation of metal ions [32] in the

recent years. Overall, this enzyme construct has a low probability to fold in artificial

environment for catalytic action so that it is suggested for appropriateness of using it in

biotechnological processes and in green chemistry applications.


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MOLECULAR EVOLUTION-DIRECTED APPROACH FOR DESIGNING OF -METHYLASPARTATE MUTASE FROM THE SEQUENCES OF...

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