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

Abstract

A good model to experimentally explore evolutionary hypothesis related to enzyme function is the ancient-like dual-substrate (ba) 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 b fi a 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 b fi a 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 b fi a 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.

Bisubstrate specificity in histidine/tryptophan biosynthesis isomerase from Mycobacterium tuberculosis by active site metamorphosis

“…Our data, however, do not support previous hypotheses on either the potential use of the twofold repeated half-barrel symmetry or general loop flexibility considerations, which present features largely shared by the bisubstrate PriA enzyme and the single-substrate HisA enzyme (12,14). These hypotheses were raised based on available apo PriA structures only, without experiment-based knowledge of the type of conformational changes associated with substrate binding.…”

“…A hisA-like gene, referred to as priA, was shown to encode an enzyme with bisubstrate specificity in Streptomyces coelicolor, rendering a bifunctional his/trp biosynthesis isomerase (11). Although some active site residues from the C-terminal ðβ∕αÞ 8 -barrel face in PriA have been shown to be involved in PriA catalysis (12)(13)(14), it was not known which molecular mechanism renders PriA a bisubstrate enzyme, in the absence of structural insight into the positioning of any of the two substrates within the active site. Here, we have investigated the PriA enzyme from Mycobacterium tuberculosis to resolve this question.…”

Exploring the Structure–Function Loop Adaptability of a (β/α)8-Barrel Enzyme through Loop Swapping and Hinge Variability

“…5a and b). As expected, despite the high structural and sequence similarity between Loop 6 scPriA and Loop 6 tmHisA, the former, which has been related to its PRA isomerase activity, 23,24 showed a larger fraction of functional variants on the ecTrpF scaffold (Fig. 5a).…”

“…22 PriA is structurally similar to HisA, and conformational changes in β/α loops 5 and 6 have been suggested as important for its PRA isomerase activity. 23,24 The loops to be used as replacements were selected with the aim of covering a range of functional, structural and evolutionary relationships with loop 6 of ecTrpF (Loop 6 Wt) ( Table 1 and Fig. 2).…”

“…Michaelis-Menten enzyme kinetics of PRA isomerase activity were determined using reported protocols 1 6 with minor modifications. 24 Each data point represents the average of at least three independent experiments using freshly purified enzyme.…”