Conversion of Aminodeoxychorismate Synthase into Anthranilate Synthase with Janus Mutations: Mechanism of Pyruvate Elimination Catalyzed by Chorismate Enzymes

Abstract

The central importance of chorismate enzymes in bacteria, fungi, parasites, and plants combined with their absence in mammals makes them attractive targets for antimicrobials and herbicides. Two of these enzymes, anthranilate synthase (AS) and aminodeoxychorismate synthase (ADCS), are structurally and mechanistically similar. The first catalytic step, amination at C2, is common between them, but AS additionally catalyzes pyruvate elimination, aromatizing the aminated intermediate to anthranilate. Despite prior attempts, the conversion of a pyruvate elimination-deficient enzyme into an elimination-proficient one has not been reported. Janus, a bioinformatics method for predicting mutations required to functionally interconvert homologous enzymes, was employed to predict mutations to convert ADCS into AS. A genetic selection on a library of Janus-predicted mutations was performed. Complementation of an AS-deficient strain of Escherichia coli grown on minimal medium led to several ADCS mutants that allow growth in 6 days compared to 2 days for wild-type AS. The purified mutant enzymes catalyze the conversion of chorismate to anthranilate at rates that are ∼50% of the rate of wild-type ADCS-catalyzed conversion of chorismate to aminodeoxychorismate. The residues mutated do not contact the substrate. Molecular dynamics studies suggest that pyruvate elimination is controlled by the conformation of the C2-aminated intermediate. Enzymes that catalyze elimination favor the equatorial conformation, which presents the C2-H to a conserved active site lysine (Lys424) for deprotonation and maximizes stereoelectronic activation. Acid/base catalysis of pyruvate elimination was confirmed in AS and salicylate synthase by showing incorporation of a solvent-derived proton into the pyruvate methyl group and by solvent kinetic isotope effects on pyruvate elimination catalyzed by AS.

Breaking a pathogen's iron will: Inhibiting siderophore production as an antimicrobial strategy

“…Both reactions occur in the same active site. The cleavage reaction has been hypothesized to be a pericyclic reaction [64, 65]; however, there is a recent resurgence for the idea that the elimination reaction is also general acid-general base catalyzed [66]. …”

Conversion of Anthranilate Synthase into Isochorismate Synthase: Implications for the Evolution of Chorismate‐Utilizing Enzymes

“…[12] Therefore,the most plausible explanation for the lack of pyruvate-elimination activity in most ICS-active stAS variants is perturbations in the IC ring conformation caused by the introduced mutations.…”

“…It is worth noting that only four of the ICS-active stAS variants also formed SA, which involves the elimination of pyruvate from IC.T his finding seems counterintuitive at first because the generic product of stAS is AA, which has,l ike SA, undergone elimination of pyruvate.H owever,i t was recently shown that the elimination of pyruvate from chorismate-derived reaction intermediates in MST enzymes is controlled by the conformation of the chorismate ring structure. [12] Therefore,the most plausible explanation for the lack of pyruvate-elimination activity in most ICS-active stAS variants is perturbations in the IC ring conformation caused by the introduced mutations.…”

Ammonium Acetate Supplement Strategy for Enhancement of Chaetominine Production in Liquid Culture of Marine-Derived Aspergillus fumigatus CY018

“…Although the detailed polymerization of alanine, anthranilic acid and tryptophan is unclear, the early steps of CHA biosynthetic pathway were verified as involving the shikimate pathway in our previous work [16]. There are some key sites for control in the shikimate pathway, such as 3-deoxy-arabino-heptulosonate-7-phosphate synthase (DAHP, dahp), chorismate synthase (cs) and anthranilate synthase (trpC) [17][18][19]. In addition, laeA, a global regulator, plays an important regulation role in the biosynthesis of secondary metabolites, such as the effect of deletion/overexpression of laeA in Aspergillus app.…”