Structural constraints in protein engineering - The coenzyme specificity of Escherichia coli isocitrate dehydrogenase

被引:20
作者
Chen, RD
Greer, AF
Dean, AM
机构
[1] FINCH UNIV HLTH SCI CHICAGO MED SCH,DEPT BIOL CHEM,N CHICAGO,IL 60064
[2] UNIV SASKATCHEWAN,COLL MED,DEPT BIOCHEM,SASKATOON,SK S7N 0W0,CANADA
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 1997年 / 250卷 / 02期
关键词
protein engineering; molecular recognition; coenzyme specificity; isocitrate dehydrogenase; isopropylmalate dehydrogenase;
D O I
10.1111/j.1432-1033.1997.0578a.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 316-325 loop, 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.
引用
收藏
页码:578 / 582
页数:5
相关论文
共 25 条
[1]   YEAST DIPHOSPHOPYRIDINE NUCLEOTIDE SPECIFIC ISOCITRATE DEHYDROGENASE - PURIFICATION AND SOME PROPERTIES [J].
BARNES, LD ;
KUEHN, GD ;
ATKINSON, DE .
BIOCHEMISTRY, 1971, 10 (21) :3939-+
[2]   MUTAGENESIS AND LAUE STRUCTURES OF ENZYME INTERMEDIATES - ISOCITRATE DEHYDROGENASE [J].
BOLDUC, JM ;
DYER, DH ;
SCOTT, WG ;
SINGER, P ;
SWEET, RM ;
KOSHLAND, DE ;
STODDARD, BL .
SCIENCE, 1995, 268 (5215) :1312-1318
[3]   A HIGHLY-ACTIVE DECARBOXYLATING DEHYDROGENASE WITH RATIONALLY INVERTED COENZYME SPECIFICITY [J].
CHEN, RD ;
GREER, A ;
DEAN, AM .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1995, 92 (25) :11666-11670
[4]   KINETIC-ANALYSIS OF NAD+-ISOCITRATE DEHYDROGENASE WITH ALTERED ISOCITRATE BINDING-SITES - CONTRIBUTION OF IDH1 AND IDH2 SUBUNITS TO REGULATION AND CATALYSIS [J].
CUPP, JR ;
MCALISTERHENN, L .
BIOCHEMISTRY, 1993, 32 (36) :9323-9328
[5]   ELECTROSTATIC AND STERIC CONTRIBUTIONS TO REGULATION AT THE ACTIVE-SITE OF ISOCITRATE DEHYDROGENASE [J].
DEAN, AM ;
KOSHLAND, DE .
SCIENCE, 1990, 249 (4972) :1044-1046
[6]  
Dean AM, 1996, PROTEIN SCI, V5, P341
[7]   KINETIC MECHANISM OF ESCHERICHIA-COLI ISOCITRATE DEHYDROGENASE [J].
DEAN, AM ;
KOSHLAND, DE .
BIOCHEMISTRY, 1993, 32 (36) :9302-9309
[8]  
ERHLICH RS, 1987, BIOCHEMISTRY-US, V26, P3461
[9]   ISOTOPE EFFECT STUDIES OF THE CHEMICAL MECHANISM OF PIG-HEART NADP ISOCITRATE DEHYDROGENASE [J].
GRISSOM, CB ;
CLELAND, WW .
BIOCHEMISTRY, 1988, 27 (08) :2934-2943
[10]   STRUCTURE OF A BACTERIAL ENZYME REGULATED BY PHOSPHORYLATION, ISOCITRATE DEHYDROGENASE [J].
HURLEY, JH ;
THORSNESS, PE ;
RAMALINGAM, V ;
HELMERS, NH ;
KOSHLAND, DE ;
STROUD, RM .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1989, 86 (22) :8635-8639