Allosteric regulation of menaquinone (vitamin K2) biosynthesis in the human pathogen Mycobacterium tuberculosis

Stephanie S. Dawes,Ghader Bashiri,Ehab N.M. Jirgis,Jodie M. Johnston,Esther M.M. Bulloch,Ngoc Anh Thu Ho,Tamsyn Stanborough,Laura V. Nigon,Edward N. Baker

doi:10.1074/jbc.ra119.012158

Stephanie S. Dawes, Ghader Bashiri + Show 7 more

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https://doi.org/10.1074/jbc.ra119.012158

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Abstract

Menaquinone (vitamin K2) plays a vital role in energy generation and environmental adaptation in many bacteria, including the human pathogen Mycobacterium tuberculosis (Mtb). Although menaquinone levels are known to be tightly linked to the cellular redox/energy status of the cell, the regulatory mechanisms underpinning this phenomenon are unclear. The first committed step in menaquinone biosynthesis is catalyzed by MenD, a thiamine diphosphate-dependent enzyme comprising three domains. Domains I and III form the MenD active site, but no function has yet been ascribed to domain II. Here, we show that the last cytosolic metabolite in the menaquinone biosynthesis pathway, 1,4-dihydroxy-2-naphthoic acid (DHNA), binds to domain II of Mtb-MenD and inhibits its activity. Using X-ray crystallography of four apo- and cofactor-bound Mtb-MenD structures, along with several spectroscopy assays, we identified three arginine residues (Arg-97, Arg-277, and Arg-303) that are important for both enzyme activity and the feedback inhibition by DHNA. Among these residues, Arg-277 appeared to be particularly important for signal propagation from the allosteric site to the active site. This is the first evidence of feedback regulation of the menaquinone biosynthesis pathway in bacteria, identifying a protein-level regulatory mechanism that controls menaquinone levels within the cell and may therefore represent a good target for disrupting menaquinone biosynthesis in M. tuberculosis.

Highlights

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis in humans, is able to adopt a persistent phenotype, resulting in long treatment times and a hard-toeradicate latent infection [1]
Various roles have been suggested for domain II in other pyruvate oxidase (POX)-family enzymes; for example, substrate and substrate-like allosteric activators bind in domain II of pyruvate decarboxylase (PDC) and phenylpyruvate decarboxylase [11, 12, 14,15,16,17,18], and nucleotides bind in other enzymes (e.g. FAD in POX and ADP in oxalyl CoA decarboxylase) [11, 19,20,21]
Mtb-MenD is situated at a key step in MK biosynthesis, the first committed step (Figure 1A/Bacillus subtilis (Bs)) [28, 29] and we hypothesized that it might be involved in controlling flux though this pathway

Summary

Introduction

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis in humans, is able to adopt a persistent phenotype, resulting in long treatment times and a hard-toeradicate latent infection [1]. To combat this latent state, there has been a growing interest in menaquinone (vitamin K2, MK), a small redox molecule that is essential for energy generation in both actively growing and persistent Mtb [2]. Like other members of the ThDP-dependent pyruvate oxidase (POX) family, which are dimers or tetramers (comprising two interfacing dimers), MenD is tetrameric, with each monomer comprising three domains [11, 12]. The function of domain II in MenD remains unexplored

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