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.
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.
Noda-García
Lianet
L
Evolution of Metabolic Diversity Laboratory, Laboratorio Nacional de Genómica para la Biodiversidad (Langebio), CINVESTAV-IPN, Km 9.6 Libramiento Norte, Carretera Irapuato-León, Irapuato, C.P. 36822, México.
Camacho-Zarco
Aldo R
AR
Verdel-Aranda
Karina
K
Wright
Helena
H
Soberón
Xavier
X
Fülöp
Vilmos
V
Barona-Gómez
Francisco
F
D019747:Aldose-Ketose Isomerases / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry; D001216:Asparagine / Q000737:chemistry; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D019143:Evolution, Molecular; D007700:Kinetics; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D020539:Sequence Analysis, Protein; D012694:Serine / Q000737:chemistry; D048372:Streptomyces coelicolor / Q000201:enzymology*; D013912:Threonine / Q000737:chemistry
D013912:Threonine; D012694:Serine; D001216:Asparagine; D001120:Arginine; D019747:Aldose-Ketose Isomerases; C029865:phosphoribosylanthranilate isomerase