|Year : 2014 | Volume
| Issue : 1 | Page : 1-9
Characterization of hydantoin racemase predicted from the genome sequence of Lactobacillus pentosus KCA1
Kingsley C Anukam
Department of Medical Laboratory Science, TWAS Genomics Research Unit, School of Basic Medical Sciences, University of Benin, Nigeria
|Date of Web Publication||1-Jul-2014|
Kingsley C Anukam
Department of Medical Laboratory Science, TWAS Genomics Research Unit, School of Basic Medical Sciences, University of Benin
Source of Support: Dr. Anukam KC research is partly supported
by the Third World Academy of Sciences (TWAS), under the
RESEARCH GRANT AGREEMENT (RGA) No.09-017RG/BIO/
AF/AC_G-UNESCOFR:3240230 312., Conflict of Interest: None
Background: Hydantoin racemase from Lactobacillus species of human origin has not been reported and characterized. The genome of Lactobacillus pentosus KCA1 has been sequenced and found to possess gene cassettes and open reading frames encoding the hydantoinase machinery, including a putative hydantoin racemase. Aims: To use bioinformatic tools to characterize the new hydantoin racemase predicted in the genome sequence of L. pentosus KCA1. Materials and Methods: Bioinformatic tools such as ClustalW algorithm was used to align hydantoin racemase from L. pentosus KCA1 with other hydantoin racemases extracted from the uniprot database. I-TASSER was used for the prediction of secondary structure, 3-D model, similarity structure in PDB, and functional active binding site residues. Results: L. pentosus KCA1 hydantoin racemase showed significant amino acid sequence identity with hydantoin racemase from the selected bacterial organisms in the protein databank (PDB). The predicted secondary structure revealed 9 alpha-helices and 8 beta-strands. Functional prediction using enzyme partners predicted EC number 18.104.22.168 as the corresponding enzyme homolog (3eq5A) showing Cys83 and Cys187 as the potential active residues in KCA1 hydantoin racemase. The 3-D structure of KCA1 hydantoin racemase has a confidence score (C-score) of 1.2 that reflects a model of better quality, based on 3qvjA from PDB. Conclusion: The in silico data presented provides new insights into the potential activity and substrate specificity of hydantoin racemase from L. pentosus KCA1 and has proposed a mechanism for racemization of hydantoin derivatives that is consistent with the two-base process observed in other members of the Aspartate/Glutamate superfamily.
Keywords: Amino acids, binding site, hydantoin racemase, Lactobacillus pentosus KCA1
|How to cite this article:|
Anukam KC. Characterization of hydantoin racemase predicted from the genome sequence of Lactobacillus pentosus KCA1. Niger J Exp Clin Biosci 2014;2:1-9
|How to cite this URL:|
Anukam KC. Characterization of hydantoin racemase predicted from the genome sequence of Lactobacillus pentosus KCA1. Niger J Exp Clin Biosci [serial online] 2014 [cited 2019 May 21];2:1-9. Available from: http://www.njecbonline.org/text.asp?2014/2/1/1/135605
| Introduction|| |
The production of optically pure D- and L- amino acids, which are utilized as intermediates in the synthesis of antibiotics, pesticides, sweeteners and biologically active peptides, involves enzymatic reactions.  The enzymatic reaction cascades are often referred to as 'hydantoinase process' whereby stereo-selective hydrolysis of DL-5-monosubstituted hydantoin substrate results in the production of optically pure amino acids.  In the second step, chemical and/or enzymatic racemization of the remaining nonhydrolyzed 5-monosubstituted hydantoin starts but the process is usually very slow, taking several hours to complete. , Total conversion and 100% optically pure D or L-amino acids are only obtained when a hydantoin racemase racemises the remaining nonhydrolyzed 5-monosubstituted hydantoin. 
Hydantoin racemase enzymes from several bacterial organisms have been purified and biochemically characterized. , Notably, the hydantoin racemase enzyme involved in the production of L-amino acids from Pseudomonas sp. strain NS671,  and Arthrobacter aurescens DSM 3747,  and more recently the production of D-amino acids from Agrobacterium tumefaciens, have been purified and biochemically characterized.
Genomic localization and genetic organization of these genes have been reported together with a hydantoinase, a carbamoylase, and a putative hydantoin transport protein. , Sequence homology has shown two highly conserved cysteine residues in the studied hydantoin racemases. The first heterologously expressed hydantoin racemase from Sinorhizobium meliloti, its purification, and its biochemical characterization has been accomplished. Sinorhizobium meliloti is an alpha-proteobacterium of the family Rhizobiaceae, as is Agrobacterium tumefaciens, which forms important Nitrogen-fixing root nodules in legumes.
However, hydantoin racemase has not been reported from Lactobacillus species. The importance of Lactobacilli in both health and synthesis of biological molecules supporting probiotic action has been reviewed.  Recently, the genome sequence of the first Lactobacillus pentosus KCA1 isolated from the vagina of a healthy Nigerian woman has been completed.  The genome has a novel five gene cluster encoding putative hydantoin racemase (KCA1_1486) with 42% amino acid identity to Thermoanaerobacter brockii subsp. finnii Ako-1, and a N-methylhydantoinase (KCA1_1489)-(ATP-hydrolyzing) with 61% amino acid identity to Enterococcus faecalis E1Soi. It appears that hydantoin racemase is present only in the genome of L. pentosus KCA1 among all the known Lactobacillus bacteria present in the uniprotein database. The gene is located within a cassette involving an ATP-hydrolyzing N-methylhydantoinase and a putative protein involved in hydantoin utilization.
The objective of this study is to use bioinformatic tools to characterize the hydantoin racemase predicted in the genome sequence of L. pentosus KCA1, with the view to providing valuable information on the binding sites of the hydantoin racemase and its potential application in the production of optically pure D- and L- amino acids.
| Materials and methods|| |
Identification of Hydantoin Racemase Gene from L. Pentosus KCA1
The Ensembl genome annotation system developed jointly by the European Buoinformatic Institute (EBI) and the Wellcome Trust Sanger Institute was used for the location and extraction of the nucleotide base sequence or open reading frame (ORF) and the amino acid translation of the hydantoin racemase from L. pentosus KCA1 (http://ensemblgenomes.org/id/EIW14007). The uniprotein database was generally searched for similarity sequences with the use of BLASTp algorithm.
Multiple Sequence Alignments
The amino acid translations from the nucleotide bases of 15 bacterial organisms were selected from the BLASTp of uniprotein database with the sequence of L. pentosus KCA1 (http://www.uniprot.org/uniprot/I9AM59) based on product annotation hit (hydantoin racemase), gene name, % identity, matrix score and E-value. The amino acids sequences were imported into the ClustalW algorithm for multiple sequence alignments. In addition, the sequences were sent for gene tree analysis using the 'one click' mode at www.phylogeny.fr.  This is a "default" mode which uses a pipeline already set up to run and connect programs recognized for their accuracy and speed (MUSCLE for multiple alignment, Gblocks for alignment curation, PhyML for phylogeny and finally TreeDyn for tree drawing) to reconstruct a robust phylogenetic tree from a set of hydantoin racemase amino acid sequences.
Prediction of Secondary Structure, 3-D Model, Similarity Structure in PDB, Functional and Binding Sites Predictions Using I-TASSER Algorithm
The iterative threading assembly refinement (I-TASSER) server is a four stage integrated platform for automated protein structure and function based on the sequence-to-structure-to-function paradigm.  This integrated algorithm was generally used for the prediction and the amino acid sequence was submitted to online,  (http://zhanglab.ccmb.med.umich.edu/I-TASSER).
Briefly, in the initial step of I-TASSER, the amino acid sequence of Hydantoin racemase from L. pentosus KCA1 was matched against a nonredundant sequence database by position-specific iterated BLAST (PSI-BLAST),  to identify evolutionary relatives. A sequence profile was then created based on multiple alignment of the sequence homologs, which was also used to predict the secondary structure using PSIPRED.  Assisted by the sequence profile and the predicted secondary structure, the KCA1 hydantoin racemase sequence was then threaded through a representative PDB structure library using LOMETS,  a locally installed meta-threading server combining seven state-of-the-art threading programs [FUGUE,  HHSEARCH,  MUSTER,  PROSPECT,  PPA,  SP3,  and SPARK.  In the threading programs, the templates are ranked by a variety of sequence-based and structure-based scores. The top template hits from each threading program are then selected for further consideration. The quality of the template alignments (and therefore the difficulty of modeling the targets) is judged based on the statistical significance of the best threading alignment, i.e., the Z-score, which is defined as the energy score in standard deviation units relative to the statistical mean of all alignments.
The second stage employs continuous fragments in threading alignments which are removed from the template structures, and are used to assemble structural conformations of the sections that aligned well, with the unaligned regions (mainly loops/tails) built by ab initio modeling. 
The third stage involves model selection and refinement. The fragment assembly simulation is performed again starting from the selected cluster centroids, whereby external constraints are pooled from the LOMETS threading alignments and the PDB structures that are structurally closest to the cluster centroids, as identified by TM-align.  The decoys generated during the second round of simulations are clustered again, and the lowest energy structures are selected as input for REMO,  which generates the final structural models by building all-atom models through the optimization of hydrogen bonding networks. In the last stage, the function of the query protein is inferred by structurally matching the predicted 3D models against the proteins of known structure and function in the PDB.
The structural analogs of the KCA1 hydantoin racemase sequence protein in the GO library are mainly matched based on the global topology using TM-align,  and a consensus is derived based on the frequency of occurrence of the GO terms. The structural analogs in the EC and binding site libraries are matched based on both global and local structural similarity.
| Results|| |
The ORF coding for KCA1 hydantoin racemase resulted in a cluster of orthologous group of protein (COG) hit -gnlǀCDDǀ33883 COG4126, belonging to COG class E involved in Amino acid transport and metabolism with e-value of 7.0 X10 -30 . The protein family has Pfam identity number as Pf01177.15, representing ASP-GLU-Racemase with e-value of 7.7X10 -15 . BLASTp from the uniprotein database revealed 250 hits for blastp blast on UNIPROTKB sorted by score descending from 489 to 258 [Table 1]. Fifteen organisms were selected based on annotation name, hydantoin racemase. T. brockii ATCC 43586 was the closest organism with 242 nucleotide base pairs and its hydantoin racemase having 42% amino acid identity to the predicted L. pentosus KCA1 hydantoin racemase. In the phylogenetic gene tree relationship [Figure 1] a confidence value of 0.914 was obtained at the node distinguishing L. pentosus KCA1 hydantoin racemase and T. brockii ATCC 43586, while a confidence value of 0.632 was observed at the node between L. pentosus KCA1 and Rhodococcus erythropolis SK121.
|Table 1: L. pentosus KCA1 hydantoin racemase BLASTp hits on UNIPROTKB sorted by score descending from 482 to 261|
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|Figure 1: Hydantoin racemase gene tree. Confidence values for the branching order were generated by bootstrapping (based on 100 replications). The number at the nodes indicates the bootstrap values. The scale bar indicates 1 amino acid substitution per 100 amino acids|
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From the clustalw multiple sequence alignments, L. pentosus KCA1 hydantoin racemase showed significant amino acid sequence identity with hydantoin racemase from the selected bacterial organisms in the protein databank [Figure 2]. Importantly four cysteine residues at position 83, 171, 187, and 216 of the L. pentosus KCA1 hydantoin racemase were identified.
|Figure 2: ClustalW multiple alignment of the amino acid sequence of hydantoin racemases. Shown are the 16 hydantoin racemases indicating the gene entry name and the position of hydantoin racemase from L. pentosus KCA1 (I9AM59). Number 83, 171, 187 and 216 shows the position of cysteine residue in KCA1 strain. Overall homology is indicated under with*|
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The predicted secondary structure with I-TASSER revealed 9 alpha-helices and 8 beta-strands as shown in [Figure 3]. The first a-helix begins at position 17 (P-Proline) and ends at position 31 (Y-Tyrosine). It appears the longest a-helix occurred between position 53 (E-Glutamic acid) and 72 (R-Arginine) while the shortest a-helix has only 4 amino acid residues at position 54 (V-Valine) and 57 (L-Leucine).
|Figure 3: Predicted secondary structure with I-TASSER showing 9 alpha helices (red color) and 8 beta-strands (blue) and protein template (3qvjA) alignment with the top rank normalized Z-score|
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The 3-D model of KCA1 hydantoin racemase has a C-score of 1.2 and an estimated accuracy of 0.88±0.07 (TM-score-template modeling score) and 3.4±2.3Ε (RMSD-root mean square deviation) based on the 10 templates used for alignments with 3qvjA-PDB having the top normalized Z-score of 4.14. The co-ordinate file of the L. pentosus KCA1 hydantoin racemase model was downloaded in PDB format and Jmol molecular visualization program  was used to view the predicted structure as shown in [Figure 4].
|Figure 4: 3D model of KCA1 hydantoin racemase as determined with I-TASSER based on alignments with 3qvjA-PDB. The co-ordinate file model in PDB format was visualized with Jmol molecular visualization program showing the position of the cysteine residues, front, top, right and left view. Red color indicates the a-helices, while yellow indicates the b-pleated sheet. Magenta (+3 turns) and White (+2 turns)|
Click here to view
Proteins with highly similar structure in PDB as identified by TM-align is shown in [Table 2]. The protein 3qvkA PDB-Hit as the top rank has a TM-score of 0.942 and a coverage of 0.955.
|Table 2: Proteins with highly similar structure in PDB as identified by TM-align|
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Five enzyme homologs were identified in PDB as having similar functions to the predicted KCA1 sequence [Table 3]. Notably, 2eq5A PDB-Hit has the top rank with confidence score of 0.663 for the Enzyme Classification (EC) number prediction. The predicted active-site residues were identified as residues at position 83 and 187 in the KCA1 hydantoin racemase sequence.
|Table 3: Function prediction using COFACTOR: Top 5 enzyme homologs in PDB|
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Predicted gene ontology (GO) terms [Table 4] associated with the KCA1 hydantoin racemase query sequence identified 2eq5A from the PDB-hit as GO:0003824, GO:0008152, GO:0016855. This protein has the top GO confidence score of 0.61 and TM-score of 0.7674.
Seven template proteins with similar binding site residues were predicted to occur at positions 14,50,83,84,123,154,186,187,188 from the KCA1 hydantoin racemase sequence [Table 5]. The template protein 3qvlB from PDB-hit has a ligand-binding site prediction confidence score of 0.74 as the top rank.
[Figure 5] shows comparative structure of 100% van der waal forces between KCA1 hydantoin racemase and the top rank proteins in the PDB for similar binding site ligands (3qvlB), functional enzyme homologs (2eq5A) and highly similar structure (3qvkA).
|Figure 5: Comparative structure of 100% van der waal forces between KCA1 hydantoin racemase and the top rank proteins in the PDB for similar binding site ligands (3qvlB), functional enzyme homologs (2eq5A) and highly similar structure (3qvkA)|
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| Discussion|| |
This is the first in silico analysis of hydantoin racemase from a Lactobacillus species (L. pentosus KCA1), which belonged to Class E in the Cluster of Orthologous Group (COG) of proteins responsible for the amino acid transport and metabolism. The protein family (Pfam) has the same domain organization similar to Asp/Glu/Hydantoin racemase. ,
The calculated molecular mass in kilo Dalton (kDa) from the sequence shows a value of 26 kDa, which falls in the range 24-27 kDa as other similar hydantoin racemases in the non-redundant protein database. It remains to be determined if the calculated molecular mass will be less or greater than the measured molecular mass. Previous study by Martinez-Rodriguez et al. reported that the molecular mass of the S. meliloti purified hydantoin racemase enzyme subunit (31 kDa) was greater than the calculated molecular mass from the amino acid sequence (27 kDa).
The highest sequence identity was observed between L. pentosus KCA1 hydantoin racemase and T. brockii ATCC 43586 and Rhodococcus erythropolis SK121. It should be noted that T. brockii ATCC 43586 was originally isolated from lake sediment found in Lake Kivu in Africa (http://www.ncbi.nlm.nih.gov/gene/10164493). The confidence value of 0.914 for the gene tree phylogeny may probably indicate relatedness of the two organisms if evolutionary location is considered.
Four cysteine residues were identified in KCA1 hydantoin racemase. However, two cysteine residues at position 83 and 187 appear to be conserved within the studied hydantoin racemases [Figure 2]. Similar studies have shown that the two cysteine residues are at the catalytic center of the hydantoin racemase for Sinorhizobium meliloti CECT 4114  and Arthrobacter aurescens DSM 3747.  Andújar-Sánchez et al.  demonstrated that activity assays with mutants showed a drastic decrease in activity of the enzyme, indicating that both cysteine 76 and 181 are essential for catalysis. More recently, mutation of either of the active-site cysteine, Cys79 or Cys184 in hydantoin racemase from Klebsiella pneumoniae (KpHpxA) to serine, inactivated this enzyme. 
Members of the aspartate/glutamate racemase superfamily to which hydantoin racemase from L. pentosus KCA1 belong are known to employ conserved cysteines for catalysis of racemization. ,
In addition to conserved cysteine residues, there are 10 other conserved residues observed in the hydantoin racemase that may serve to strengthen the structure. These conserved residues include, using hydantoin racemase from KCA1 as reference, proline (Pro13, Pro209), isoleucine (Ile50), glycine (Gly46, Gly88, Gly102, Gly186, Gly189), arginine (Arg93), valine (Val154), and alanine (Ala174). Interestingly, the cysteine residue at position 187 of hydantoin racemase from KCA1 appears to be conserved with GCTG motif in seven of the aligned sequences, while GCAG was observed in six sequences, including hydantoin racemase from S. meliloti. Also GCGG motif is present in three of the studied sequences. The functional differences of these motifs are yet to be determined.
The secondary structure of hydantoin racemase from KCA1 strain indicates prediction with higher confidence as the confidence scores for both alpha helices and beta strands are high [Figure 3]. The confidence values are shown for each residue ranging between 0 and 9, in which a higher score indicates a prediction with strong confidence.  The 3-D structure of KCA1 hydantoin racemase has a C-score of 1.2 that reflects a model of better quality [Figure 3] and [Figure 4]. This C-score was based on allantoin racemase 3qvjA PDB and 3qvkA PDB (245 base pairs) from Klebsiella pneumoniae with active-site cysteine identified at position Cys79 or Cys184. C-score is typically in the range of − 5, to 2. In general, models with C-score > − 1.5 have a correct fold. C-score is an estimate of the quality of the predicted models, and is calculated based on the significance (Z-score) of the threading alignments in LOMETS and the convergence (cluster density) of the I-TASSER simulations.  The active-site cysteine are predicted at position Cys83 and Cys187 of the hydantoin racemase from L. pentosus KCA1 based on Clustal Waal alignment, and model prediction. The I-TASSER, on function prediction using cofactor, predicted EC number 22.214.171.124 as the corresponding enzyme homologs showing Cys83 and Cys187 as the potential active residues. All the five PDB hits (2eq5A, 1jflA, 2jfuA, 2jfqA, and 1zuwC) gave the same active-site prediction [Table 3], indicating that the crystal structure of hydantoin racemase (2eq5A PDB) from Pyrococcus horikoshii OT3 has the top rank for C-score and TM-score relative to KCA1 strain. The predicted gene ontology (GO) terms also indicated that 2eq5A PDB-hit has the highest GO-C-score (GO:0003824, GO:0008152, GO:0016855) [Table 4]. These GO terms describes catalysis of biochemical reactions at physiological temperatures with enzymes possessing specific binding sites for substrates and are usually composed largely of proteins. The seven templates with similar binding sites to KCA1 hydantoin racemase showed that 3qvlB PDB-hit from allantoin racemase (Klebsiella pneumoniae) has the top rank for ligand-binding sites with 5HY (Hydantoin-5-Acetic acid) as the ligand name [Table 5]. Comparatively, the KCA1 hydantoin racemase structure is very similar to the 3qvlB PDB as shown in the display of atoms by 100% van der waal forces [Figure 5].
In conclusion, bioinformatic tools have characterized the hydantoin racemase predicted in the genome sequence of L. pentosus KCA1 as a protein possessing 9 a-helices and 8 β-strands. The protein may employ putative conserved cysteine residues (Cys83 and Cys187) for catalysis of racemization of 5-monosubstituted hydantoin derivatives leading to the production of optically pure D and L-amino acids. In recent times, the demand for atomic-level structural refinements for generation of models for use in drug screening and biochemical function inference is becoming more germane than ever. Hopefully, the structure of KCA1 hydantoin racemase may serve as a new template that would be an addition to structural genomics and traditional structural biology studies.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]