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ISSN: 2153-0602
Journal of Data Mining in Genomics & Proteomics
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Motif Design for Nitrilases

Nikhil Sharma and Tek Chand Bhalla*

Sub- Distributed Information Centre, Himachal Pradesh University, Summer-Hill, Shimla-171005, India

*Corresponding Author:
Dr. Tek Chand Bhalla
Sub-Distributed Information Centre
Himachal Pradesh University
Summer-Hill, Shimla-171005, India
E-mail: [email protected]

Received date: August 06, 2012; Accepted date: September 24, 2012; Published date: October 01, 2012

Citation: Sharma N, Bhalla TC (2012) Motif Design for Nitrilases. J Data Mining Genomics Proteomics 3:119. doi: 10.4172/2153-0602.1000119

Copyright: © 2012 Sharma N, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Nitrilase is one of the nitrile metabolizing enzymes that catalyses the conversion of nitriles to corresponding acids which has gained importance in green chemistry. Nitrilase being substrate specific yet it acts over a wide range of nitriles (aliphatic/aromatic) has drawn attention due to its utility in mild hydrolysis. Most of the nitrilases reported hitherto have been physically extracted characterized from the microbial/plant sources. In order to identify sequences for nitrilases two groups of motif were designed i.e. aliphatic nitrilase motif’s (MDMAl) and aromatic nitrilase motif’s (MDMAr) each with four motifs specifically belonging to nitrilase with conserved catalytic triad (Glu-48, Lys-131, Cys-165) which can be used as marker for nitrilase. Conserved regions were identified by performing Multiple Sequence Alignment (MSA) using Multiple EM for Motif Elicitation (MEME). The Manually Designed Motifs (MDM’s) were validated by ScanProsite and their presence is also confirmed by PRATT, Gblocks and MEME. The ScanProsite search against the MDMAr exhibited some new sources of aromatic nitrilase from plant, animals and microbes whereas MDMAl only exhibited nitrilase from microbes. Besides identifying unique motifs in order to confirm their substrate specificity for nitriles, randomly selected sequences were validated by studying some important physiochemical parameters and position specific amino acids.

Keywords

Gblock; Manually designed motif (MDM); Multiple alignments; Nitrilase; PROSITE; Pratt; Multiple EM for motif elicitation (MEME); ScanProsite; Substrate specificity

Background

Nitrilases are responsible to catalyze the hydrolysis of nitriles into corresponding acids and ammonia which are used in chemistry for the production of industrially important acids. Nitrilases frequently exhibit enantioselectivity under mild reaction conditions, making them ideal tools for green chemistry from conversion of nitrile or in environment management for remediation of nitrile contaminated soil, water and air. Based on substrate specificity they are grouped as aromatic and aliphatic nitrilase.

A large number of microbial plant and animal genome have been sequenced in the past decade and genome sequence data have tremendously expanded over those years. Screening of genome/ proteome databank will be worthwhile to find out novel sources of enzymes. Many tools and techniques such as BLAST, Hidden Markov Model (HMM), neural network classification are used for in silico screening of genome/proteome sequence database till date. Among these, motif design has been found to be one of the most reliable strategy for efficient screening of database [1-3] as motifs of a particular protein sequence signifies its specific structure and functionally which is useful to characterize and classify that protein.

Nitrile metabolizing microorganisms are mostly isolated from soil or water using enrichment culture method and these are cultured and tested for the nitrilase activity during screening of microbial isolates. This is a classical method for isolation of the nitrile metabolizing microorganisms and subsequent screening for nitrilases activity is a time consuming and cost intensive process [4-7]. Presently bioinformatical tools and techniques such as Blast [1], HMM (Hidden Markov Model) [8-9] the neural network classification [10,11] and Motif Identification Neural Design (MOTIFIND) [12] are used for screening of genome and sequence database for searching gene coding for novel enzymes. The present communication aims at Manually Designed Motif (MDM) for aliphatic and aromatic nitrilase and their validation using Prosite, ScanProsite, BLAST, PRATT and G-block is being reported. On the basis of the earlier studies on in silico analysis of amino acid sequence of the aromatic and aliphatic nitrilases [13] and using bioinformatics approaches, motifs were manually designed to differentiate and identified aromatic and aliphatic nitrilases. Computational analysis of amino acid sequences and study pertaining to physiochemical properties [14] of nitrilases have revealed differences between aromatic and aliphatic nitrilases [13].

Methodology

Retrieval of sequences and designing of motifs

The protein sequences were obtained from the protein server UniprotKB/SwissProt (release 2011-12) database. To design motifs manually, six microbial aliphatic and aromatic nitrilases were selected on the basis of our earlier study (Table 1) [13].

Microorganism Substrate specificity Accession Number (ExPASy)
R. rhodochrous J1 Aliphatic Q03217
R. rhodochrous K22 Aliphatic Q02068
Methylibium petroleiphilum Aliphatic A2SEG6
Pseudomonas syringae pv. syringae Aliphatic Q500U1
Synechococcus sp. ATCC 27144 Aliphatic Q5N478
Bradyrhizobium sp ORS278 Aliphatic A4YWK0
Burkholderia cepacia J2315 Aliphatic B4EE44
Pseudomonas entomophila Aliphatic Q1I7X1
Janthinobacterium Marseille Aliphatic A6T0X3
Microscilla marina Aliphatic A1ZD79
Bordetella bronchiseptica Aliphatic Q7WNC4
Shewanella sediminis Aliphatic A8FQL4

Table 1: Names of various microrganisms and accession number of the some microbial nitrilases (aliphatic and aromatic).

Multiple Sequence alignment and phylogenetic analyses

Multiple sequence alignments were performed using CLUSTAL W [15] and CLUSTAL X (version 2.1) software with default settings and verified with MAFFT (Multiple Alignment with Fast Fourier Transform) and MEME (Multiple Em for Motif Elicidation). Nitrilase (aliphatic and aromatic) sequences were used for phylogenetic relationship inferences. Phylogenetic tree was generated by Neighbor Joining (NJ) using CLUSTAL X (Figure 2). After multiple alignments and also on the basis of presence of catalytic triad i.e glutamine, lysine, and cysteine (Glu-48, Lys-131, and Cys-165) motifs were manually designed (MDM) for aliphatic (MDMAl) and aromatic nitrilases (MDMAr). In order to verify the manually designed motif and to eliminate poorly aligned or divergent regions of aligned protein Gblock software was used [16,17]. Bootstrap value was calculated by using default value (1000).

In-silico physiochemical characterization and validation of motifs

After confirmation through MEME version 4.8.1 (Figure 1a and Figure 1b), Gblock tool, Manually Designed Motif ’s (MDM’s) were subjected to database search through ScanProsite to validate and determine the specificity of these motifs in identifying nitrilase sequences. ScanProsite tool was used to search the databases both from UniprotKB/Swiss-Prot and UniprotKB/TrEMBL with match mode of “not greedy, not overlaps” and “no includes”. ScanProsite analysis resulted in protein sequences which were counted and were grouped into plants, bacteria and uncultured organisms (Table 2).

Nitrilases Manually Designed Motif Protein Sequence Obtained Hits obtained form MDM Motif Presence UniProtKB/TrEMBL Entries
Aliphatic [FL]-[ILV]-[AV]-F-P-E-[VT]-[FW]-[IL]-P-[GY]-Y-P-[WY] 84 F - 17
B- 38
UO-29
34 - 68 84
R-R-K-[LI]-[KRI]-[PA]-T-[HY]-[VAH]-E-R 115 F - 31
B- 53
UO-31
125 - 177 115
C-W-E-H-[FLX]-[NQ]-[PT]-L 248 F - 49
B- 132
UO-67
157 - 215 248
[VA]-A-X-[AV]-Q-[AI]-X-P-[VA]-X-[LF]-[SD] 130 F - 06
B- 87
UO-37
1 - 30 130
Aromatic [ALV]-[LV]-[FLM]-P-E-[AS]-[FLV]-[LV]-[AGP]-[AG]-Y-P 55 F-01
B-42
P-10
UO-2
26 - 55 55
[AGN]-[KR]-H-R-K-L-[MK]-P-T-[AGN]-X-E-R 104 F-09
B-68
P-18
UO-8
A-01
165 - 206 104
C-W-E-N-[HY]-M-P-[LM]-[AL]-R-X-X-[ML]-Y 128 F-3
B-67
A-01
UO-23
125 - 180 128
A-X-E-G-R-C-[FW]-V-[LIV] 105 F-19
B-74
A-05
UO-08
191 - 216 105

Table 2: Manually designed motif’s with number of hits obtained from UniProt KB and TrEMBL and their position. F-Fungus, B-Bacteria, UO-Uncultured Organism, P-Plants and A-Animal.

data-mining-genomics-Nitrilase-validated

Figure 1a: Aliphatic Motif’s for Nitrilase validated by MEME version 4.8.1(2012) (the height of the motif “block” is proportional to -log(p-value), truncated at the height for a motif with a p-value of 1e-10).

data-mining-genomics-MEME-version

Figure 1b: Aromatic Motif’s for Nitrilase validated by MEME version 4.8.1(2012) (the height of the motif “block” is proportional to -log(p-value), truncated at the height for a motif with a p-value of 1e-10).

MDM were further verified by using Pratt tool to identify conserved pattern in protein sequences. In order to find the nature of the nitrilase sequences i.e. aliphatic/aromatic we also studied some of the physiochemical properties of randomly selected protein sequences. The physicochemical parameters studied included negatively charged residue (Asp+Glu), molecular weight, alanine content (%) and instability index. The values of these parameters for the predicted/ reported nitrilase sequences were deduced from the ProtParam (http://web.expasy.org/protparam/) of Expert Protein Analysis System (ExPASy) i.e. the proteomic server of Swiss Institute of Bioinformatics (SIB). Fasta format of sequences were used for analysis.

Results

Manually Designed Motifs were validated for the two groups of nitrilases i.e. aliphatic and aromatic and the results are shown in Table 1. Hits obtained from each manually designed motif (MDM) of aliphatic and aromatic nitrilases in SwissProt and TrEMBL databases are obtained through a motif search. These motifs are conserved for aliphatic and aromatic nitrilases (Figure 1a and Figure 1b). MEME version 4.8.1 (2012) software also confirmed the presence of all the MDM, which were analyzed through ScanProsite and resulted in protein sequences only for nitrilase family. These sequences have conserved catalytic triad i.e. glutamine, lysine, and cysteine (Glu-48, Lys-131, Cys-165) which are essential for the nitrilases activity [18-21].

Designing and validation of motifs for aliphatic nitrilase

In the present study manually designed motifs for aliphatic nitrilases (MDMAl) were (i)[FL]-[ILV]-[AV]-F-P-E-[VT]-[FW]-[IL]-P- [GY]-Y-P-[WY]; (ii)R-R-K-[LI]-[KRI]-[PA]-T-[HY]-[VAH]-E-R; (iii) C-W-E-H-[FLY]-[NQ]-[PT]-L and (iv) [VA]-A-X-[AV]-Q-[AI]-XP-[ VA]-X-[LF]-[SD]. ScanProsite analysis of these MDMAl resulted in 85 (i) 115 (ii) 248 (iii) 130 and (iv) hits of amino acid sequences respectively. All the 578 protein sequences those were obtained after ScanProsite analysis of all the four MDMAl pertained to microbes only. In these 578 protein sequences, there were 164 nitrilase sequences from the uncultured microorganisms as shown in Table 2.

MDMAl were found between amino acid (i) 40-55, (ii) 125-140, (iii) 160-180, (iv) 1-30. All these results except the fourth MDMAl were further confirmed by the Pratt analysis. According to Pratt analysis first, second, third and fourth MDMAl were found between 40-55, 125-140, 160-180 and 1-30 amino acid numbers respectively (see additional file 1). Presence of MDMAl through Pratt analysis showed that the first, second and third DMP (design motif pattern) were in between 34 to 68, 125 to177 and 157 to 215 numbered amino acids, respectively (see additional file 1).

Total amino acid numbers of all resulted sequences were found to be in between 330-382. Molecular masses were from 37226 to 41971 daltons, alanine content was in between 10-16%, negative charged residue were in between 37-52 and instability index ranged from 30.32 to 44.13 for the aliphatic nitrilase sequences (Table 3a).

Pattern 1  ( [FL]-[ILV]-[AV]-F-P-E-[VT]-[FW]-[IL]-P-[GY]-Y-P-[WY] )
S. No   Organism Access. No. Mol wt. Amino Acid No. NCR* Alanine (%) Instability index
1 Uncultured organism Q6RWK6 378791 354 39 16.9 36.02
2 Nocardia sp. Q5D4V8 41971.9 381 51 11.5 30.81
3 Sclerotinia sclerotiorum A7F824 41160.5 368 49 6.8 35.31
4 Nectria haematococa C7YVF8 41018.6 366 53 7.7 33.84
5 Silicibacter pomeroyi Q5LLB2 37226.1 344 44 10.8 35.63
Pattern 2  ( R-R-K-[LI]-[KRI]-[PA]-T-[HY]-[VAH]-E-R )
1 Uncultured organism Q6RWS5 37242.6 345 40 15.4 37.02
2 Uncultured organism Q6RWS0 38048.3 349 36 12.3 35.09
3 Rhodococcus rhodochorus A4LA85 40370.4 366 46 10.9 39.90
4 B.japonicum Q89GE3 36203.3 334 39 13.5 33.69
5 Aspergillus niger. A9QXEO 40022.2 356 49 7.6 30.71
Pattern 3  ( C-W-E-H-[FLX]-[NQ]-[PT]-L )
1 Burkholderia multivorans B9BNU9 37461.8 345 42 11.9 36.48
2 Uncultured organism Q6RWF2 37809.2 344 44 11.0 39.69
3 Uncultured organism Q6RWF9 38029.4 353 40 17.0 37.41
4 Methylibium petroleiphilum A2SEG6 37969.1 357 39 16.0 42.17
5 Paracoccidioides brasiliensis C1H5H4 37871.1 356 39 10.1 37.47
Pattern 4  ( [VA]-A-X-[AV]-Q-[AI]-X-P-[VA]-X-[LF]-[SD] )
1 Rhodococcus rhodochrous Q03217 40189.2 366 46 11.2 39.36
2 Uncultured organism Q6RWK6 37879.1 354 39 16.9 36.02
3 Sphingomonas wittichi B4WRX6 35995.2 334 42 12.6 38.51
4 Silicibacter pomeroyi Q5LLB2 37226.1 344 44 10.8 35.63
5 Providencia alcalifaciens B6XD88 37552.7 343 43 10.8 30.32

Table 3a: In silico analysis of some physiochemical properties of some aliphatic nitrilase (sequences obtained from ScanProsite) from Manually Designed Motif analysis.

Designing and validation of motifs for aromatic nitrilase

Manually designed motifs for aromatic nitrilase (MDMAr) were (i) [ALV]-[LV]-[FLM]-P-E-[AS]-[FLV]-[LV]-[AGP]-[AG]-Y-P; (ii) [AGN]-[KR]-H-R-K-L-[MK]-P-T-[AGN]-X-E-R; (iii) C-W-E-N- [HY]-M-P-[LM]-[AL]-R-X-X-[ML]Y and (iv) A-X-E-G-R-C-[FW]-V- [LIV]. ScanProsite analysis of all the four MDMAr resulted in 55, 104, 128 and 105 protein sequences respectively. Out of these 392 protein sequences, 251 were of microbes, 28 of higher plants and 7 from animals. Forty one sequences belonged to uncultured organisms. Plant protein sequences were only obtained from first and second MDMAr and not from (iii) and (iv) manually designed motifs (Table 3). The designed motif pattern was found to be in between 40-60; 130-165; 160- 185; 200-220 amino acid respectively (see additional file 2). In order to confirm the ScanProsite results, Pratt analysis regarding the presence of MDM was done and it was found that first, second, third and fourth designed motif pattern (DMP) were in between 26 to 55, 125 to 180, 165 to 206 and 191 to 216 amino acids, respectively.

In silico physiochemical analysis of the protein sequences retrieved from ScanProsite using first, second, third and fourth MDMAr exhibited the total number of amino acid ranged between 306-331, molecular sizes were found to be between 32273-36940 dalton’s, alanine content was between 4.9-11.0%, negative charged residues ranged from 34-44 and instability index was found to be 39.82-48.68 for the aromatic nitrilase sequences (Table 3b).

Pattern 1 ( [ALV]-[LV]-[FLM]-P-E-[AS]-[FLV]-[LV]-[AGP]-[AG]-Y-P )
S. No Organism Access. No. Mol wt. Amino Acid No. NCR* Alanine (%) Instability index
1 Burkholderia capacia HI2424 A0K4N0 32737.0 307 36 11.4 41.50
2 Microscilla marina A1ZD79 33583.0 302 31 7.9 42.07
3 Magnoparthe grisea A4R7D5 34847.0 324 42 9.0 44.68
4 Populus trichocarpa B9HBW3 28464.0 266 28 10.5 41.42
5 Pseudomonas aeruginosa A6V5Q2 33417.2 310 35 10.6 40.34
Pattern 2 ( [AGN]-[KR]-H-R-K-L-[MK]-P-T-[AGN]-X-E-R )
1 Shewanella sediminis(strain HAW-EB3) A8FQL4 34872.8 317 41 8.5 41.24
2 Actinobacillus minor NM305 C5RYV4 33992.0 307 39 7.5 43.43
3 Pirellula staleyiDSM 6068 D2R9H8 32521.3 302 35 10.6 49.18
4 Pantoea sp. At-9b. C8Q5J0 33273.0 306 35 10.5 40.97
5 Burkholderia cenocepacia(strain AU 1054) Q1BZ21 32737.4 307 36 11.4 41.50
Pattern 3 ( C-W-E-N-[HY]-M-P-[LM]-[AL]-R-X-X-[ML]Y )
1 Aspergillus niger A2QYH5 34964.7 320 36 8.8 41.75
2 Shewanella pealeana A8H6J4 34786.8 314 39 9.2 39.82
3 Syntrophobacter fumaroxidans A0LKP2 36011.4 328 44 11.0 41.02
4 Burkholderia ambifaria B1FFB0 32904.5 307 38 11.1 40.05
5 Algoriphagus sp. PR A3HXT3 34738.1 305 43 4.9 41.30
Pattern 4 ( A-X-E-G-R-C-[FW]-V-[LIV] )
1 Syntrophobacter fumaroxidans A0LKP2 36011.4 328 44 11.0 41.02
2 Talaromyces stipitatus B8MN39 35560.5 325 41 10.2 46.51
3 Uncultured organism Q6RWR5 33711.5 310 36 11.0 41.86
4 Microscilla marina A1ZHQ2 34937.1 311 38 9.0 41.02
5 Rhodopseudomonas palustris D2MB94 33698.4 317 40 13.9 39.93

Table 3b: In silico analysis of some physiochemical properties of some aromatic nitrilase (sequences obtained from ScanProsite) from Manually Designed Motif analysis.

Phylogenetic analysis

Twelve protein sequences for nitrilases (aliphatic/aromatic) were subjected for the phylogenetic tree construction. Tree topology generated by Neighbor Joining Method (NJ) revealed that aliphatic and aromatic nitrilases were clustered with well distinct groups and well supported bootstrap value of 1000 (Figure 2).

data-mining-genomics-aromatic-bacterial-source

Figure 2: Phylogenetic tree of some nitrilase protein sequences from aliphatic and aromatic bacterial source organism constructed by NJ method, where aliphatic and aromatic is shown in two different groups.

Discussion

The expansion of molecular sequences and genomic databases has made the area of genome sequence data analysis more challenging and interesting to develop tools for rapid and reliable searching and analysis of the data. In this endeavor, database searching against gene/protein families with motifs has emerged as important strategy for efficient similarity searching [22]. While several domain/motif databases are being compiled, it is important to develop database search tools that fully utilize the conserved structural and functional information embedded in those sequence data to enhance the reliability of the search. Sequence motifs typically occur in a specific and known order in a sequence family. The ordering and spacing of motifs therefore, provide powerful additional criteria for classifying sequences into families. In this paper, we have manually designed groups of motifs (MDM) i.e. MDMAl and MDMAr each with four motifs for rapid and reliable nitrilase identification and compared it to the currently available methods, including the BLAST search, the PROSITE pattern search, Gblock [23] MEME version 4.8.1 (Multiple Em for Motif Elicitation) and the HMM method. All the designed motifs have one amino acid residue of active site, therefore when MDM were analyzed and validated through ScanProsite, resulting protein sequences were specifically belonging to nitrilase with conserved catalytic triad Glu-48, Lys-131, Cys-165 [15,18,20,24] and total number of hits were found to be significantly higher (578 aliphatic & 392 aromatic) when compared to databases such as BLAST and PROSITE.

Gblock eliminated poorly aligned and divergent region of the protein sequences for better phylogenetic analysis. Pratt results confirmed that the Manually Designed Motif for aliphatic nitrilase (MDMAl ) ranged between 40-55 amino acid for pattern 2; 125- 141 amino acid for pattern 3; 160-172 amino acid for pattern 4 (see additional file 1 & 2). This revealed that Pratt analysis for MDM are reliable and accurate tool for conducting search of similar sequence from databases as its results were similar to the Scan Prosite analysis as other similar sequence from databases as its results were similar to the ScanProsite analysis. One motif i.e. [VA]-A-X-[AV]-Q-[AI]-X-P-[VA]-X-[LF]-[SD] was not confirmed by the Pratt analysis as Pratt started the analysis after 30th amino acid and we have designed this MDM between 1-30 amino-acid (Table 2).

Multiple sequence alignment (MSA) in present study revealed that there are specific amino acids present at specific position which are responsible for the activity of nitrilases. Phylogenetic analysis by the Neighbor Joining (NJ) method clearly makes a distinction between the two groups of nitrilases i.e. aliphatic and aromatic nitrilases (Figure 2).

In our previous communication [13] we have reported that some of the physiochemical properties play a significant role in the substrate specificity of aliphatic and aromatic nitrilases. The physicochemical parameters analysis of these sequences further confirmed that all the sequences having a total number of amino acid between 330-382, molecular weight between 37226–41971 daltons, alanine content 10- 16%, negatively charged residue (Asp+Glu) 37-52 and instability index 30.32-44.13 belonged to aliphatic nitrilases. Similarly sequences having a total number of amino acid between 300-330, molecular weight between 32273–36940 daltons, alanine content (%) 8-11%, negative charged residue (Asp+Glu) 37-52 and instability index 39.82-48.68 were of aromatic nitrilases (Table 3a and 3b).

It is well known that uncultured organisms are the new sources of enzymes, antibiotics and drug discovery [25,26] In the present study, we have found nitrilase sequences belonging to uncultured organisms from the database (aliphatic-164, aromatic-41). The protein sequences of these uncultured organisms have all the specific properties responsible for the nitrilase activity. Further studies on these sequences may lead to find out specific nitrilases needed for transformation of nitriles in organic chemistry and industry.

Conclusion

Nitrilases are distributed among microorganisms, plants and some plant species. We here present computational analysis of two classes of nitrilase enzyme which includes the study of motifs, physiochemical and phylogenetic analysis which could be used as tool to predict and differentiate nitrilases on basis of their substrate specificity. The present analysis predicts new sources of nitrilases with study of common and important physicochemical/biochemical properties as they share a common ancestry and use of these observations will be useful to predict molecular function of the randomly selected nitrilases. Computational analysis of various properties of nitrilases revealed differences and also MEME, Pratt and Gblock analysis have confirmed the presence of four motifs. Additionally this approach has also led us to find new sources of nitrilase across SwissProt/TrEMBL databases. MDM for aliphatic/ aromatic nitrilases, have been validated with the results and these motifs will be of potential application for screening genome/protein sequence databases to find novel sources of nitrilases.

Acknowledgements

The authors are grateful to Department of Biotechnology, Ministry of Science and Technology, Govt. of India for funding.

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