Evolving the naturally compromised chorismate mutase from Mycobacterium tuberculosis to top performance

Abstract

Chorismate mutase (CM), an essential enzyme at the branch point of the shikimate pathway, is required for the biosynthesis of phenylalanine and tyrosine in bacteria, archaea, plants, and fungi. MtCM, the CM from Mycobacterium tuberculosis, has less than 1% of the catalytic efficiency of a typical natural CM and requires complex formation with DAHP synthase for high activity. To explore the full potential of MtCM for catalyzing its native reaction, we applied diverse iterative cycles of mutagenesis and selection, thereby raising kcat/Km 270-fold to 5 × 105 M-1s-1, which is even higher than for the complex. Moreover, the evolutionarily optimized autonomous MtCM, which had 11 of its 90 amino acids exchanged, was stabilized compared to its progenitor, as indicated by a 9 °C increase in melting temperature. The 1.5 Å crystal structure of the top-evolved MtCM variant reveals the molecular underpinnings of this activity boost. Some acquired residues, (e.g. Pro52 and Asp55) are conserved in naturally efficient CMs, but most of them lie beyond the active site. Our evolutionary trajectories reached a plateau at the level of the best natural enzymes suggesting that we have exhausted the potential of MtCM. Taken together, these findings show that the scaffold of MtCM, which naturally evolved for mediocrity to enable inter-enzyme allosteric regulation of the shikimate pathway, is inherently capable of high activity.

Investigate Natural Product Indolmycin and the Synthetically Improved Analogue Toward Antimycobacterial Agents

“…M1-1, M2-2, M2-8, and M2-10 genomes encode mutations in an intracellular chorismate mutase (CM). The corresponding mutations S49P, R18H, G84V, and V32G are located within or near the active site in Mtb CM (Rv0948c). , CM catalyzes the conversion of chorismate to prephenate, and the reaction lies at the branch point of the shikimate pathway where biosynthesis of two other aromatic amino acids (Phe and Tyr) splits from Trp biosynthesis. Thus, reduction of CM activity in the mutants may lead to increasing Trp biosynthesis at a sacrifice of Phe/Tyr.…”

What drives chorismate mutase to top performance? Insights from a combinedin silicoandin vitrostudy

“…Nevertheless, pre-organization and pre-stabilization appear to be of crucial importance for the catalytic prowess of MtCM. V55D is the single substitution among the MtCM V mutations that resulted in the largest boost in catalytic activity (12-fold enhancement) (12). This residue is located on the C-terminal side of the H1-H2 loop (Fig.…”

“…This is mainly caused by an increase in kcat rather than an altered substrate affinity. Interestingly, the L88D exchange together with T52P and V55D in the MtCM Triple variant does not lead to a significant increase in kcat/Km compared to MtCM 3p3 (12), which just carries the two loop mutations T52P and V55D.…”

“…The resulting combination of large-impact and more subtle residue substitutions in MtCM V (Fig. 1D-E) gave a kcat/Km about 500 times greater than that of the parental starting point (12).…”

“…Natural CMs belong to two main classes with two distinct folds AroH and AroQ, which are equally efficient, with typical kcat/Km values in the range of 1-5  10 5 M -1 s -1 (12). The AroH fold, exemplified by the Bacillus subtilis CM, has a trimeric pseudo α/β-barrel structure (13,14), whereas the structures of AroQ enzymes have all-α-helical folds (15)(16)(17)(18)(19)(20)(21).…”