Comparative Studies on Alcohol Dehydrogen for Mesophilic (Bsubbls) and Hyperthermphylic (pyrabaalum aolidytohs) Source

Abstract

Alcohol dehydrogenases are very important and have critical role in pharmaceutical, food, chemical industry, second generation biofuel and white biotechnology. The present study describes the cloning and characterization of alcohol dehydrogenases from mesophilic bacterial (Bacillus subtilis R5) and hyperthermophilic archaeal (Pyrobaculum calidifontis VA1) sources. The genes encoding alcohol dehydrogenases were identified by the genome wide search of the respective organism. Complete genome of B. subtilis (GeneBank accession number NC_000964) and P. calidifontis (GeneBank accession number NC_009073) were searched for alcohol dehydrogenase gene. BSU26970 (B. subtilis), Pcal-0882, Pcal-1311 and Pcal-1581 (P. calidifontis) were selected for the present study. The alcohol dehydrogenase gene (ADHR5) from Bacillus subtilis R5 was annotated as glutathione dependent formaldehyde dehydrogenase/alcohol dehydrogenase was 1137 nucleotides in length which encoded a protein of 378 amino acids with a calculated molecular mass of 41.2 kDa. ADHR5 was cloned in pET-21a(+) and expressed in Escherichia coli. Recombinant ADHR5 was produced as inclusion bodies in E. coli. Efforts were made to produce it in soluble form by expressing the gene at lower temperature but ADHR5 remained in inclusion bodies. Then ADHR5 was refolded by denaturing with urea and gradual removal of urea. Although the protein was in soluble form after removal of urea but it did not display any activity. Therefore ADHR5 was coexpressed with a chaperonin GroEL4S in E. coli by using pETDuet-GroEL4S vector. His-tag was introduced at the N-terminal to facilitate the purification of recombinant ADHR5. By coexpression with GroEL4S, ADHR5 was produced as soluble protein by lowering the cultivation temperature and purified by nickel affinity and gel filtration column chromatography. Recombinant ADHR5 was found to be a homotetramer. ADHR5 showed highest activity 885 nmol min-1mg-1 against 1-propanol. Although ADHR5H was cloned from a mesophilic source but the optimum temperature for ADHR5 was found to be 60 °C and no change in the secondary structure was observed by circular dichroism spectroscopy at 60 °C. Sequence comparison showed thar ADHR5 belonged to zinc dependent medium chain alcohol dehydrogenases, however highest activity and secondary structure stability was observed when ADHR5 was produced in the presence of copper or iron instead of zinc. v Apart from the mesophilic source, I studied three alcohol dehydrogenases from the hyperthermophilic source P. calidifontis. Three open reading frames that were annotated as alcohol dehydrogenase include Pcal-0882, Pcal-1311 and Pcal-1581. Pcal-0882 contained 996 nucleotides encoding a protein 331 amino acids an approximate molecular mass of 35 kDa. The gene encoding Pcal-0882 was annotated as an alcohol dehydrogenase belonging to zinc dependent medium chain alcohol dehydrogenases. Pcal-0882 was cloned in pET-21a(+) and expressed in E. coli. Recombinant Pcal-0882 was produced in soluble form and it was a thermostable protein. Recombinant Pcal-0882 was purified by heat treatment at 80 °C and anion exchange chromatography. Recombinant Pcal-0882 was assayed for alcohol dehydrogenase activity but no activity could be detected against any substrate. The detailed amino acid sequence analysis showed that Pcal-0882 has conserved Zn2+ binding and cofactor binding domains except alcohol binding amino acids. The substrate binding amino acids were identical to acrolyl-CoA reductase from Sulfolobus tokodaii. Substrate binding amino acids and no detection of alcohol dehydrogenase activity indicate that Pcal-0882 is not an alcohol dehydrogenase but may be an acrolyl-CoA reductase. Pcal-1311 was composed of 1038 nucleotides, encoding protein of 37.5. Pcal-1311 gene was cloned in pET-21a(+) and expressed in Escherichia coli. Pcal-1311 was produced as soluble protein. Pcal-1311 exhibited maximum activity as 80 °C. Pcal-1311 was found to have optimum pH of 9.5 for oxidation reaction and 6 for reduction. Pcal-1311 was found to be active against a broad range of substrates with a clear preference for primary and aliphatic alcohols over secondary and branched chained alcohols. Maximum activity in the oxidation direction was observed with 1,4 butanediol (4.22± 0.4 U mg-1 min-1). Pcal-1311 was found to be more active in the reduction direction and maximum activity of (150.3±8 U mg-1 min-1) was observed against gluteraldehyde. Pcal-1311 exhibited higher reduction activity as compared to the oxidation activity. Pcal-1311 is zinc dependent medium chain alcohol dehydrogenase and it requires supplementation of zinc in the growth medium for proper activity of the recombinant protein. EDTA does not affect the activity of recombinant Pcal-1311 unless it is incubated at higher temperature with EDTA. Usually protein are reported to lose their activities in the presence of high concentration of urea, however when effect of urea was studied on Pcal-1311, instead of losing activity, enhancement of the enzyme activity was observed. The fluorescence spectra of Pcal-1311 revealed that protein remain stable in 6 M of urea at least for 10 h. vi The most interesting point was genomic location of Pcal-1311. It was observed that the complete operon consists of seven genes consisting enoyl Co-A hydratase (Pcal-1306), hydrolase (Pcal- 1307), acyl CoA-domain dehydrogenase (Pcal-1308), acetyl-CoA C-acetyltransferase (Pcal- 1309), protein of unknown function DUF35 (Pcal-1310), alcohol dehydrogenase (Pcal-1311) and 3-hydroxyacyl-ACP reductase (Pcal-1312). These genes may be involved either in butanol synthesis pathway or beta oxidation of fatty acid. So the whole operon was cloned and biochemical properties of these genes were studied, which are described in detail in 3rd and 4th chaperter. Pcal-1581 was composed of 1047 nucleotides, encoding proteins of 37 kDa. Pcal-1581 gene was cloned in pET-21a(+) and expressed in Escherichia coli. Pcal-1581 was produced as insoluble and inactive protein In conclusion, the results of these studies demonstrate that ADHR5 is not a glutathione dependent alcohol dehydrogenase but a metal dependent alcohol dehydrogenase. Pcal-1311 is a true alcohol dehydrogenase. Presence of the metal ions is essential for proper folding of recombinant ADHR5 and Pcal-1311 in E. coli. The results of this study demonstrate that for proper activity of zinc dependent ADH, incorporation of metal ions in protein is essential at the time protein production. Pcal-1311 is an alcohol dehydrogenase exhibited significant stability against denaturants. The genomic location of Pcal-1311 makes it an attractive clue for identification and characterization of novel pathways in archaea.