In Silico Analysis of Amino Acid Sequences in Relation to Specificity and Physiochemical Properties of Some Microbial Nitrilases

Mehta

Biotech and App Biochem

et al. 2014

Self Cite

Arylacetonitrile-hydrolyzing nitrilase (E.C. 3.5.5.5) of Alcaligenes sp. MTCC 10675 has been purified by up to 46-fold to homogeneity and 32% yield using ammonium sulfate fractionation, Sephacryl S-300 gel permeation, and anion exchange chromatography. The molecular weight of the native enzyme was estimated to be 520 ± 60 kDa. The subunit has a molecular weight of 60 ± 14 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature of the purified enzyme were 6.5 and 50 °C, respectively. The purified arylacetonitrilase has a half-life of 3 H 20 Min at its optimum temperature. The value for Vmax, Km , kcat , and ki of enzyme for mandelonitrile as a substrate was 50 ± 05 µmol/Min/mg, 13 ± 02 mM, 26 ± 03 Sec(-) , and 32.4 ± 03 mM, respectively. Alcaligenes sp. MTCC 10675 arylacetonitrilase amino acid sequence has variations from other reported arylacetonitrilase, namely, A11G, N21H, D149N, S170T, P171R, S179A, Q180N, and S191A, and it has a high thermal stability and catalytic rate as compared with the already purified arylacetonitrilase.

Section: Discussionsupporting

confidence: 75%

Purification and characterization of arylacetonitrile‐specific nitrilase of Alcaligenes sp. MTCC 10675

Mehta

Biotech and App Biochem

et al. 2014

Self Cite

“…The predicted NitC nitrilase of the strain CECT5344 has four conserved residues (Arg129, His168, Glu182 and His185) present in other nitrilases that use aliphatic nitriles (Fig. S3), but lacks the residues His129, Asn168 and Met170 conserved in aromatic nitrilases (Sharma et al ., 2009). This agrees with the substrate specificity found for this enzyme (Table 1).…”

Section: Resultsmentioning

confidence: 99%

The nit1C gene cluster of Pseudomonas pseudoalcaligenes CECT5344 involved in assimilation of nitriles is essential for growth on cyanide

Estepa¹,

Luque-Almagro²,

Manso³

et al. 2012

Environ Microbiol Rep

A proteomic approach was used to identify several proteins induced by cyanide in the alkaliphilic bacterium Pseudomonas pseudoalcaligenes CECT5344, two of them, NitB and NitG, encoded by genes that belong to the nit1C gene cluster. The predicted products of the nit1C gene cluster are a Fis-like σ(54) -dependent transcriptional activator (NitA), a nitrilase (NitC), an S-adenosylmethionine superfamily member (NitD), an N-acyltransferase superfamily member (NitE), a trifunctional polypeptide of the AIRS/GARS family (NitF), an NADH-dependent oxidoreductase (NitH) and two hypothetical proteins of unknown function (NitB and NitG). RT-PCR analysis suggested that nitBCDEFGH genes were co-transcribed, whereas the regulatory nitA gene was divergently transcribed. Real-time RT-PCR revealed that expression of the nitBCDEFGH genes was induced by cyanide and repressed by ammonium. The P. pseudoalcaligenes CECT5344 nit1C gene cluster was found to be involved in assimilation of free and organic cyanides (nitriles) as deduced for the inability to grow with cyanides showed by the NitA, NitB and NitC mutant strains. The wild-type strain CECT5344 showed a nitrilase activity that allows growth on cyanide or hydroxynitriles. The NitB and NitC mutants only presented low basal levels of nitrilase activity that were not enough to support growth on either free cyanide or aliphatic nitriles, suggesting that nitrilase NitC is specific and essential for cyanide and aliphatic nitriles assimilation.

“…Aliphatic and aromatic nitrilases can also be differentiated based on their amino acid sequence and physiochemical properties (48). The physicochemical properties of the UW4 nitrilase were deduced from an in silico analysis of the amino acid sequence using the ProtParam subroutine of the Expert Protein Analysis System (ExPASy) from the proteomic server of the Swiss Institute of Bioinformatics, in order to predict aromaticity or aliphaticity.…”

Section: Resultsmentioning

confidence: 99%

Characterization of a Nitrilase and a Nitrile Hydratase from Pseudomonas sp. Strain UW4 That Converts Indole-3-Acetonitrile to Indole-3-Acetic Acid

Duca

Rose

Glick

2014

Appl Environ Microbiol

Indole-3-acetic acid (IAA) is a fundamental phytohormone with the ability to control many aspects of plant growth and development. Pseudomonas sp. strain UW4 is a rhizospheric plant growth-promoting bacterium that produces and secretes IAA. While several putative IAA biosynthetic genes have been reported in this bacterium, the pathways leading to the production of IAA in strain UW4 are unclear. Here, the presence of the indole-3-acetamide (IAM) and indole-3-acetaldoxime/indole-3-acetonitrile (IAOx/IAN) pathways of IAA biosynthesis is described, and the specific role of two of the enzymes (nitrilase and nitrile hydratase) that mediate these pathways is assessed. The genes encoding these two enzymes were expressed in Escherichia coli, and the enzymes were isolated and characterized. Substrate-feeding assays indicate that the nitrilase produces both IAM and IAA from the IAN substrate, while the nitrile hydratase only produces IAM. The two nitrile-hydrolyzing enzymes have very different temperature and pH optimums. Nitrilase prefers a temperature of 50°C and a pH of 6, while nitrile hydratase prefers 4°C and a pH of 7.5. Based on multiple sequence alignments and motif analyses, physicochemical properties and enzyme assays, it is concluded that the UW4 nitrilase has an aromatic substrate specificity. The nitrile hydratase is identified as an iron-type metalloenzyme that does not require the help of a P47K activator protein to be active. These data are interpreted in terms of a preliminary model for the biosynthesis of IAA in this bacterium.