scholarly journals Phe-140 Determines the Catalytic Efficiency of Arylacetonitrilase from Alcaligenes faecalis

2020 ◽  
Vol 21 (21) ◽  
pp. 7859
Author(s):  
Jung-Soo Kim ◽  
Sanjay K. S. Patel ◽  
Manish K. Tiwari ◽  
Chunfen Lai ◽  
Anurag Kumar ◽  
...  
Keyword(s):  
Amino Acids ◽  
Catalytic Efficiency ◽  
Thiol Group ◽  
Alcaligenes Faecalis ◽  
Nucleophilic Attack ◽  
Charged Amino Acids ◽  
Molecular Determinant ◽  
Systematic Strategy

Arylacetonitrilase from Alcaligenes faecalis ATCC8750 (NitAF) hydrolyzes various arylacetonitriles to the corresponding carboxylic acids. A systematic strategy of amino acid residue screening through sequence alignment, followed by homology modeling and biochemical confirmation was employed to elucidate the determinant of NitAF catalytic efficiency. Substituting Phe-140 in NitAF (wild-type) to Trp did not change the catalytic efficiency toward phenylacetonitrile, an arylacetonitrile. The mutants with nonpolar aliphatic amino acids (Ala, Gly, Leu, or Val) at location 140 had lower activity, and those with charged amino acids (Asp, Glu, or Arg) exhibited nearly no activity for phenylacetonitrile. Molecular modeling showed that the hydrophobic benzene ring at position 140 supports a mechanism in which the thiol group of Cys-163 carries out a nucleophilic attack on a cyanocarbon of the substrate. Characterization of the role of the Phe-140 residue demonstrated the molecular determinant for the efficient formation of arylcarboxylic acids.

scholarly journals Characterization of symbiotic and associated bacteria from entomopathogenic nematode Heterorhabditis sp. (nematode: Heterorhabditidae) isolated from India

2020 ◽  
Vol 30 (1) ◽  
Author(s):  
Rashid Pervez ◽  
Showkat Ahmad Lone ◽  
Sasmita Pattnaik
Keyword(s):  
Insect Pests ◽  
Entomopathogenic Nematode ◽  
Alcaligenes Faecalis ◽  
Symbiotic Bacteria ◽  
Rrna Gene ◽  
Main Body ◽  
Associated Bacteria ◽  
Molecular Approaches

Abstract Background Entomopathogenic nematodes (EPNs) harboring symbiotic bacteria are one of the safest alternatives to the chemical insecticides for the control of various insect pests. Infective juveniles of EPNs locate a target insect, enter through the openings, and reach the hemocoel, where they release the symbiotic bacteria and the target gets killed by the virulence factors of the bacteria. Photorhabdus with Heterorhabditis spp. are well documented; little is known about the associated bacteria. Main body In this study, we explored the presence of symbiotic and associated bacteria from Heterorhabditis sp. (IISR-EPN 09) and characterized by phenotypic, biochemical, and molecular approaches. Six bacterial isolates, belonging to four different genera, were recovered and identified as follows: Photorhabdus luminescens, one each strain of Providencia vermicola, Pseudomonas entomophila, Alcaligenes aquatilis, and two strains of Alcaligenes faecalis based on the phenotypic, biochemical criteria and the sequencing of 16S rRNA gene. Conclusion P. luminescens is symbiotically associated with Heterorhabditis sp. (IISR-EPN 09), whereas P. vermicola, P. entomophila, A. aquatilis, and A. faecalis are the associated bacteria. Further studies are needed to determine the exact role of the bacterial associates with the Heterorhabditis sp.

scholarly journals Structural and biochemical characterization of the environmental MBLs MYO-1, ECV-1 and SHD-1

2020 ◽  
Vol 75 (9) ◽  
pp. 2554-2563 ◽  
Author(s):  
Christopher Fröhlich ◽  
Vidar Sørum ◽  
Sandra Huber ◽  
Ørjan Samuelsen ◽  
Fanny Berglund ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Homology Modelling ◽  
Catalytic Efficiency ◽  
Hydrolytic Activity ◽  
Human Pathogens ◽  
E Coli ◽  
X Ray Crystallography ◽  
Environmental Reservoir

Abstract Background MBLs form a large and heterogeneous group of bacterial enzymes conferring resistance to β-lactam antibiotics, including carbapenems. A large environmental reservoir of MBLs has been identified, which can act as a source for transfer into human pathogens. Therefore, structural investigation of environmental and clinically rare MBLs can give new insights into structure–activity relationships to explore the role of catalytic and second shell residues, which are under selective pressure. Objectives To investigate the structure and activity of the environmental subclass B1 MBLs MYO-1, SHD-1 and ECV-1. Methods The respective genes of these MBLs were cloned into vectors and expressed in Escherichia coli. Purified enzymes were characterized with respect to their catalytic efficiency (kcat/Km). The enzymatic activities and MICs were determined for a panel of different β-lactams, including penicillins, cephalosporins and carbapenems. Thermostability was measured and structures were solved using X-ray crystallography (MYO-1 and ECV-1) or generated by homology modelling (SHD-1). Results Expression of the environmental MBLs in E. coli resulted in the characteristic MBL profile, not affecting aztreonam susceptibility and decreasing susceptibility to carbapenems, cephalosporins and penicillins. The purified enzymes showed variable catalytic activity in the order of <5% to ∼70% compared with the clinically widespread NDM-1. The thermostability of ECV-1 and SHD-1 was up to 8°C higher than that of MYO-1 and NDM-1. Using solved structures and molecular modelling, we identified differences in their second shell composition, possibly responsible for their relatively low hydrolytic activity. Conclusions These results show the importance of environmental species acting as reservoirs for MBL-encoding genes.

scholarly journals Role of S3 and S4 Transmembrane Domain Charged Amino Acids in Channel Biogenesis and Gating of KCa2.3 and KCa3.1

2008 ◽  
Vol 283 (14) ◽  
pp. 9049-9059 ◽  
Author(s):  
Yajuan Gao ◽  
Cavita K. Chotoo ◽  
Corina M. Balut ◽  
Fei Sun ◽  
Mark A. Bailey ◽  
...  
Keyword(s):  
Amino Acids ◽  
Transmembrane Domain ◽  
Charged Amino Acids

Role of trinuclear Zn(II) complexes in promoting the hydrolysis of 4-nitrophenyl acetate

2004 ◽  
Vol 82 (3) ◽  
pp. 409-417 ◽  
Author(s):  
Qing-Chun Ge ◽  
Yan-He Guo ◽  
Hai Lin ◽  
Dong-Zhao Gao ◽  
Hua-Kuan Lin ◽  
...  
Keyword(s):  
Rate Constants ◽  
Volume Fraction ◽  
Bound Water ◽  
Catalytic Efficiency ◽  
Active Species ◽  
Nucleophilic Attack ◽  
Carboxyl Oxygen Atom ◽  
Ph Range ◽  
Hydrolysis Of

Potentiometric determination shows that trinuclear Zn(II) complexes of the four tripods 1,3,5-tri(2′,5′-diazahexyl)benzene (L1), 1,3,5-tri(2′,5′-diazaheptyl)benzene (L2), 1,3,5-tri(2′,5′-diazaoctyl)benzene (L3), and 1,3,5-tri(2′,5′-diazanonyl)benzene (L4) could be potential hydrolytic catalysts. CH3CN solutions containing [3Zn:L]T (0.5~2 × 10–3 mol·dm–3) with I = 0.10 mol·dm–3 of KNO3 and Good's buffer (10% volume fraction) were studied for the catalyzing hydrolysis of p-nitrophenyl acetate (NA, 0.5~2 × 10–3 mol·dm–3), at 298 K, in the 6.5–8.2 pH range. The observed rate constants, kobs, fit the equilibrium equation kobs = kcom [3Zn:L]T + kOH[OH–] + k0. The sigmoid pH~kcom profiles for NA hydrolysis suggest that either the Zn(II)-bound hydroxyl or the Zn(II)-bound water forms of the catalysts can be the active species. The observed second-order rate constants are 0.0082, 0.011, 0.0059, and 0.0019 mol–1·dm3·s–1 for the four Zn3L–H2O complexes (kA) and 0.342, 0.257, 0.382, and 0.091 mol–1·dm3·s–1 for the four Zn3L–OH- groups (kB), respectively. However, under the condition that [NA] = 0.5 × 10–3 mol·dm–3 and [3Zn:L1]T = 2~4 × 10–2 mol·dm–3 at pH 7.6, the observed rate constants, kobs, obey the equilibrium kobs = kcom[3Zn:L]T/(1/K′ + [3Zn:L]T). This indicates that the 3:1 complex (or its deprotonated hydroxide form) mediates NA hydrolysis by nucleophilic attack of the carboxyl center with the pre-formation of a coordination bond between the carboxyl oxygen atom and the Zn(II) ion. Comparison with other models was made, and the reasons for the high catalytic efficiency of the tripodal complexes were given.Key words: tripod, Zn(II), catalysis, NA hydrolysis, polynuclear.

Discovery and Biocatalytic Application of a PLP-Dependent Amino Acid γ-Substitution Enzyme that Catalyzes C-C Bond Formation

2020 ◽  
Author(s):  
Mengbin Chen ◽  
Chun-Ting Liu ◽  
Yi Tang
Keyword(s):  
Amino Acids ◽  
Biosynthetic Pathway ◽  
Pyridoxal Phosphate ◽  
Biochemical Characterization ◽  
Indole Alkaloid ◽  
Building Blocks ◽  
Nucleophilic Attack ◽  
Keto Esters ◽  
Key Enzyme

Pyridoxal phosphate (PLP)-dependent enzymes can catalyze various transformations of amino acids at alpha, beta, and gamma positions. These versatile enzymes are prominently involved in the biosynthesis of nonproteinogenic amino acids as building blocks of natural products, and are attractive biocatalysts. Here, we report the discovery of a two-step enzymatic synthesis of (2<i>S, </i>6<i>S</i>)-6-methyl pipecolate <b>1</b>, from the biosynthetic pathway of indole alkaloid citrinadin. The key enzyme CndF is PLP-dependent and catalyzes synthesis of (<i>S</i>)-2-amino-6-oxoheptanoate <b>3</b> that is in equilibrium with the cyclic Schiff base. The second enzyme CndE is a stereoselective imine reductase that gives <b>1</b>. Biochemical characterization of CndF showed this enzyme performs gamma-elimination of <i>O</i>-acetyl L-homoserine to generate the vinylglycine ketimine, which is subjected to nucleophilic attack by acetoacetate to form the new C<sub>gamma</sub>-C<sub>delta</sub> bond in <b>3 </b>and complete the gamma-substitution reaction. CndF displays substrate promiscuity towards different beta-keto carboxylate and esters. Using a recombinant <i>Aspergillus </i>strain expressing CndF and CndE, feeding various alkyl-beta-keto esters led to the biosynthesis of 6-substituted L-pipecolates. The discovery of CndF expands the repertoire of reactions that can be catalyzed by PLP-dependent enzymes.

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