title: Benzyl alcohol dehydrogenase from Acinetobacter calcoaceticus
Creators: name: Gillooly, David John
Publisher: name: University of Glasgow
Year: 1996
Subjects: subject: Microbiology
subject: FOS: Biological sciences

description: 1. Acinetobacter calcoaceticus NCIB8250 can grow on benzyl alcohol as sole carbon and energy source. The benzyl alcohol pathway involves the NAD- dependent enzymes benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II. These two enzymes had previously been purified from A. calcoaceticus and xylC, the gene encoding benzaldehyde dehydrogenase II, had been sequenced. This thesis describes work which completed the cloning of xylB, the gene encoding benzyl alcohol dehydrogenase, the sequencing of xylB and further studies of this enzyme with particular' emphasis on its substrate specificity and proton relay mechanism. 2. A clone from an A. calcoaceticus lambda genomic DNA library which in a previous study had been proposed to harbour the full lengths of both xylC and xylB was sub-cloned into pBluescript SK II and a series of sub-clones developed so that benzyl alcohol dehydrogenase could be expressed in Escherichia coli DH5alpha with and without benzaldehyde dehydrogenase II. 3. The nucleotide sequence of xylB was determined. The xylB structural gene is 1110 bp in length. The nucleotide sequence, together with the observation that both xylC and xylB are transcribed together from the lac promoter of pBluescript SK II after induction with IPTG, suggests that the two genes form part of an operon transcribed in the direction xylC→xylB. A possible a factor-independent transcription terminator sequence was identified downstream of xylB. 4. The nucleotide sequence of xylB indicated that benzyl alcohol dehydrogenase is a 370 amino acid protein with a relative molecular mass (Mr) of 38,923. Benzyl alcohol dehydrogenase could be purified from E. coli DH5alpha/pDG20 using the method previously described for purification of the enzyme from A. calcoaceticus. N-Terminal amino acid analysis confirmed that the purified enzyme was the xylB product. The Mr of purified benzyl alcohol dehydrogenase was determined to be 38,929 +/- 8 by electrospray mass spectometry, thus confirming the nucleotide sequence. 5. A xylB clone was constructed using PCR so that a T7 RNA polymerase expression system could be used to over-express benzyl alcohol dehydrogenase. This system enabled benzyl alcohol dehydrogenase to be expressed at a level of at least 50% of the total soluble protein in E. coli JM109(DE3) and enabled the purification of greater than 10 mg quantities of enzyme from less than 10 g of cells. 6. Alignment of the primary sequence of benzyl alcohol dehydrogenase with members of the group of NAD(P)-dependent long-chain zinc-dependent alcohol dehydrogenases showed benzyl alcohol dehydrogenase to be a member of this group of enzymes. In particular, the zinc ligands of horse liver alcohol dehydrogenase are all conserved in benzyl alcohol dehydrogenase and this suggests that the enzyme probably binds two zinc atoms per enzyme subunit one catalytic and one structural. The A. calcoaceticus enzyme shares 54% sequence identity with benzyl alcohol dehydrogenase encoded by the TOL-plasmid pWWO of P.seudomonas putida mt-2 and greater than 30% sequence identity with several mammalian and plant zinc-dependent alcohol dehydrogenases. These enzymes all appear to share a common evolutionary ancestor. (Abstract shortened by ProQuest.).