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Titolo:
STRUCTURAL CONSTRAINTS IN PROTEIN ENGINEERING - THE COENZYME SPECIFICITY OF ESCHERICHIA-COLI ISOCITRATE DEHYDROGENASE
Autore:
CHEN RD; GREER AF; DEAN AM;
Indirizzi:
FINCH UNIV HLTH SCI CHICAGO MED SCH,DEPT BIOL CHEM,3333 GREEN BAY RD N CHICAGO IL 60064 FINCH UNIV HLTH SCI CHICAGO MED SCH,DEPT BIOL CHEM N CHICAGO IL 60064 UNIV SASKATCHEWAN,COLL MED,DEPT BIOCHEM SASKATOON SK S7N 0W0 CANADA
Titolo Testata:
European journal of biochemistry
fascicolo: 2, volume: 250, anno: 1997,
pagine: 578 - 582
SICI:
0014-2956(1997)250:2<578:SCIPE->2.0.ZU;2-0
Fonte:
ISI
Lingua:
ENG
Soggetto:
3-ISOPROPYLMALATE DEHYDROGENASE; THERMUS-THERMOPHILUS; COFACTOR SPECIFICITY; ACTIVE-SITE; ENZYME; MECHANISM; PHOSPHORYLATION; DETERMINANTS; MUTAGENESIS; RESOLUTION;
Keywords:
PROTEIN ENGINEERING; MOLECULAR RECOGNITION; COENZYME SPECIFICITY; ISOCITRATE DEHYDROGENASE; ISOPROPYLMALATE DEHYDROGENASE;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
25
Recensione:
Indirizzi per estratti:
Citazione:
R.D. Chen et al., "STRUCTURAL CONSTRAINTS IN PROTEIN ENGINEERING - THE COENZYME SPECIFICITY OF ESCHERICHIA-COLI ISOCITRATE DEHYDROGENASE", European journal of biochemistry, 250(2), 1997, pp. 578-582

Abstract

In a previous study we reported on the successful inversion of coenzyme specificity in isocitrate dehydrogenase (IDH) from NADP to NAD [Chen, R., Greer, A. & Dean, A, M. (1995) A highly active decarboxylating dehydrogenase with rationally inverted coenzyme specificity, Proc. Natl Acad. Sci. USA 92, 11666-11670]. Here, we explore alternative means to generate NAD dependence in the NADP-dependent scaffold of Escherichia coli IDH. The results reveal that engineering a preference for NAD is constrained by the architecture of the IDH coenzyme binding pocket and confirms that the substituted Asp344 in the engineered enzyme is the major determinant of coenzyme specificity. Mutations in the 316-325loop, which forms part of the coenzyme binding site, reduce activity through transmission of long-range conformational changes into the active site some 14 Angstrom distant. Conformational changes seen upon substituting Cys332-->Tyr are not directly involved with improving activity, Replacements at Cys201 reveal that subtle changes in the packing of hydrophobic residues (Met and Ile versus Leu) can elicit markedly different responses. We caution against using sequence alignments as the sole guide for mutagenesis and show how a combination of rational design of active-site residues based on X-ray structures and random substitutions at surrounding residues provides an efficient means to improve enzyme preference and catalytic efficiency towards novel substrates.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 27/10/20 alle ore 05:15:49