Chloroacetaldehyde dehydrogenase from Ancylobacter aquaticus UV5: Cloning, expression, characterization and molecular modeling

1,2-Dichloroethane (1,2-DCE) is oxidatively converted to a carcinogenic intermediate compound, chloroacetaldehyde by chloroacetaldehyde dehydrogenase (CAldA) during its biodegradation by many bacterial strains, including Xanthobacter autotrophicus and Ancylobacter aquaticus. In this study, a 55 kDa NAD-dependent CAldA expressed by chromosomally encoded aldA gene, is reported in an indigenous Ancylobacter aquaticus UV5. A. aquaticus UV5 aldA gene was found to be 99% homologous to the plasmid (pXAU1) encoded aldA gene reported in X. autotrophicus GJ10. Pulse-field gel electrophoresis (PFGE) and PCR experiments revealed the absence of pXAU1 in A. aquaticus UV5 and that aldA was chromosomal encoded. A 6× His-tag fused CAldA cloned in pET15b, overexpressed and purified on Co-agarose affinity column using AKTA purification system showed M_r of 57,526. CAldA was active optimally at pH 9 and 30 °C. The K_m and v_max for the substrate, acetaldehyde were found to be 115 μM and 650 mU/mg, respectively. CAldA substrate specificity was found to be low for chloroacetaldehyde, formaldehyde, propionaldehyde, butyraldehyde, benzaldehyde and glutaraldehyde as compared to acetaldehyde. Computational modeling revealed a predicted structure of CAldA consisting of five β-sheets that comprise seven antiparallel β-strands and 11 mix strands. The Molecular Dynamics and Docking studies showed that acetaldehyde bind to CaldA more tightly as compared to chloroacetaldehyde.