Computational Analysis of Amino Acid Sequences in Relation toThermostability of Interspecific Nitrile Degrading Enzyme (Amidase) fromVarious Thermophiles/Hyperthermophiles<br>

Computational Analysis of Amino Acid Sequences in Relation toThermostability of Interspecific Nitrile Degrading Enzyme (Amidase) fromVarious Thermophiles/Hyperthermophiles

Research Article

Open Access

Reena Kanwar, Nikhil Sharma and Tek Chand Bhalla^*

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

^*Corresponding author:

Tek Chand Bhalla
Sub-Distributed Information Centre
Himachal Pradesh University
Summer-Hill, Shimla-171005, India
E-mail: bhallatc@rediffmail.com

Received November 08, 2012; Published December 24, 2012

Citation: Kanwar R, Sharma N, Bhalla TC (2012) Computational Analysis of Amino Acid Sequences in Relation to Thermostability of Interspecific Nitrile Degrading Enzyme (Amidase) from Various Thermophiles/Hyperthermophiles. 1:556 doi:10.4172/scientificreports.556

Copyright: Â© 2012 Kanwar R, 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.

Abstract

Computational analysis of amino acid sequences of some thermophilic/hyper thermophilic microbial amidases for various physiochemical properties and amino acid has been done. Forty thermophilic and hyperthermophilic bacteria and archaea sequences were retrieved from NCBI database. These sequences were analyzed using ProtParam (ExPASy) tool for various physiochemical properties, whereas statistical significance was calculated using P value for the same, which indicates a clear distinction between thermophiles and hyperthermophiles. Physiochemical parameters, such as number of amino acid, molecular weight and negatively charged residues were found to be significantly higher in case of thermophiles. The number of amino acids, Leu, Arg and Ser were observed to be highly significant (1.15, 1.18 and 1.17 fold) for thermophiles, whereas in case of hyperthermophiles, Cys, Thr, His and Trp were found to be highly significant (2.29, 1.33, 1.56 and 1.93 fold), which makes a clear cut distinction between the thermostability of two groups (thermophiles/hyperthermophiles) of amidases.

Keywords

Amidase; Hyperthermophiles; Physiochemical properties; Thermophiles; Amino acid

Introduction

Various cyclic amide-metabolizing systems occur in nature and play significant roles in a variety of metabolisms, such as amino acid, antibiotic and pyrimidine/purine metabolisms [1]. The ability to degrade nitriles is quite common among micro-organisms. Amidase or amidohydrolase is an interesting member of the family hydrolase and superfamily nitrilase [2,3], used for the metabolism of amides. They act on the amide bonds (carbon-nitrogen (C-N) bond, other than peptide bonds), which are of considerable importance in biochemistry because many C-terminal amino acids act as hormones. Amidases participate in various metabolic pathways such as urea cycle, metabolism of amino groups, phenylalanine metabolism, tryptophan metabolism, cyanoamino acid metabolism, benzoate degradation and styrene degradation. Amidases have been used as biocatalyst for the production of ammonia, acrylic acid, and several other important compounds of industrial importance [4-6]. In industries, they are employed in combination with nitrile hydratase for the production of commercially important organic acids, such as acrylic acid, p-amino benzoic acid, pyrazinoic acid, nicotinic acid through biotransformation of nitriles [4,7].

There are special classes of enzymes which are stable at high temperature called thermostable enzymes (thermozymes), and explored as excellent biocatalyst in industrial processes [8-11]. There are certain micro-organisms which catalyses reaction even at the temperature above 100Â°C, such as Pyrococcus furiosus, Methanopyrus kandleri AV19 and Thermatoga maritima by hydrolyzing variety of substrates. The first hyperthermophilic microorganism Sulfolobus species was discovered in 1972 from hot acidic springs in Yellowstone Park [8,12]. During the past four decades, the molecular basis of thermal stability of protein has expanded as a vast research area. The importance of amidases in biotechnology is growing rapidly because of their potential applications in chemical and pharmaceutical industries, as well as in bioremediation [13]. Microbial amidases are a class of enzymes that have potential applications in the development of commercial bioprocesses [14,15]. They are used in the detoxification of industrial effluents containing toxic amides, such as acrylamide and formamide [16,17], and their acyl transferase activity is exploited mainly for the synthesis of pharmaceutically active hydroxamic acids [18-20].

For efficient industrial applications, various parameters such as the number of amino acid residues, molecular mass, theoretical pI, amino acid composition, negatively charged residues, positively charged residues and extinction coefficients are needed to be studied carefully, which are considered to be important in exploring and comparing thermostability feature of enzymes, including amidases. These properties can be determined by In silico analysis of amino acid sequences available in the database, which provides meaningful information about the structure and function of proteins. In the present study, In silico analysis of some important, a physiochemical property of thermophilic/hyperthermophilic amidases, has been carried out, which has not been done so far.

Materials and Methods

Data collection and analysis

The information about micro-organisms (thermophilic/ hyperthermophilic) producing amidases was searched from National Centre for Biotechnology Information (NCBI, https://www.ncbi.nlm. nih.gov/protein) and BRaunschweig ENzyme DAtabase (BRENDA, https://www.brenda-enzymes.info/). Various bacteria and archaea were searched from these databases, and their accession numbers were obtained. Amino acid sequences for both thermophilic and hyperthermophilic microorganisms which produce amidases were taken and these sequences are not fragmented, putative, pseudo and hypothetical. Protein sequences of amidases (aliphatic and aromatic) of thermophiles and hyperthermophiles was searched from Expert Protein Analysis System (ExPASy) proteomics server of the Swiss Institute of Bioinformatics (SIB) and UniProt (https://www.uniprot.org/uniprot/). FASTA format of protein sequences were applied or saved for subsequent analysis of microorganisms, given in the tables. Different tools in the proteomic server (ProtParam, ProtScale, and Protein calculator) were applied to calculate various physiochemical properties of bacterial and archaeal amidases from their protein sequences. The physiochemical parameters computed by ProtParam included number of amino acid, molecular weight, theoretical pI, negatively charged residues (Asp & Lys), positive charge residues (Arg & Lys), extinction co-efficient (which depends upon the tyrosine residue, tryptophan residue and cysteine molecules), instability index and aliphatic index for thermostability, and grand average of hydropathicity (GRAVY). The extinction co-efficient of various amidases was calculated using the equation [21].

E (prot)=Numb (Tyr)*Ext (Tyr)+Numb (Trp)*Ext (Trp)+Numb (cysteine)*Ext (cysteine)

The values of the aliphatic index of various amidase sequences were obtained using the ProtParam (ExPASy) tool [22]. The instability index and grand average of hydropathicity (GRAVY) were assessed by following the method [23]. The molecular weight in kilodaltons (kda) and the pI of amidases were deduced using pK values of amino acid. Atomic composition and amino acid percent count were also estimated for all protein sequences.

Results

In the present studies, using ProtParam (https://web.expasy. org/protparam/) tool, various physicochemical properties of amino acid sequences of amidases in thermophiles/hyperthermophiles of bacteria and archaea were determined. The significant differences in the physiochemical properties of the two groups (thermophiles and hyperthermophiles) have been observed (Table 1.1 a and b). The total number of amino acid residues were found to be significantly higher (1.02 fold) in thermophilic bacteria and archaea, as when compared to hyperthermophilic bacteria and archaea, as shown in table 1.1 a and b. Theoretical pI varied between 4.66-9.32; 4.64-8.75; 4.59-10.05 and 4.06- 8.28, respectively for hyperthermophilic and thermophilic bacteria and archea. Negative charge residues (Glu and Asp) were found to be significantly higher, i.e 1.16 fold in hyperthermophiles, in comparison to thermophilic archaeal and bacterial amidases. Positively charged (Arg and Lys) residues were found higher in hyperthermophilic amidases, in comparison to thermophiles. Instability index was also found to be higher for amidases of thermophiles, in comparison to hyperthermophiles.

Table 1.1(a): Comparative analysis of physiochemical properties of amidases of bacteria and archaebacteria (hyperthermophiles).

Table 1.1(b): Comparative analysis of physiochemical properties of bacteria and archaebacteria (thermophiles).

Aliphatic index is defined also regarded to increase the thermostability of proteins, and was found to be higher in case of hyperthermophiles, when compared to thermophilic bacteria and archea. The values for grand average of hydropathicity (GRAVY) were substantially higher for amidase sequences of hyperthermophiles than that of thermophiles (1.07 fold). The results of amino acid analysis hyperthermophilic and thermophilic (bacteria and archea) amidase are shown in table 1.2a and b. The comparison of amino acid composition for both groups of amidases (thermophiles/hyperthermophiles) has revealed that glycine (Gly), one of the simplest amino acid was found to be the predominant residue in hyperthermophiles; its percentage was more 1.01 folds higher in case of hyperthermophiles. The amino acid cysteine (Cys) was considered to be an important parameter in the calculation of extinction co-effieient of proteins, and its content was 1.03 fold higher in hyperthermophiles. The amino acid Ala, Cys, Glu, Gly, Pro, His and Trp (1.02, 1.07, 1.07, 1.10, 1.03 and 1.36 fold) were found to be significantly higher for hyperthermophilic amidases, whereas thermophilic amidases contained Asn, Asp, Gln, Lys, Met, Phe and Ser (1.14, 1.15, 1.31, 1.0, 1.08, 1.06 and 1.07 fold). The comparison of atomic composition revealed that the sulphur content was observed significantly higher in amidases of hyperthermophilic origin.

Table 1.2(a): Comparison between amino acids percent count of amidases of bacteria and archaebacteria (hyperthermophiles).

Table 1.2(b): Comparison between amino acids percent count of bacteria and archaebacteria (thermophiles).

Discussion

The present study aims to compare the parameters responsible for thermostability of amidases present in thermophiles and hyperthermophiles (bacteria and archaea), on the basis of their amino acid sequences and physiochemical properties. The variation in total number of amino acid residues and molecular weight might be playing some role in thermostability of proteins, in both groups of amidases producing thermophiles/hyperthermophiles (bacteria and archaea). Significant difference for extinction co-efficient and aliphatic index was found between these two groups of microorganisms. Investigation of aliphatic index is important as the hydrophobicity of aliphatic amino acids is a potentially attractive measure of the stability of proteins at high temperature, as well as against denaturants, such as urea and initial scanning of amino acid. Composition of several thermostable proteins invariably showed a high content of Gly, Ala, Glu, and Leu [24]. The distribution of pI of proteins exhibited a clear relationship with subcellular localization, ecology, length of proteins and taxonomy of organisms [25]. The proteins with instability index less than 40 were considered stable, and greater than 40 are considered to be unstable as shown in table 1.1a and b. The present study revealed that greater the number of cystine and more will be the formation of disulphide bonds (S-S bonds), which imparts stability for hyperthermophilic amidases. Thus, the majority of the thermophilic archaea are sulphur dependent [26]. In contrast to sulphur reducing microorganisms, mainly Sulfolobus, Acidianus, Metallosphaera and Sulfurisphaera, undergo highly exothermic reaction of bio-oxidation, which significantly improve the leaching kinetics, accelerate the reaction rate and shorten the leaching cycle because of the inherent advantages of tolerating the high temperature [1,27-29]. Disulfide bonds are an important factor concerning the structural stabilization of intracellular proteins, which are found in oxidizing environment, while in chemically reducing conditions; it favours thiol form of cysteine, which is not thermodynamically responsible for stability of proteins in hyperthermophilic archaea and bacteria [30].

In the present investigations, the structural basis for thermal stability in thermophilic and hyperthermophilic archaeal and bacterial amidases was deduced. Glu and Asp participate in the formation of salt bridges, which provide extra stability to thermal proteins [31,32]. Methionine is sulphur containing amino acids form salt bridges, which are strong bonds, and thus provide stability to proteins. Methionine is an aliphatic amino acid, which shows that by increasing the length of the aliphatic side chain of amino acid in the protein, thermostability increases [33]. Comparative analysis of complete proteomes showed extremely strong bias toward arginine-to-lysine replacement in hyperthermophilic organisms, and overall much greater content of lysine than arginine in hyperthermophiles [34,35].

Proline being one of the responsible residues for the stability, maintaining a common fold conformation in the polypeptide chain had less conformational freedom, when compared with other amino acids, as the pyrrolidine ring of proline imposes N-C rotation. Glutamine was significantly higher in hyperthermophilic bacteria and archaea,which are responsible for stability of amidases at higher temperature. Some researchers found that the decreased Gln content may minimize deamidation, which results in increased thermostability of proteins. Alanine is the best helix-forming residue [36,37], and is found to be more in case of hyperthermophilic bacteria. Cysteine (Cys) on other hand was found to be more in case of hyperthermophiles, when compared to thermophiles.

In the present studies, aromatic amino acids (tryptophan, tyrosine and phenylalanine) were found to be significantly higher in thermophilic amidases, as thermophilic amidases are found to be more stable than hyperthermophilic amidases. Aromatic amino acids are bulkier and hydrophobic in nature, and form complex with a histidine residue, which provided extra stability to heat-resistant proteins [38,39], and is found to increase in thermophilic amidases. Hydrophobic effect plays a crucial role in protein folding and considered to be a major factor responsible for protein stability [40,41]. In both heatâ€“resistant archaeal and bacterial proteins, mostly Glu and Asp (negatively charged residues) are higher in amount, which participates in the formation of salt bridges, responsible for thermostability of proteins. However, the present investigation revealed that higher amount of Glu was found in hyperthermophilic amidases, in comparison to thermophilic amidases (1.07 fold). Investigations in recent years have indicated that there are disparities in the heat-resistant mechanism between archaeal proteins and bacterial proteins [42,43,31]. Archaeal organisms resist high temperature by substituting non-charged polar amino acids, with Glu, Lys and non-polar amino acids with Ile on protein surfaces [44].

The ratio of salt bridge network is higher in hyperthermophilic archaea than thermophilic archaea. Accordingly, salt bridges significantly contribute to the ability of the organism to withstand high temperatures. These results demonstrate that salt bridges are the most important factor determining the heat resistance in archaeal or bacterial proteins. Previous studies have shown that salt bridge networks are major factors that affect the thermostability of protein [2,45-47]. In addition, there was high Ser and Thr content in hyperthermophilic amidases, in comparison to thermophilic or mesophilic amidases [48]. Ser and Thr are more flexible, so increasing flexibility of proteins to withstand the extreme heat. Due to higher number of sulphur atoms in hyperthermophiles, more salt bridges can be formed, which increase the thermostability of proteins, in comparison to thermophiles [49].

Conclusion

A number of amino acid sequences of thermophilic/ hyperthermophilic amidases were analyzed In silico, for some of the physiochemical properties. The hyperthermophilic amidases are found to be more stable than thermophilic amidases. The hyperthermophilic amidases and thermophilic amidases show significant difference in amino acid residues, molecular weight, and percent count of some amino acids. Amino acids which are responsible for thermostability of these amidases such as Cys, Glu, Ala, Arg, Pro, Tyr, Trp, His and Val, which have been found to be significantly higher in case of hyperthermophilic amidases. The results of present investigation will be quite useful in the prediction of extent of thermostability among amidases of thermophiles and hyperthermophiles of various forms of bacteria.

References