Identification and analysis of residues contained on β → α loops of the dual‐substrate (βα) 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 (βα) 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 β → α 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 β → α 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 Å herein strongly implicates the occurrence of conformational changes in this β → α 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.