Dataset Information

Facile immobilization of pyridoxal 5'-phosphate using p-diazobenzoyl-derivatized Sepharose 4B.

ABSTRACT: Pyridoxal 5'-phosphate (PLP) is a ubiquitous and versatile cofactor utilized by numerous enzymes involved in amino acid biosynthetic pathways. Immobilized PLP is a valuable tool to isolate unknown PLP-dependent enzymes in nature or to perform in vitro selection or directed evolution on existing or de novo PLP-dependent enzymes. The C-6 position is preferred for covalent immobilization of PLP because it maintains all important functional groups in their native, unmodified form. Previously reported diazonium derivatization methods for C-6 immobilization utilized an azide linker compound that is hazardous and not readily available. Here we report a safer and more accessible method to synthesize p-diazobenzoyl-derivatized Sepharose 4B using the N-hydroxysuccinimide (NHS) ester chemistry. The derivative was used to immobilize PLP, and the resulting C-6 immobilized PLP had a loading of ~2.6 μmol PLP per mL of resin, comparable to commercially available products of other immobilized cofactors.

SUBMITTER: Lee KH

PROVIDER: S-EPMC10735239 | biostudies-literature | 2023 Dec

REPOSITORIES: biostudies-literature

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Facile immobilization of pyridoxal 5'-phosphate using p-diazobenzoyl-derivatized Sepharose 4B.

Lee Kun-Hwa KH Distefano Mark D MD Seelig Burckhard B

Results in chemistry 20230720

Pyridoxal 5'-phosphate (PLP) is a ubiquitous and versatile cofactor utilized by numerous enzymes involved in amino acid biosynthetic pathways. Immobilized PLP is a valuable tool to isolate unknown PLP-dependent enzymes in nature or to perform <i>in vitro</i> selection or directed evolution on existing or <i>de novo</i> PLP-dependent enzymes. The C-6 position is preferred for covalent immobilization of PLP because it maintains all important functional groups in their native, unmodified form. Prev ...[more]

PMID: 38131063

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Project description: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.

Dataset Information

Facile immobilization of pyridoxal 5'-phosphate using p-diazobenzoyl-derivatized Sepharose 4B.

Publications

Facile immobilization of pyridoxal 5'-phosphate using p-diazobenzoyl-derivatized Sepharose 4B.

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