Antimicrobial resistance is a significant cause of mortality globally due to infections, a trend that is expected to continue to rise. As existing treatments fail and new drug discovery slows, the urgency to develop novel antimicrobial therapeutics grows stronger. One promising strategy involves targeting bacterial systems exclusive to pathogens, such as the transcription regulator protein GabR. Expressed in diverse bacteria including Escherichia coli, Bordetella pertussis, and Klebsiella pneumoniae, GabR has no homolog in eukaryotes, making it an ideal therapeutic target. Bacillus subtilis GabR (bsGabR), the most studied variant, regulates its own transcription and activates genes for GABA aminotransferase (GabT) and succinic semialdehyde dehydrogenase (GabD). This intricate regulatory system presents a compelling antimicrobial target with the potential for agonistic intervention to disrupt bacterial gene expression and induce cellular dysfunction, especially in bacterial stress responses. To explore manipulation of this system and the potential of this protein as an antimicrobial target, an in-depth understanding of the unique PLP-dependent transcription regulation is critical. Herein, we report the successful structural modification of the cofactor PLP and demonstrate the biochemical reactivity of the PLP analog pyridoxal-5 '-tetrazole (PLT). Through both spectrophotometric and X-ray crystallographic analyses, we explore the interaction between bsGabR and PLT, together with a synthesized GABA derivative (S)-4-amino-5-phenoxypentanoate (4-phenoxymethyl-GABA or 4PMG). Most notably, we present a crystal structure of the condensed, external aldimine complex within bsGabR. While PLT alone is not a drug candidate, it can act as a probe to study the detailed mechanism of GabR-mediated function. PLT employs a tetrazole moiety as a bioisosteric replacement for phosphate in PLP. In addition, the PLP-4PMG adduct observed in the structure may serve as a novel chemical scaffold for subsequent structure-based antimicrobial design.

Dihydropyrimidine dehydrogenase (DPD) is a flavin dependent enzyme that catalyzes the reduction of the 5,6-vinylic bond of pyrimidines uracil and thymine with electrons from NADPH. DPD has two active sites that are separated by ∼60 Å. At one site NADPH binds adjacent to an FAD cofactor and at the other pyrimidine binds proximal to an FMN. Four Fe4S4 centers span the distance between these active sites. It has recently been established that the enzyme undergoes reductive activation prior to reducing the pyrimidine. In this initial process NADPH is oxidized at the FAD site and electrons are transmitted to the FMN via the Fe4S4 centers to yield the active state with a cofactor set of FAD•4(Fe4S4)•FMNH2. The catalytic chemistry of DPD can be studied in transient-state by observation of either NADPH consumption or charge transfer absorption associated with complexation of NADPH adjacent to the FAD. Here we have utilized both sets of absorption transitions to find evidence for specific additional aspects of the DPD mechanism. Competition for binding with NADP+ indicates that the two charge transfer species observed in activation/single turnover reactions arise from NADPH populating the FAD site before and after reductive activation. An additional charge transfer species is observed to accumulate at longer times when high NADPH concentrations are mixed with the enzyme•pyrimidine complex and this data can be modelled based on asymmetry in the homodimer. It was also shown that, like pyrimidines, dihydropyrimidines induce rapid reductive activation indicating that the reduced pyrimidine formed in turnover can stimulate the reinstatement of the active state of the enzyme. Investigation of the reverse reaction revealed that dihydropyrimidines alone can reductively activate the enzyme, albeit inefficiently. In the presence of dihydropyrimidine and NADP+ DPD will form NADPH but apparently without measurable reductive activation. Pyrimidines that have 5-substituent halogens were utilized to probe both reductive activation and turnover. The linearity of the Hammett plot based on the rate of hydride transfer to the pyrimidine establishes that, at least to the radius of an iodo-group, the 5-substituent volume does not have influence on the observed kinetics of pyrimidine reduction.