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Noda-García, L, Camacho-Zarco, AR, Verdel-Aranda, K, Wright, H, Soberón, X, Fülöp, V and Barona-Gómez, F (2010) Identification and analysis of residues contained on beta --> alpha loops of the dual-substrate (beta alpha)8 phosphoribosyl isomerase A specific for its phosphoribosyl anthranilate isomerase activity. Protein Sci. 19:535-43


A good model to experimentally explore evolutionary hypothesis related to enzyme function is the ancient-like dual-substrate (beta alpha)(8) phosphoribosyl isomerase A (PriA), which takes part in both histidine and tryptophan biosynthesis in Streptomyces coelicolor and related organisms. In this study, we determined the Michaelis-Menten enzyme kinetics for both isomerase activities in wild-type PriA from S. coelicolor and in selected single-residue monofunctional mutants, identified after Escherichia coli in vivo complementation experiments. Structural and functional analyses of a hitherto unnoticed residue contained on the functionally important beta --> alpha loop 5, namely, Arg(139), which was postulated on structural grounds to be important for the dual-substrate specificity of PriA, is presented for the first time. Indeed, enzyme kinetics analyses done on the mutant variants PriA_Ser(81)Thr and PriA_Arg(139)Asn showed that these residues, which are contained on beta --> alpha loops and in close proximity to the N-terminal phosphate-binding site, are essential solely for the phosphoribosyl anthranilate isomerase activity of PriA. Moreover, analysis of the X-ray crystallographic structure of PriA_Arg(139)Asn elucidated at 1.95 A herein strongly implicates the occurrence of conformational changes in this beta --> alpha loop as a major structural feature related to the evolution of the dual-substrate specificity of PriA. It is suggested that PriA has evolved by tuning a fine energetic balance that allows the sufficient degree of structural flexibility needed for accommodating two topologically dissimilar substrates--within a bifunctional and thus highly constrained active site--without compromising its structural stability.


PubMed PMC2866278 Online version:10.1002/pro.331


Aldose-Ketose Isomerases/chemistry; Aldose-Ketose Isomerases/genetics; Amino Acid Sequence; Arginine/chemistry; Asparagine/chemistry; Catalytic Domain; Crystallography, X-Ray; Evolution, Molecular; Kinetics; Protein Conformation; Protein Structure, Secondary; Sequence Analysis, Protein; Serine/chemistry; Streptomyces coelicolor/enzymology; Threonine/chemistry


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