Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing nitrogen-containing organic compound
Patent
1997-04-01
1998-08-25
Wax, Robert A.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing nitrogen-containing organic compound
435136, 435146, 4351721, 435189, 435190, 435280, C12P 1300, C12P 740, C12N 1500, C12N 902
Patent
active
057982341
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention is relative to a method for the directed modification of enzymes, modified enzymes and their use.
2. Background Information
Enzymes can usually be modified either by chemical modification of the amino acids forming the enzyme or by mutation of the gene coding the enzyme. Chemical modifications are frequently non-specific, so that the directed modifying of the enzyme structure by directed mutation of the gene has advantages over the chemical modification.
Modified enzymes frequently exhibit improved properties as regards activity, specificity or stability in comparison to the unmodified enzymes. Previous activities in this area were frequently limited to improving global properties of the enzyme such as e.g. stability vis-a-vis reaction media. Thus, for example, alkaline proteases were stabilized against the oxidizing action of bleaching agents. However, the problem of changing the specificity of enzymes in order to convert previously defined substrates has hardly been treated. One exception is the work of H. M. Wilks et al. (H. M. Wilks et al., Science 1988, 242, 1541-1544) in which the substrate specificity of a lactate dehydrogenase with lactate as preferred substrate is transformed to a malate dehydrogenase with malate as preferred substrate.
The selectivity for producing enantiomerically pure compounds is especially desirable for enzymes. The enantioselectivity is especially prominent in the case of amino-acid dehydrogenases, among others. Thus, amino-acid dehydrogenases were screened from a number of organisms. The most important enzymes for use in organic synthesis are alanine dehydrogenase (AlaDH, E.C. 1.4.1.1.), phenylalanine dehydrogenase (PheDH; still no E.C. number) and, especially, leucine dehydrogenase (LeuDH, E.C. 1.4.1.9.). However, the best-investigated enzyme of the group is the ubiquitous glutamate dehydrogenase (GluDH, E.C. 1.4.1.2.-4.), which forms an important branch point between carbon metabolism and nitrogen metabolism. GluDH catalyses the NAD (P).sup.+ -dependent, oxidative deamination of L-glutamate to 2-oxoglutarate and ammonia: ##STR1##
Amino-acid dehydrogenases generally catalyze the reversible reductive amination of prochiral keto acids to L-amino acids ((S) configuration) and the reverse reaction of the oxidative deamination of L-amino acids to oxo acids.
In most instances, including all NAD.sup.+ -dependent GluDH'es, the enzyme has six identical subunits of approximately 48 kD each. A considerable homology is found in the polypeptide chain of the hexameric GluDH'es; in particular, the glutamate binding pocket and the active center display a remarkable similarity (Britton, K. L., Baker, P. J., Rice, D. W. and Stillman, T. J., Eur. J. Biochem. 1992, 209, 851-859).
The degree of similarity between the structures of the members of the family of amino-acid dehydrogenases from different organisms is so great that a single superfamily of enzymes is assumed in the case of amino-acid dehydrogenases, which superfamily was produced by divergent evolution (S. Nagata, K. Tanisawa, N. Esaki, Y. Sakamoto, T. Ohshima, H. Tanaka and K. Soda, Biochemistry 1988, 27, 9056-62; H. Takada, T. Yoshimura, T. Ohshima, N. Esaki and K. Soda, J. Biochem. 1991, 109, 371-6).
The three-dimensional structure of the glutamate dehydrogenase from Clostridium symbiosum is known (Baker, P. J. et al., Proteins 1992, 12, 75-86). The gene of this enzyme was cloned and overexpressed in Escherichia coli (Teller, J. K. et al., Eur. J. Biochem. 1992, 286, 151-159). However, it has not been successful in the past to modify the enzymatic structure in such a manner by directed mutation of the gene that even certain substrates selected before the mutation were converted.
SUMMARY OF THE INVENTION
The invention therefore has the problem of making available a method for the directed modification of enzymes in which desired substrates can be converted independently of the natural substrate specificity of the enzyme. A further object of the invention is to
REFERENCES:
Kataoka et al: "Site-directed mutagenesis of a hexapeptide segment involved in substrate recognition of Phenylalanine Dehydrogenase from Thermoacetinomyces intermedius", J. Biochem., vol. 114, No. 1, Jul. 1993, pp. 69-75.
Wilks et al: "Designs for broad substrate specificity keto acid dehydrogenase", Biochemistry, vol. 29, No. 37, 1990, pp. 8587-8591.
Green et al: "Inversion of the substrate specificity of Yest Alcohol Dehydrogenase", J.Biochem., vol. 268, No. 11, Apr. 15, 1993, pp. 7792-7798.
Wilson et al: "Computational method for the design of enzymes with altered substrate specificity", J.MolBiol., vol. 220, 1991, pp. 495-506.
Power et al: Biotechnology, vol. 7b, "Gene Technology"; vol. eds. Jacobsen, G.K. & Jolly, S.O., chapter 6b: The Engineerying of structural and catalytic properties of proteins, pp. 2620276, VCH Publishers, ISBN 0-89573-561-X.
J.E. Rife and W.W. Cleland (1980) Biochemistry, 19, 2328-2333.
K.S. Lilley and P.C. Engel (1988) Biochemical society transactions, 16, 875-876.
Engel Paul C.
Rice David
Degussa - Aktiengesellschaft
Slobodyansky Elizabeth
Wax Robert A.
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