Molecular Characterisation of Microbial NAD(P)-Dependent Alcohol Dehydrogenases

Reid, Matthew Francis (1993) Molecular Characterisation of Microbial NAD(P)-Dependent Alcohol Dehydrogenases. MSc(R) thesis, University of Glasgow.

Abstract

1. Alcohol oxidoreductases have been isolated from various sources and classified into five distinct evolutionary categories: Groups I, II and III NAD(P)-dependent alcohol dehydrogenases, NAD(P)-independent alcohol dehydrogenases and alcohol oxidases. Examples of all of these groups are found in microorganisms. The first part of this thesis consists principally of a critical review of information covering the primary, secondary', tertiary and quaternary structures of the microbial NAD(P)-dependent alcohol dehydrogenases and their catalytic mechanisms, in an attempt to clarify the evolutionary relationships amongst them. The second part of the thesis is a description of research into the molecular characterisation of benzyl alcohol dehydrogenase from Acinetobacter calcoaceticus NCIB 8250. 2. (a) Primary structure analysis and three-dimensional modelling of the group I alcohol dehydrogenases has shown that the coenzyme specificity is determined by a single residue, Aspanate-223. There does not appear to be a relationship between primary structure, presence of a second structural zinc atom and quaternary' structure of the group I alcohol dehydrogenases. There are 14 strictly conserved residues amongst the microbial group I alcohol dehydrogenases, compared to nine amongst the whole of the group I alcohol dehydrogenases. Identity of primary' amino acid structure amongst pairs of microbial group I alcohol dehydrogenases ranges from 2A% to 94%. In some instances higher sequence identity is seen between prokaryote and eukaryote members than between members of the same cell class. All microbial group I alcohol dehydrogenases are tetrameric except for the Tol-plasmid (pWW0) encoded benzyl alcohol dehydrogenase from Pseudomonas putida and the trimeric nicotinoproiein, 4-nitroso-N,N-dimethylaniline-dependent alcohol/aldehyde oxidoreductase from Amycolatopsis methanolica . The benzyl alcohol dehydrogenase from Pseudomonas putida is dimeric, like plant and mammalian group I alcohol dehydrogenases; the benzyl alcohol dehydrogenase from Acinetobacter colcoaceticus NCIB 8250 is tetrameric. Whether the trimeric nitroso-N,N-dimethylaniline-dependent aldehyde/alcohol oxidoreductase from Amycolatopsis methanolica is a member of a group I alcohol dehydrogenase sub-group or possibly the first member of a novel group of alcohol dehydrogenase will not be known until its full amino acid sequence is determined. (b) The three dimensional structure of 3alpha,20beta-hydroxysteroid dehydrogenase (a group II alcohol dehydrogenases) from Streptomyces hydrogenans has been solved. The catalytic mechanism is less well understood than is that of the group I alcohol dehydrogenases, although there is no evidence to suggest that coenzyme preference is determined by a single residue as is seen in the group I alcohol dehydrogenases. Overall, the degree of amino acid identity amongst the group II alcohol dehydrogenases is lower than that seen amongst the group I alcohol dehydrogenases, and ranges from 19% to 59%. (c) The characteristics of the group III alcohol dehydrogenases are far less conserved than those of either group I or II enzymes. So far, all group III alcohol dehydrogenases have been isolated from micro-organisms, there is no three dimensional structure is available and very little is known about their catalytic mechanism. The degree of identity amongst the group III alcohol dehydrogenases ranges from 18% to 53%. With some members there is a requirement for various metal ions for catalytic activity and in one case an activator protein has been suggested to be involved. Subunit sizes are conserved amongst this group at approximately 40 kDa, but native structure varies considerably from dimers to tetramers and decamers. Like the group I alcohol dehydrogenases, there are nicotinoproteins which on the basis of partial amino acid sequence analysis have been proposed to be members of the group III alcohol dehydrogenases. Whether these nicotinoproteins form a novel type of alcohol dehydrogenase or constitute a sub-group of the group III alcohol dehydrogenases will only be determined once their full primary sequences are solved. (d) N.AD(P)-independent alcohol dehydrogenases are divided into two functionally distinct groups; the methanol dehydrogenases and the ethanol dehydrogenases. Their exact catahtic mechanism has not yet been defined but it is believed to involve the passing of reducing equivalents to pyrroloquinoline and cytochrome c. (e) Alcohol oxidases differ from alcohol dehydrogenases in that they irreversibly oxidise alcohols to aldehydes and ketones. They have been characterised as flavoproteins and they are located in the peroxisomes of methylotrophic yeasts and filamentous fungi.

• Item Type: Thesis (MSc(R))
• Qualification Level: Masters
• Additional Information: Adviser: Charles Fewson
• Keywords: Biochemistry, Molecular biology
• Date of Award: 1993
• Depositing User: Enlighten Team
• Unique ID: glathesis:1993-76360
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 19 Nov 2019 15:22
• Last Modified: 19 Nov 2019 15:22
• URI: https://theses.gla.ac.uk/id/eprint/76360