In Silico Analysis of Glutaminase from Different Species of Escherichia and Bacillus.

BACKGROUND: Glutaminase (EC 3.5.1.2) catalyzes the hydrolytic degradation of L-glutamine to L-glutamic acid and has been introduced for cancer therapy in recent years. The present study was an in silico analysis of glutaminase to further elucidate its structure and physicochemical properties.
METHODS: Forty glutaminase protein sequences from different species of Escherichia and Bacillus obtained from the UniProt Protein Database were characterized for homology search, physiochemical properties, phylogenetic tree construction, motif, superfamily search, and multiple sequence alignment.
RESULTS: The sequence level homology was obtained among different groups of glutaminase enzymes, which belonged to superfamily serine-dependent β-lactamases and penicillin-binding proteins. The phylogenetic tree constructed indicated 2 main clusters for the glutaminases. The distribution of common β-lactamase motifs was also observed; however, various non-common motifs were also observed.
CONCLUSION: Our results showed that the existence of a conserved motif with a signature amino-acid sequence of β-lactamases could be considered for the genetic engineering of glutaminases in view of their potential application in cancer therapy. Nonetheless, further research is needed to improve the stability of glutaminases and decrease their immunogenicity in both medical and food industrial applications.

What’s Known

Glutaminase is one of the important enzymes in food and pharmaceutical industries. In recent years, glutaminase has been identified from various biological sources such as bacteria, fungi, yeasts, and mammals. But Escherichia and Bacillus spp. are the major producers of glutaminase.

What’s New

Escherichia and Bacillus spp. are the main producers of glutaminase. We performed a comprehensive in silico study of bacterial glutaminase producers, especially various Bacillus and Escherichia strains, regarding their physicochemical properties and phylogenetic relations in order to find new enzyme sources.

Introduction

Glutaminase or glutamine amidohydrolase (EC 3.5.1.2) catalyzes the hydrolytic deamination of L-glutamine, leading to the generation of L-glutamate and ammonium.[1,2] In recent years, glutaminase has attracted much attention given its proposed applications in both food and pharmaceuticals industries.

Glutaminase has been recognized in bacteria, fungi, yeasts, and mammals.[2-4] It plays an essential role in nitrogen metabolism, involving glutaminolysis. While mitochondrial glutaminase is elevated in some tumor types and is frequently upregulated in MYC-transformed cells,[5] it is thought to be a potential chemotherapeutic target.[6] Moreover, Achromobacter glutaminase exerted antileukemic influences in patients with acute myeloid leukemia or acute lymphoblastic leukemia in a preliminary clinical trial.[7] Glutaminase–asparaginase obtained from Pseudomonas 7A showed considerable antitumor activity in some studied mice,[7] particularly when applied together with glutamine antimetabolites. Glutaminase has provided hope as an encouraging therapeutic agent for the healing of diseases caused by retroviruses. It has also attracted significant attention from the pharmaceutical industry on the strength of its potential applications as an anticancer agent. In this regard, there are 2 genes for the glutaminase available in Escherichiacoli, namely yneH (308 aa) and ybaS (310 aa). The ybaS gene encodes an enzyme that is only active in acidic pH but not in physiological pH. On the other hand, the yneH gene has optimum activity in physiological pH and is suitable for cancer therapy purposes.

Alongside its demonstrated potential as an antileukemic agent, glutaminase is generally regarded as a key enzyme in controlling the taste of fermented foods such as soy sauce, especially in Asian countries.[8,9] Most of the essential flavor components of fermented condiments are amino acids generated by the enzymatic hydrolysis of proteins contained in raw food materials; and among them, L-glutamic acid is a broadly recognized flavor-enhancing amino acid.[8] Moreover, L-glutamate (monosodium glutamate) is a prominent umami taste factor. Hence, the deamination of glutamine is an important route in the food industry with the aim of enhancing the umami taste. For instance, the distinctive taste of fermented soy sauce is ascribed chiefly to glutamic acid (concentrations of 0.7 to 0.8% per total nitrogen).[2] The activity of glutaminase, accountable for the fabrication of glutamic acid, renders it a chief supplement for the period of soy-sauce fermentation. Efforts to enhance the glutamate content of soy sauce by means of salt and thermotolerant glutaminases have attracted much attention.[10]

Among glutaminase producers, Escherichia and Bacillus spp. are well studied microorganisms in all aspects, especially some Bacillus spp. have been given GRAS (generally regarded as safe) status by the Food and Drug Administration (FDA). Furthermore, therapeutic enzymes such as asparaginase have been obtained from Escherichia spp.[11] Asparaginase also has relative glutaminase activity, which makes it a good candidate for therapeutic purposes.

In the context of increased practical applications for glutaminase, we performed an in silico analysis of 40 glutaminase protein sequences from Escherichia and Bacillus spp. To our knowledge, this is the first research to analyze glutaminase protein sequences using bioinformatics approaches. Drawing upon a variety of bioinformatics tools, we sought to characterize glutaminase protein sequences in terms of biochemical traits, multiple sequence alignment (MSA), homology search, motif, phylogenetic tree construction, and superfamily allocation.

Materials and Methods

The amino-acid sequences of glutaminase from various Escherichia and Bacillus spp. were obtained from the UniProt Protein Database and the Expert Protein Analysis System (ExPASy) proteomics server. Physiochemical data were provided through ProtParam via the ExPASy server (the proteomics server of the Swiss Institute of Bioinformatics). The Fast Adaptive Shrinkage/Thresholding Algorithm (FASTA) format of the sequences was utilized for subsequent analyses. Various tools in the proteomics server (ProtParam, ClustalW, Compute pI/Mw, Protein Calculator, and ProtScale)[12] were implemented to calculate/deduce different physiochemical features of the glutaminases from the protein sequences. The molecular weights (kDa) of the various glutaminases were computed by adding the mean isotopic mass of the amino acid in the enzyme and deducting the mean isotopic mass of 1 water molecule. The pI of the enzyme was computed using the pKa value of the amino acid according to Bjellqvist et al.[13] (1993). The atomic compositions of the glutaminases were obtained using ProtParam, available at ExPASy. The aliphatic index values of the various glutaminase protein sequences were determined using ProtParam (ExPASy).[12] The grand average of hydropathicity (GRAVY) and the instability index were estimated using the Kyte and Doolittle[12] and Guruprasad[14] methods, respectively. CLC Sequence Viewer 7 was used[15] for dendrogram construction via the neighbor-joining method (NJ).[16] For domain search, Pfam (http://sanger.ac.uk/software/Pfam/search.html) and InterPro (http://www.ebi.ac.uk/interpro) were used. Motif analysis was done using MEME (http://meme.sdsc.edu/meme/meme.html) and MOTIF search (http://www.genome.jp/tools/motif). The protein conserved motifs deduced by MEME were subjected to biological functional analysis using protein BLAST, and the motifs were studied using InterProScan to find the best possible match based on the highest similarity score. Forty glutaminase protein sequences with accession numbers showing different species of Escherichia and Bacillus are listed in table 1.

Table 1

Biochemical features of glutaminase protein sequences from different species of Escherichia and Bacillus

S. no	Accession number	Source organisms	Number Of amino acids	Molecular weight	Theoretical pI	Total number of negatively charged residues (Asp+Glu)	Total number of positively charged pesidues (Arg+Lys)	Instability index	Aliphatic index	Grand average of hydropathicity (GRAVY)
1	P0A6W0	E. coli (strain K12)	308	33515.5	5.98	29	25	43.79	98.80	0.133
2	P0A6W2	E. coli O157:H7	308	33515.5	5.98	29	25	43.79	98.80	0.133
3	P0A6W1	E. coli O6:H1	308	33515.5	5.98	29	25	43.79	98.80	0.133
4	C3T9T2	E. coli	308	33515.5	5.98	29	25	43.79	98.80	0.133
5	B1LF91	E. coli (strain SMS35)	308	33515.5	5.98	29	25	43.79	98.80	0.133
6	B7N4U9	E. coli O17:K52:H18	308	33514.6	6.17	28	25	44.06	98.80	0.133
7	D3QT73	E. coli O55:H7	308	33515.5	5.98	29	25	43.79	98.80	0.133
8	D3GSG1	E. coli O44:H18	308	33515.5	5.98	29	25	43.79	98.80	0.133
9	B7URQ1	E. coli O127:H6	308	33515.5	5.98	29	25	43.79	98.80	0.133
10	C8TRP0	E. coli O26:H11	308	33515.5	5.98	29	25	43.79	98.80	0.133
11	B7L7M4	E. coli (strain 55989)	308	33515.5	5.98	29	25	43.79	98.80	0.133
12	E3PM25	E. coli O78:H11	308	33515.5	5.98	29	25	43.79	98.80	0.133
13	B7LRC7	E. fergusonii (ATCC 35469)	308	33532.6	6.25	29	26	38.87	96.30	0.120
14	Q1RBP3	E. coli (strain UTI89)	308	33515.5	5.98	29	25	43.79	98.80	0.133
15	C8U8J5	E. coli O103:H2	308	33515.5	5.98	29	25	43.79	98.80	0.133
16	B6IAS7	E. coli (strain SE11)	308	33515.5	5.98	29	25	43.79	98.80	0.133
17	A7ZLY0	E. coli O139:H28	308	33515.5	5.98	29	25	43.79	98.80	0.133
18	C8UPW4	E. coli O111:H	308	33515.5	5.98	29	25	43.79	98.80	0.133
19	B7MMY8	E. coli O45:K1	308	33515.5	5.98	29	25	43.79	98.80	0.133
20	D8BUE3	E. coli MS 1961	308	33515.5	5.98	29	25	43.79	98.80	0.133
21	W0B1N6	E. albertii KF1	308	33542.6	6.24	28	25	41.24	97.86	0.146
22	A0A0F6C473	E. coli Xuzhou21	308	33515.5	5.98	29	25	43.79	98.80	0.133
23	K4WSD4	E. coli O111:H8	308	33515.5	5.98	29	25	43.79	98.80	0.133
24	W8ZS57	E. coli ST131	308	33515.5	5.98	29	25	43.79	98.80	0.133
25	A0A0E0V4K9	E. coli O7:K1	308	33515.5	5.98	29	25	43.79	98.80	0.133
26	S1HXQ0	E. coli KTE108	308	33515.5	5.98	29	25	43.79	98.80	0.133
27	S1HI46	E. coli KTE103	308	33515.5	5.98	29	25	43.79	98.80	0.133
28	K5CT98	E. coli AD30	308	33515.5	5.98	29	25	43.79	98.80	0.133
29	H5V2J6	E. hermannii NBRC 105704	308	33312.5	6.31	28	26	40.55	104.87	0.210
30	A0A090V5E8	E. vulneris NBRC 102420	308	33760.6	5.85	32	26	52.44	95.03	0.020
31	B1ELZ3	E. albertii (strain TW07627)	308	33489.6	6.07	28	24	41.24	97.86	0.169
32	O07637	B. subtilis (strain 168)	309	34012.3	5.78	36	33	23.17	94.01	0.026
33	A8FCU0	B. pumilus (strain SAFR032)	309	33600.8	5.90	35	32	33.86	87.18	0.011
34	A7Z4A6	B. amyloliquefaciens subsp. Plantarum	309	33989.2	5.65	38	33	34.97	87.73	0.046
35	G4NWR9	B. subtilis subsp. spizizenii	309	34001.4	5.77	36	33	26.55	94.66	0.016
36	D4FW74	B. subtilis subsp. natto	309	34040.4	6.03	36	34	24.04	94.01	0.022
37	R9TT78	B. licheniformis 9945A	309	33601.7	5.03	39	29	17.37	92.49	0.047
38	A0A060LRN5	B. lehensis G1	309	33947.8	4.85	42	28	28.24	91.91	0.079
39	I3E8E0	B. methanolicus MGA3	309	33751.4	8.31	32	35	29.15	96.28	0.088

Results

Forty glutaminase protein sequences from different species of Escherichia and Bacillus retrieved from the UniProt Protein Database were characterized for homology search, MSA, biochemical features, phylogenetic tree construction, superfamily, and motif search using a variety of bioinformatics tools.

The biochemical features for these glutaminases are listed in table 1. The total number of amino-acid residues was 308 for the Escherichia spp. and 310 for the Bacillus spp., with variable molecular weights. The pI value ranged from 4.85 to 8.31. The variability was also observed among these glutaminases in terms of other physiochemical parameters such as positively charged amino-acid residues, negatively charged residues (Asp and Glu), and hydropathicity (GRAVY) (table 1). The sequence-based analysis of the aliphatic index among these glutaminases in the Escherichia spp. revealed homogeny with a range of ~98 with the exception of E. hermannii, which had a value of 104.87. As for the Bacillus spp., a variety of aliphatic indices were observed, from 87.18 to 96.28.

The MSA and homology search of these 40 glutaminase protein sequences disclosed a stretch of conserved regions (figure 1). However, a few highly conserved amino acids were also observed for many of the sequences (figure 1).

Figure 1

Multiple sequence alignment of glutaminase protein sequences shows maximum homology from amino-acid residues 60–120. The represented accession numbers of the bacteria and their complete details are provided in table 1.

The phylogenetic tree constructed based on the glutaminase protein sequences using the NJ method revealed 2 major clusters for the Escherichia and Bacillus spp., denoting the sequence-level similarity of the glutaminase protein sequences (figure 2). Several Escherichia species-specific clusters for glutaminase, namely E. fergusonii, E. albertii, E. hermannii, and E. vulneris, were also observed (figure 2). A similar profile was achieved from the phylogenetic tree constructed using the unweighted pair group method with arithmetic mean (UPGMA) and the minimum-evolution method (data not shown).

Figure 2

Phylogenetic tree constructed using the neighbor-joining (NJ) method based on the glutaminase protein sequences from different species of Escherichia and Bacillus is depicted here. The represented accession numbers of the bacteria and their comprehensive details are provided in table 1.

These glutaminases, when subjected to the SUPERFAMILY tool on the ExPASy server,[17] revealed their identity: They belonged to superfamily serine-dependent β-lactamases and penicillin-binding proteins. The motif analysis of the glutaminases from the Escherichia and Bacillus spp. revealed the existence of more than 40 absolutely conserved residues including the predicted β-lactamase motif 1,[18] a catalytic diad Ser-X-X-Lys. Moreover, β-lactamase sequence motif 3 (Lys/Arg-Ser/Thr-Gly) was identifiable in the glutaminases (Lys259-Ser260- Gly261), while only Ser (Ser160) could be identified for the Ser-Asp-Asn triad of class A β-lactamase motif 2 (figure 1).

In addition to the conserved β-lactamase sequences, varied motifs were also obtained. The MotD (flagellar motor protein) motifs (LETILRQVRPLIGQGKVADYIPALATVEG SRLGIAICT VDGQ LFQ AGDA QERF SIQSISKV) along with WisP family C-Terminal Region (RGLSGVSDIAYDTVVAR SEFEH SARNAAIA WLMKSFGNFHHD VTTVLQNYFHYC) were observed among the Escherichia spp., but not in the Bacillus spp. However, various motifs were observed in the Bacillus spp., including aminoacyl-tRNA ligase (QEPTGDPFNSIIKLETVN PSKPLNPMINAGALVVTSLIRGRT VKERLDYLLSFIRRLTN) motif in B. subtilis spp. (strain 168, spizizenii and natto) and TENA motif (IRILTFVQELAGNSN VAYSQE VAKSEFESS FLNRSLCY) in B. methanolicus, which may help to secret proteases such as glutaminase into the extracellular environment.

Discussion

Glutaminase (EC 3.5.1.2) catalyzes the hydrolytic deamination of L-glutamine to L-glutamic acid and has a vital task in cellular nitrogen metabolism. In mammals, both kidney and liver types of glutaminase are present. However, it is widely distributed in almost all organisms, including bacteria. In this regard, Escherichia and Bacillus spp. attract a great deal of attention due to their potential in medical and industrial applications. Nowadays, many bioinformatics tools are harnessed in different biological fields such as protein engineering and vaccinology to lower the costs and improve the accuracy of experimental investigations.[19-21]

In this research, we performed an in silico study of glutaminases from 2 bacteria, namely Escherichia and Bacillus spp. The biochemical traits for these glutaminase enzymes are depicted in table 1. The sequence-based analysis of the aliphatic index among these glutaminases in the Escherichia spp. revealed homogeny with a range of ~98 with the exception of E. hermannii, which had a value of 104.87. As for the Bacillus spp., a variety of aliphatic indices were observed, from 87.18 to 96.28. The aliphatic index of a protein sequence is an extent of the relative volume occupied by aliphatic side chain of valine, alanine, isoleucine, and leucine amino acids. An increase in the aliphatic index is considered to represent an elevation in the thermostability of globular proteins.[15] The glutaminases of Escherichia and Bacillus spp. appear to be thermostable given the high value of their aliphatic index.[15] The instability index is considered for the measurement of the in vivo half-life of a protein.[14] It has been reported that proteins that possess an in vivo half-life >16 hours have an instability index <40, while those that possess an in vivo half-life <5 hours display an instability index >40.[22] The computed instability index of the glutaminases from the Escherichia spp. was found to be half-life <5 hours, with the exception of E. fergusonii (half-life >16 h). In contrast, all the Bacillus spp. represented an instability index <40, which showed a half-life >16 hours and indicated that they were good candidates for medical and industrial applications.

The MSA and homology search divulge several homologies. The presence of conserved small sequence patches with important roles in the authentication of protein and helix-coil transition has been previously stated.[23,24] Structural and sequence homology methods principally represent the global similarities between the compared glutaminases.[25] However, in general terms, the molecular role of a glutaminase is confined to its known active site, which may include in an interaction with the peptide linkage of proteins. Keeping the core structural constituent of the active site is necessary for maintaining the functional activity of the enzyme. Therefore, protein comparisons that focus on structural similarities in a global sequence may fail to spot proteins with conserved active sites but divergent structures and sequences.[26] The conserved region observed between these glutaminases could be utilized for designing degenerate primers for polymerase chain reaction (PCR)-based amplification and cloning of reputed glutaminase genes from the diverse species of Escherichia and Bacillus.

Our sequence analysis of the glutaminases using the SUPERFAMILY tool on the ExPASy server revealed that they belonged to superfamily serine-dependent β-lactamases and penicillin-binding proteins. Poorly characterized glutaminases belong to the huge cluster of serine penicillin-binding proteins and β-lactamases, which have a shared evolutionary origin and apportion the protein fold, catalytic mechanism, and structural motifs.[18] This huge set of enzymes comprises DD-peptidases, glutaminases, 3 classes of well-characterized serine β-lactamases (A, C, and D), and transpeptidases.[27] β-Lactamase (EC 3.5.2.6) catalyzes the hydrolysis of an amide bond (N–CO) in the β-lactam ring of the antibiotics of the penicillin/cephalosporin family contributing to the most common mechanism of bacterial resistance to β-lactam antibiotics, while penicillin-binding proteins encompass transpeptidase, carboxypeptidase, and transglycosylase activities and have a part in the biosynthesis of the bacterial cell wall.[18,28,29] The representatives of all DD-peptidase and β-lactamase families have been described together biochemically and structurally, and the molecular mechanisms of the catalytic activity have been recognized.[30-33] Motif analysis represents 2 major β-lactamase motifs, including class C β-lactamases. Class C β-lactamases include a conserved Tyr residue (Tyr150 in AmpC from Enterobacter cloacae) in place of Ser in motif 2,[18] which also has no apparent counterpart in glutaminase sequences. Consequently, motif analysis denotes that glutaminases keep motifs 1 and 3 of β-lactamases but vary in motif 2. These sequences could be exploited for the expression and diversity analysis of glutaminase enzymes and confer valuable data for a better understanding of the structure and function of glutaminase. To that end, further research is required to assess the immunogenicity and thermal tolerance of glutaminase and improve its stability in different environmental conditions.

Conclusion

Our in silico evaluation of glutaminase protein sequences from diverse species of Escherichia and Bacillus clearly disclosed a sequence level similarity which could be helpful in cloning putative genes using degenerate primers designed from the conserved sequences. The phylogenetic clustering, conserved motif sequences, and discrepancy between the biochemical traits of the different glutaminases in this study could be deemed critical information for investigating new glutaminases and comparing them with other types of β-lactamases for the further classification and application of diverse β-lactamases. The operational characterization of amino-acid residues in the conserved domains of glutaminases is needed to identify their role in enzyme catalysis. Overall, this in silico analysis can be considered significant for the genetic engineering of glutaminases in light of their application in food and pharmaceutical industries as well as cancer therapy.