Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Patent
1991-10-24
1994-10-11
Wax, Robert A.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
930240, C12N 904
Patent
active
053546768
DESCRIPTION:
BRIEF SUMMARY
This invention relates to novel modified NAD-dependent dehydrogenases.
Proteins are extremely effective as catalysts for chemical conversions end have evolved to be highly selective. One possibility now emerging from the ability to chemically synthesise genes is the design end construction of proteins with properties not available in nature.
NAD-dependent dehydrogenases are frequently used in assay systems end in stereospecific syntheses of organic compounds. These enzymes catalyse both the hydrogenation of .alpha.-keto acids to .alpha.-hydroxy acids and the oxidation (dehydrogenation) of .alpha.-hydroxy acids to give .alpha.-karo acids. The native NAD-dependent dehydrogenase obtained from Bacillus stearothermophilus has activity as a lactate dehydrogenase, but has little activity as a dehydrogenase for other substrates. A feature of this particular enzyme is that it is highly thermophilic, i.e. it has a half-life of five minutes at 90.degree. C.
There is a need for modified NAD-dependent dehydrogenases, particularly ones which are thermally stable, and which have enzymic activity on selected substrates.
Thermophilic enzymes which have that property are more desirable than mesophilic ones for use in the production of long chain S-lactates which have value as chiral intermediates in pharmaceutical synthesis. Chiral lactates are useful intermediates for the synthesis of both off-patent generic and potential new pharmaceutical products. Many such compounds are currently marketed as Pacemates. However the fear of US and eventually European legislation in favour of single compound drags, and their higher audistoic ratios has caused an increase in the proportion of enantiomerically pure compounds being submitted for screening by Committee on Safety of Medicines. There is thus commercial advantage in an enzyme system which can be engineered to synthesize such chiral intermediates.
In GB 8806358 we identified residues contributing to the substrate recognition site of NAD-dependent dehydrogenases and showed that the site is present, at least in part, in a particular amino acid sequence. We further described certain modifications to that sequence which resulted in the formation of NAD-dependent dehydrogenases having, inter alia, different substrate specificities and other commercially desirable properties. Specifically, we have demonstrated that it is possible to alter the substrate specificity of the nicotinamide adenine dinucleotide (NAD)-dependent L-lactate dehydrogenase (LDH) from B. stearothermophilus from lactate to malate, producing a highly specific catalyst for the new substrate.
To alter or broaden the specificity of an enzyme it is necessary to have a detailed understanding of the mechanism so that the "central" chemistry can be retained. In the case of enzymes with small substrates, the catalytic and specificity sites are necessarily close together, making it difficult to alter substrate specificity without affecting catalysis.
We have now developed a mutation strategy which has enabled the production of NAD-dependent dehydrogenases which are thermophilic and which have a particularly desirable substrate specificity.
Specifically the novel enzymes of the invention are effective in catalysing the dehydrogenation of homologues of pyruvic acid of formula C.sub.n H.sub.2n+1 COCOOH wherein n is>1, to homologues of lactic acid of formula CH.sub.n H.sub.2n+1 CHOHCOOH wherein n is>1. Examples of resiues C.sub.n H.sub.2n+1 include straight and branched chain alkyl rsidues having 2 to 6 carbon atoms, including ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl end sec-butyl.
The wild type lactate dehydrogenases have very poor rates of reaction with long, branched .alpha.-karo acids and the corresponding .alpha.-hydroxy acids. The new enzymes we have synthesized have much more desirable properties with the higher homologues referred to above.
Such enzymes are of particular value in the field of, for example, synthetic chemistry where it is desired to convert a keto compound stereospecifically to a corresponding
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Atkinson Anthony
Chia William N.
Clarke Anthony R.
Holbrook Joseph J.
Wilks Helen M.
Public Health Laboratory Service Board
Schmickel David B.
The University of Bristol
Wax Robert A.
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