Homology Modeling of Bifunctional Enzyme Alanine Racemase from Taibaiella chishuiensis

Alanine Racemase from Taibaiella chishuiensis bacteria is one of the bifunctional enzymes that catalyze the Land D-alanine racemization of peptidoglycan biosynthesis in bacteria and ligation (UDP-N-acetylmuramoyl-Tripeptide-D-alanyl-D-alanine ligase). It had two EC numbers 5.1.1.1 and 6.3.2.10 respectively. This enzyme is an important target for antimicrobial drug productions or inhibitor design. However, the 3D structure of Alanine Racemase from Taibaiella or UDP-N-acetylmuramoyl-tripeptide-D-alanyl-D-alanine ligase/ alanine racemase has remained unknown. Thus, this study modeled and validated the 3D structure of the enzyme in the query. The bioinformatics tools/databases and software such as BRENDA, NCBI, Uni Prot, Clustal Omega, Prot Param, Swiss model, Phyre2, GOR, PROCHECK, and PyMOL were used for modeling, validation, and structural comparison. From the sequence and 3D structure analysis, it is indicated that Alanine racemase from Taibaiella had the same active and binding sites with the reference enzymes. Thus, we were able to study the similarities and differences in the sequence and structural properties of alanine racemase in two different bacteria. Finally, it was found that our enzyme has two parts for two different functions (racemization and ligation). The predicted model of alanine racemase of T. chishuiensis from this study could serve as a useful model for further study regarding the other bifunctional enzymes structure and function as well as drug design projects.

This enzyme has a significant role in the growth of the bacteria by providing D-alanine, the peptidoglycan layer constitutive of the bacterial cell wall. (Liu et al., 2018). In both gram-positive and Gram-negative bacteria, the peptidoglycan layer of the bacterial cell wall prepares resistivity to osmotic lysis (Islam et al., 2017). Alanine racemase is special to bacteria. There are few exceptions to the fact that certain eukaryotes have the enzyme D-alanine-containing peptides biosynthesis in fungi, D-alanine metabolism in yeast, as well as osmotic regulationin a crayfishplusa bivalve mollusk (Nomura et al., 2001).
This enzyme is absent in humans and is aunique enzyme that transforms L-alanine and D-alanine in most bacteria thus, it is an important target for antimicrobial drug productions (Im et al., 2011;Wei et al., 2016). For instance, alanine racemase was chosen as a target to discover new drugs to treat tuberculosis or for the inhibitor design (Anthony et al., 2011). Based on previous studies, most of previously studied inhibitors suffer from the lack of specificity, and further investigation is required to solve this issue (Azam and Jayaram, 2016). To target an enzyme for effective antibacterial drugs, and to develop new and selective inhibitors, which improves treatment in public health systems, it is important to know about the structure and characteristics of the enzyme in various living organisms. The 3D structure of the alanine racemase of several bacteria is available in the PDB. But there is no 3D structure of the Taibaiellachi shuiensis alanine racemase. Strain AY17 T of Taibaiella chishuiensis contained MK-7 as the predominant respiratory quinone, plus the main polar lipid phosphatidylethanolamine, and hydrolyses the aesculin, casein, and gelatin (Tan et al., 2014). Therefore, in the current study, we perform modelling of alanine racemase in the mentioned bacteria. The present investigation is the first study of the sequence and 3D structural characterization of the Taibaiella chishuiensis alanineracemase enzyme.This study modeled the structure of the novel bifunctional, Taibaiella chishuiens-based alanine racemase, which belongs to the family of isomerases. A bacterial strain, AY17 T of Taibaiella chishuiensis, was isolated from the Chishui River in Guizhou Province, Southwest China.
Based on phenotypic, phylogenetic, plus genetic evidence, theAY17T strain was categorized as a novel representative species of the Taibaiella genus for which the Taibaiella chishuiensis sp.Nov., the name has been suggested. This bacteria belongs to the Chitinophagaceae family (Wei et al., 2016). The structure of the alanine racemase was first performed on an enzyme isolated from Bacillus stearothermophilus. This enzyme is a homodimeric enzyme with each monomer consisted of á/â barrel domain at the N-terminus and a C-terminal domain consisting mainly of â strands. The location of the active site is at the interface of the á/â barrel andthe â domain, close to the PLP cofactor, forming  To clarify the bifunction potency of the alanine racemase enzyme, a comparative study was performed between the primary sequence and the predicted structure of the new bifunctional alanine racemase enzyme and similar, clostridium difficile (PDB: 4LUT) from the family, Peptostreptococcaceaeas as well as T. maritime (PDB: 3zl8) from the family, thermotogaceae (Couñago et al., 2009;Tan et al., 2014;Dong et al., 2018).   Finally, several analyses were performed to provide useful details on the bifunctionality of this enzyme. This study wishes to provide useful information for modeling/designing other unknown structures of the bifunctional enzymes as well as drug design projects.

Research methods Sequence retrieval and multiple sequence alignment(MSA)
The information about the enzyme alanine racemase of T.chishuiensis is obtained by searching the EC number (5.1.1.1) in BRENDA (Schomburg et al., 2004). So, we found out the optimum temperature, PH, metabolic pathway reaction. Also, the FASTA format and other informationregarding the PDB structure is received from UNIPROT (Ju et al., 2011). Then, the Blastp from NCBI database was done to obtain a similar sequence of alanine racemase from different organism to compare and analyze the structure (Asojo et al., 2014). Based on the data, it was found that our enzyme is very similar to bifunctional enzymes "UDP-Nacetylmuramoyl-tripeptide-D-alanyl-Dalanine ligase/alanine racemase" of different bacteria The primary structure analysis model of enzyme alanine racemase from T. chishuiensis was done using the ProtParam tool (Gasteiger et al., 2003;Hassan et al.,2020).This step was carried out to identify the estimated molecular weight, the theoretical pI value, the composition of the amino acid, the total amount of positively charged plus negatively charged residues, the atomic composition, the chemical formula, the aliphatic index, and the hydropathicity value of the model protein.

Secondary structure prediction plus 3D structure-modeling
The secondary structure of enzyme alanine racemase in Taibaiella chishuiensis was predicted using the GOR database (Hassan et al., 2020). The 3D structure of the alanine racemase of T. chishuiensis was modeled using the SWISS-MODEL (Biasini et al., 2014). The best model generated from SWISS-MODEL was selected on the basis of a high sequence identity score. Our enzyme (alanine racemase of Taibaiella chishuiensis) was similar to two different enzymes: UDP-N-acetylmuramoyl-tripeptide-Dalanyl-Dalanine ligase (PDB ID: 3zl8) plus monomer of alanine racemase (PDB ID: 4lut). Also, PHYRE2 modeler was used for our sequence to build the 3D model (Kelley et al.,2015).

Homology model validation
The predicted 3D structure was validated using the PROCHECK software and Ramachandran plot to investigate the psi-phi angles to determine the accuracy of the predicted structure. Verify 3D  is also used to evaluate the 3D protein structure model (Meo and Cozzetto, 2006).

Structural comparison
The structure of alanine racemase from T. chishuiensis was analyzed and compared with alanine racemase from C. difficile. PyMol was used to superimpose the modeled structure of the alanine racemase of T. chishuiensis and the known structure of the alanineracemase of C. difficile. Since our enzyme is bifunctional, act also as a ligase, thus, we compared with the known structure of UDPN-acetylmuramoyl-tripeptide-D-alanyl-Dalanine ligase (PDB ID: 3zl8). For comparison of the surface structure and determining active site and binding site, we used also the PyMol (Meo and Cozzetto,2006).

Multiple Sequence Alignment
From the BLAST results obtained from NCBI,alanineracemase was chosen from the five different species to perform the multiple sequences alignment ( Table 2). These 5 enzymes were aligned by using Clustal Omega whichis shown in Figure 2. Positions with a single, completely conserved residue have been demonstrated by an asterisk (*), conservation between groups with highly similar characteristics were indicated by a colon(:), conservation between groups with less similar characteristics were indicated by a period (.) and the variable regions were indicated by a gap in between. From the obtained result, it can be revealed that the active sites of all of 5 enzymes were highly conserved (Mcwilliam et al.,2013).

Prediction of the Secondary Structure
Analysis of secondary structure prediction from GOR on alanine racemase showed that there are 335 amino acid residues involving in the formation of the helix, 176 amino acids for extended strands (beta-sheet) formation and 300 amino acid residues in the formation of the coil, consisting of 42.72 %, 21.18 %, and 36.

The 3D Structure Modeling
The 3D structure of the alanine racemase was modeled using the SWISS-MODEL database. Our enzyme (alanine racemase of Taibaiella chishuiensis) was similar to two different enzymes: UDPN-acetylmuramoyl-tripeptide-D-alanyl-Dalanine ligase (3zl8.1.A) and monomer of alanine racemase (4lut.1.A). So that our sequence from residue 18 up to 468 is similar to a template model of 3zl8, and from 468 upto 829 residues are very similar to the template model of 4lut (Figure 4).
At which about half of our sequence (from N-terminal) identity was 27.10% (with 3zl8.1.A model template), and another half of sequence (from C-terminal) identity was 39.33% (with 4lut.1.A model template).So when we combine these two template models, it will complete all residues of our sequence and it will reveal a reliable Table 3 The active sites of the model enzyme and reference enzymes.  Table 4. The Sarface of modeled enzyme alanine racemase of T. chishuiensis and reference enzymes of alanine racemase C. difficile as well as T. maritima

Enzymes Sarface
Alanine racemase of T. chishuiensis Alanine racemase of C. difficel UDP-N-acetylmuramoyl-tripeptide-D-alanyl-D-alanine ligase (3zl8) ofT. maritima model for our sequence. Also, we modeled our sequence using Phyer2 (Figure 4). The generated structure, which was consists of one chain is very similar to the combined model of SWISS-MODELER Homology Model Validation Ramachandran Plot Ramachandran plot was used to validate the accuracy of the 3D structure by visualizing the phi (Ô) and psi (q) dihedral angles of the residues of amino acids in the protein structure (Mcwilliam et al., 2013) (Figure 5).
In this case, there are 568 amino acid residues in the preferred zone, which provide 76.6 % accuracy, 127 amino acid residues (16.9 %) in the additional allowed regions, as well as 30 amino acid residues (4.0 %) in the generously allowed regions. There are 26 (3.5 %) residues of amino acid in the disallowed zones.

Verify 3D
The Verify3D software has been used to assess the consistency of the degree of every amino acid in a structure to the environment which includes the polarity. The alanine racemase  . 13. Major differences between modeled enzyme alanine racemase of T. chishuiensis (green) and a reference model of enzyme UDP-Nacetylmuramoyltripeptide-D-alanyl-D-alanine ligase of T. maritima (3zl8, blue), highlighted by red color predicted structure possesses an 84% of the residues with the averaged 3D-1D score >=0.2.
From the obtained results of validation software, it is revealed that the model generated possesseda high resolution and high accuracy. Thus, the 3D model generated for alanine racemase is highly reliable (Figure6).

3D structure comparison
A comparative study was performed to investigate the structural differences of alanine racemase and the contribution to the ligand-binding cofactor on its function, based on the sequence alignment with different alanine racemase. The generated 3D structure from Phyre2 for alanine racemase of T.chishuiensis was super imposed with the structure of the templates obtained from SWISS-Model (3zl8, UDP-N-acetylmuramoyltripeptide-D-alanyl-D-alanine ligase, also 4lut, alanine racemase of C. difficile) (Figure7). From the superimposed structure shown in Figure 8, A it can berevealed that the catalytic residues of alanine racemase of T. chishuiensis is conserved with alanine racemase of C. difficile (PDB ID: 4lut). Also, the superimposed structure is shown in Figure 8 (Bhardwaj et al.,2018).
These observations show that the enzyme alanine racemase of T. chishuiensis is a bifunctional enzyme. This enzyme works with racemization of L-alanine and D-alanine as well as ligation of amino acid (Favini-stabile et al., 2013). Therefore, the enzyme alanine racemase of T. chishuiensis functionally is the same with alanine racemase of C. difficile and also has the same function with UDP-Nacetylmuramoyl-tripeptide-D-alanyl-Dalanine ligase of T. maritima. So, this enzyme has two functions that we can call it a bifunctional enzyme.
Structurally, there are differences between alanine racemase of T. chishuiensis and alanine racemase of C. difficile as well as some difference between alanine racemase and UDP-Nacetylmuramoyl-tripeptide-D-alanyl-D-alanine ligaseof T.maritima. These two parts of our enzyme are connected at the loop region: I465, H466, K467, and T468.
The superimposed structure of alanine racemase with binding site labeled with the reference structure (UDP-N-acetylmuramoyltripeptide-D-alanyl-D-alanine ligase (3zl8)) shows that the binding site was found at the similar region, overlapping each other (Figure 8, B and Figure11). This specifies that the binding site for both alanine racemase and UDP-N-acetylmuramoyltripeptide-D-alanyl-D-alanine ligase (3zl8) was conserved and possessed similar functions.
Also, there are two binding regions. The first binding region shows less conservation and it is because of some changes of amino acids such as, the change of amino acid S to amino acid N, but both of them are small and polar so functionally both of them are the same, and also the change of amino acid T to amino acid I, which have the same characteristics, they are hydrophobic. The second binding region is strongly conserved.
The major different spots between alanine racemase of T. chishuiensis and alanine racemase of C. difficile were highlighted by using PYMOL (Figure 12, Table3 and Table4) and shown in red color.
The main different spots between alanine racemase of T.chishuiensis and reference enzyme UDP-Nacetylmuramoyl-tripeptide-D-alanyl-Dalanine ligase of T. maritima (3zl8) are highlighted by using PYMOL (Figure 13 and Table 4). Major different spots between two sequences are highlighted in redcolor.
The predicted model of alanine racemase of T. chishuiensis from this study could serve as a useful model for further study of the structure and function of other bifunctional enzymes. Based on this study, the name of alanineracemase of T.chishuiensis can be bifunctional UDP-Nacetylmuramoyl-tripeptide: D-alanyl-D-alanine ligase/alanine racemase in the UniProt database. Besides, the structural information about alanine racemase of T. chishuiensis especially the active sitesand binding sites could help drug designers to do further research. However, many scientists have been studied for the design of more specific alanine racemase inhibitors by targeting the active sites and binding sites and mutations in the enzyme strain in various microorganisms (Anthony et al., 2011;Azam and Jayaram, 2016). Due to the lack of specificity of most of the previous inhibitors, further study is required for highly selective alanine racemase inhibitors (Azam and Jayaram, 2016). Since the current study is the first study of 3D structural characterization of bifunctional alanine racemase in T. chishuiensis, it may provide useful information about the structure and function of this enzyme for drug designers to do more investigation for more specific inhibitors of alanine racemases and provide effective treatment.

concluSion
In the currentstudy, the bioinformatics approaches have been used to predict the 3D structure of alanineracemase (EC:5.1.1.1) from T. chishuiensis. Due to the absence of a 3D structure of the bifunctional enzyme (UDP-Nacetylmuramoyl-tripeptide: D-alanyl-D-alanine ligase/alanine racemase) in PDB database, we have done the homology modeling with template models of SWISS-MODELLER database (UDP-N-acetylmuramoyl-tripeptide-D-alanyl-D-alanine ligase (3zl8) plus alanine racemase (4lut)).Our enzyme (alanine racemase of Taibaiella chishuiensis) was similar to two different enzymes: UDP-N-acetylmuramoyl-tripeptide-D-alanyl-Dalanine ligase (PDB ID: 3zl8, EC: 6.3.2.10) and of alanine racemase (PDB ID: 4lut, EC: 5.1.1.1). From the sequence and 3D structure analysis, we realized that the first half of the sequence of our modeled enzyme, from residue 18 upto 468 was 27.10% identical with the existing template 3zl8, and the identity of the second half of our query from 468 upto 829 residues was 39.33%, with the template 4lut. Hence, the combination of these two template models has made a reliable model for our sequence. Both, alanine racemase from Taibaiella chishuiensis and alanine racemase of C.difficile (4lut) have the same active site at Lys (499), Lys (39), Tyr (499), Tyr (725) (Tan etal.,2014). On the other hand, bothalanine racemase from Taibaiella chishuiensis and UDP-N-acetylmuramoyltripeptide-D-alanyl-D-alanine ligase (3zl8) from T. maritima also have the same binding site at Asn (295), Asn (264), Arg(326), Arg(293), Lys(447), Lys (405) (Dong et al., 2018). Besides, verify 3D shows good quality with a percentage of 80. Based on these observations, our enzyme (alanine racemase from T. chishuiensis) is bifunctional. Therefore, it has two commission numbers (EC: 5.1.1.1 and EC: 6.3.2.10). Findings from this study may help to contribute to the rational design of the remaining unknown structure of the same enzyme in other organisms and assist in designing proteins model with enhanced properties.

AcKnoWleDGeMenTS
I wish to express my sincere appreciation to my main lecturer, Prof. Dr. Mohd Shahir Shamsir Omar, for encouragement, guidance, critics, and friendship. Without his continued support and interest, this study would not have been the same as presented here.I am also indebted to the Ministry of Higher Education Afghanistan and Higher Education Development Program (HEDP), for supporting and funding this study. Special thanks to the University Technology Malaysia (UTM) for providing the opportunity to study at this prestigious university.