Directed evolution of a (beta alpha)8-barrel enzyme to catalyze related reactions in two different metabolic pathways.

Enzymes participating in different metabolic pathways often have similar catalytic mechanisms and structures, suggesting their evolution from a common ancestral precursor enzyme. We sought to create a precursor-like enzyme for N'-[(5'-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (ProFAR) isomerase (HisA; EC ) and phosphoribosylanthranilate (PRA) isomerase (TrpF; EC ), which catalyze similar reactions in the biosynthesis of the amino acids histidine and tryptophan and have a similar (betaalpha)(8)-barrel structure. Using random mutagenesis and selection, we generated several HisA variants that catalyze the TrpF reaction both in vivo and in vitro, and one of these variants retained significant HisA activity. A more detailed analysis revealed that a single amino acid exchange could establish TrpF activity on the HisA scaffold. These findings suggest that HisA and TrpF may have evolved from an ancestral enzyme of broader substrate specificity and underscore that (betaalpha)(8)-barrel enzymes are very suitable for the design of new catalytic activities.

[1]  B. Hall.  Experimental evolution of Ebg enzyme provides clues about the evolution of catalysis and to evolutionary potential. , 1999, FEMS microbiology letters.

[2]  G. Cohen.  Align : A program to superimpose protein coordinates, accounting for insertions and deletions , 1997 .

[3]  R. Sauer,et al.  Evolution of a protein fold in vitro. , 1999, Science.

[4]  W. P. Schneider,et al.  Procedure for production of hybrid genes and proteins and its use in assessing significance of amino acid differences in homologous tryptophan synthetase alpha polypeptides. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[5]  M. Gerstein,et al.  The relationship between protein structure and function: a comprehensive survey with application to the yeast genome. , 1999, Journal of molecular biology.

[6]  M. Hennig,et al.  Crystal structure at 2.0 A resolution of phosphoribosyl anthranilate isomerase from the hyperthermophile Thermotoga maritima: possible determinants of protein stability. , 1997, Biochemistry.

[7]  Alan R. Fersht,et al.  Directed evolution of new catalytic activity using the α/β-barrel scaffold , 2000, Nature.

[8]  V. Davisson,et al.  Imidazole glycerol phosphate synthase: the glutamine amidotransferase in histidine biosynthesis. , 1993, Biochemistry.

[9]  V. Davisson,et al.  A plasmid-based approach for the synthesis of a histidine biosynthetic intermediate , 1994 .

[10]  F. Arnold,et al.  Directed evolution of biocatalysts. , 1999, Current opinion in chemical biology.

[11]  C. Yanofsky,et al.  Improving the catalytic activity of a thermophilic enzyme at low temperatures. , 2000, Biochemistry.

[12]  G. Sarkar,et al.  The "megaprimer" method of site-directed mutagenesis. , 1990, BioTechniques.

[13]  P C Babbitt,et al.  Mechanistically diverse enzyme superfamilies: the importance of chemistry in the evolution of catalysis. , 1998, Current opinion in chemical biology.

[14]  M. Wilmanns,et al.  Efficient expression, purification and crystallisation of two hyperthermostable enzymes of histidine biosynthesis , 1999, FEBS letters.

[15]  N H Horowitz,et al.  On the Evolution of Biochemical Syntheses. , 1945, Proceedings of the National Academy of Sciences of the United States of America.

[16]  R. Sterner,et al.  (Beta alpha)8‐barrel proteins of tryptophan biosynthesis in the hyperthermophile Thermotoga maritima. , 1995, The EMBO journal.

[17]  Eleanor Lawrence,et al.  The encyclopedia of molecular biology , 1995 .

[18]  R. Jensen.  Enzyme recruitment in evolution of new function. , 1976, Annual review of microbiology.

[19]  G. Petsko.  Enzyme evolution: Design by necessity , 2000, Nature.

[20]  K. Kirschner,et al.  Phosphoribosyl anthranilate isomerase catalyzes a reversible amadori reaction. , 1995, Biochemistry.

[21]  H. Vogel,et al.  Acetylornithinase of Escherichia coli: partial purification and some properties. , 1956, The Journal of biological chemistry.

[22]  P C Babbitt,et al.  Evolution of an enzyme active site: the structure of a new crystal form of muconate lactonizing enzyme compared with mandelate racemase and enolase. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[23]  W. Stemmer.  Rapid evolution of a protein in vitro by DNA shuffling , 1994, Nature.

[24]  T. Jardetzky,et al.  Three-dimensional structure of the bifunctional enzyme N-(5'-phosphoribosyl)anthranilate isomerase-indole-3-glycerol-phosphate synthase from Escherichia coli. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[25]  A. Lustig,et al.  Phosphoribosyl anthranilate isomerase from Thermotoga maritima is an extremely stable and active homodimer , 1996, Protein science : a publication of the Protein Society.

[26]  D. Marshak,et al.  Dimethyl sulfoxide-mediated primer Tm reduction: a method for analyzing the role of renaturation temperature in the polymerase chain reaction. , 1993, Analytical biochemistry.