Mechanism-based Inactivation by Aromatization of the Transaminase BioA Involved in Biotin Biosynthesis in Mycobaterium tuberculosis

Abstract

BioA catalyzes the second step of biotin biosynthesis and this enzyme represents a potential target to develop new antitubercular agents. Herein we report the design, synthesis, and biochemical characterization of a mechanism-based inhibitor (1) featuring a 3,6-dihydropyrid-2-one heterocycle that covalently modifies the pyridoxal 5′-phosphate (PLP) cofactor of BioA through aromatization. The structure of the PLP adduct was confirmed by MS/MS and X-ray crystallography at 1.94 Å resolution. Inactivation of BioA by 1 was time- and concentration-dependent and protected by substrate. We used a conditional knock-down mutant of M. tuberculosis to demonstrate the antitubercular activity of 1 correlated with BioA expression and these results provide support for the designed mechanism of action.

Genetic Approaches to Facilitate Antibacterial Drug Development

“…Underexpression of gyrase subunit A (GyrA, target of quinolones [Sugino et al 1977], Alr [Caceres et al 1997 (Duckworth et al 2011;Kim et al 2010;Wei et al 2011). Similar observations were made with Mtb, in which partial genetic inactivation of the biotin synthesis enzyme BioA or DHFR increased its susceptibility toward the novel inhibitors of these enzymes (Shi et al 2011;Kumar et al 2012). For many of these mutants, it was also shown that they were most strongly sensitized to inhibitors of the partially depleted protein.…”

Inhibition of Mycobacterium tuberculosis Transaminase BioA by Aryl Hydrazines and Hydrazides

“…Ser125, Asp254, Lys-283 and Thr-318’ all make specific interactions with the PLP, are strictly conserved in all Class I transaminases, [24] and adopt a conserved conformation in all BioA structures reported to date. While residues 25–33 are disordered in the original BioA structure reported by Dey et al (PDB-id 3BV0), [7] Tyr25 is well-ordered in the structure we reported as a “pre-reaction” conformation in our study of an irreversible inhibitor of BioA, [11] and the side chain hydroxyl of the tyrosine lies H-bonded to Asp-160 and poised to interact with substrates. This structure (PDB-id 3TFT) will stand to represent the holo enzyme (PLP-bound resting state) conformation in the remainder of this discussion.…”

“…There is no obvious difference between the two active sites that may readily explain this, but we can relate the empirical observation that we have frequently seen differences in the sensitivity toward binding of small molecules in the two “equivalent” positions in this crystal form, [7] even with respect to the covalent adduct we have previously described. [11] The two binding environments are not identical, and they do not always behave so.…”

“…Mtb BioA has been structurally characterized with bound PLP co-factor (PDB-id 3BV0, 3TFT). [7, 11] Based on a comparison of a H315R mutated Mtb BioA complex with the unreactive SAM analog sinefungin (PDB-id 3LV2) [7] and an E. coli BioA complex with KAPA (PDB-id 1QJ3), [12] Sacchettini and co-workers have proposed that Mtb BioA catalyzes the transamination by an induced fit mechanism that relies upon active site conformational changes that accommodate structurally different substrates. [7] This necessary capacity for change in the active site conformation makes it challenging to design Mtb BioA inhibitors and predict ligand binding using computational modeling methods, particularly based on homology models derived from structures from other species.…”

Targeting protein biotinylation enhances tuberculosis chemotherapy

“…We previously reported the design and characterization of potent inhibitors of biotin protein ligase (BPL), the enzyme responsible for covalently ligating biotin onto the ACCs (12)(13)(14)(15)(16)(17)(18)(19). This led to 5′-[N-(d-biotinoyl)sulfamoyl]amino-5′-deoxyadenosine (Bio-AMS), a BPL inhibitor with potent on-target whole-cell activity against drugsensitive and drug-resistant Mtb (17).…”