Complete amino acid sequence and characterization of the reaction mechanism of a glucosamine-induced novel alcohol dehydrogenase from Agrobacterium radiobacter (tumefaciens).
A glucosamine-induced novel alcohol dehydrogenase has been isolated from Agrobacterium radiobacter (tumefaciens) and its fundamental properties have been characterized. The enzyme catalyzes NAD-dependent dehydrogenation of aliphatic alcohols and amino alcohols. In this work, the complete amino acid sequence of the alcohol dehydrogenase was determined by PCR method using genomic DNA of A. radiobacter as template. The enzyme comprises 336 amino acids and has a molecular mass of 36 kDa. The primary structure of the enzyme demonstrates a high homology to structures of alcohol dehydrogenases from Shinorhizobium meliloti (83% identity, 90% positive) and Pseudomonas aeruginosa (65% identity, 76% positive). The two Zn(2+) ion binding sites, both the active site and another site that contributed to stabilization of the enzyme, are conserved in those enzymes. Sequences analysis of the NAD-dependent dehydrogenase family using a hypothetical phylogenetic tree indicates that these three enzymes form a new group distinct from other members of the Zn-containing long-chain alcohol dehydrogenase family. The physicochemical properties of alcohol dehydrogenase from A. radiobacter were characterized as follows. (1) Stereospecificity of the hydride transfer from ethanol to NADH was categorized as pro-R type by NMR spectra of NADH formed in the enzymatic reaction using ethanol-D(6) was used as substrate. (2) Optimal pH for all alcohols with no amino group examined was pH 8.5 (of the C(2)-C(6) alcohols, n-amyl alcohol demonstrated the highest activity). Conversely, glucosaminitol was optimally dehydrogenated at pH 10.0. (3) The rate-determining step of the dehydrogenase for ethanol is deprotonation of the enzyme-NAD-Zn-OHCH(2)CH(3) complex to enzyme-NAD-Zn-O(-)CH(2)CH(3) complex and that for glucosaminitol is H(2)O addition to enzyme-Zn-NADH complex.
Iwamoto
Ryoko
R
Department of Chemistry, Nara Women's University, Kitauoya Nishimachi, Nara, 630-8506, Japan. kurushima@cc.nara-wu.ac.jp
Kubota
Humie
H
Hosoki
Tomoko
T
Ikehara
Kenji
K
Tanaka
Mieko
M
D000426:Alcohol Dehydrogenase / Q000032:analysis* / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D004790:Enzyme Induction / Q000187:drug effects; D004795:Enzyme Stability; D000431:Ethanol / Q000378:metabolism; D005944:Glucosamine / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012231:Rhizobium / Q000201:enzymology*; D013379:Substrate Specificity
D000431:Ethanol; D000426:Alcohol Dehydrogenase; D005944:Glucosamine