title: Two structurally different dienelactone hydrolases (TfdEI and TfdEII) from Cupriavidus necator JMP134 plasmid pJP4 catalyse cis- and trans-dienelactones with similar efficiency.
abstract: In this study, dienelactone hydrolases (TfdEI and TfdEII) located on plasmid pJP4 of Cupriavidus necator JMP134 were cloned, purified, characterized and three dimensional structures were predicted. tfdEI and tfdEII genes were cloned into pET21b vector and expressed in E. coli BL21(DE3). The enzymes were purified by applying ultra-membrane filtration, anion-exchange QFF and gel-filtration columns. The enzyme activity was determined by using cis-dienelactone. The three-dimensional structure of enzymes was predicted using SWISS-MODEL workspace and the biophysical properties were determined on ExPASy server. Both TfdEI and TfdEII (Mr 25 kDa) exhibited optimum activity at 37°C and pH 7.0. The enzymes retained approximately 50% of their activity after 1 h of incubation at 50°C and showed high stability against denaturing agents. The TfdEI and TfdEII hydrolysed cis-dienelactone at a rate of 0.258 and 0.182 µMs(-1), with a Km value of 87 µM and 305 µM, respectively. Also, TfdEI and TfdEII hydrolysed trans-dienelactone at a rate of 0.053 µMs(-1) and 0.0766 µMs(-1), with a Km value of 84 µM and 178 µM, respectively. The TfdEI and TfdEII kcat/Km ratios were 0.12 µM(-1) s(-1) and 0.13 µM(-1) s(-1) and 0.216 µM(-1) s(-1) and 0.094 µM(-1) s(-1) for for cis- and trans-dienelactone, respectively. The kcat/Km ratios for cis-dienelactone show that both enzymes catalyse the reaction with same efficiency even though Km value differs significantly. This is the first report to characterize and compare reaction kinetics of purified TfdEI and TfdEII from Cupriavidus necator JMP134 and may be helpful for further exploration of their catalytic mechanisms.
authors: Discipline of Microbiology, School of Life Sciences, College of Agriculture, Engineering and Science, University of KwaZulu-Natal (Westville Campus), Durban, Republic of South Africa.
authors: Ajit
authors: A
authors: Kumar
authors: Discipline of Microbiology, School of Life Sciences, College of Agriculture, Engineering and Science, University of KwaZulu-Natal (Westville Campus), Durban, Republic of South Africa.
authors: Balakrishna
authors: B
authors: Pillay
authors: Discipline of Microbiology, School of Life Sciences, College of Agriculture, Engineering and Science, University of KwaZulu-Natal (Westville Campus), Durban, Republic of South Africa.
authors: Ademola O
authors: AO
authors: Olaniran
mesh_terms: D000595:Amino Acid Sequence; D055162:Biocatalysis; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020118:Cupriavidus necator / Q000201:enzymology* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D007783:Lactones / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D007527:Isoenzymes; D007783:Lactones; C084081:dienelactone; D002265:Carboxylic Ester Hydrolases; C046130:carboxymethylenebutenolidase