1H, 13C, 15N backbone and side-chain NMR assignments of the C-terminal domain of Mycobacterium Tuberculosis ribosome maturation factor RimM

ABSTRACTCoenzyme F420plays a key role in the redox metabolisms of various archaea and bacteria, includingMycobacterium tuberculosis. InM. tuberculosis, F420-dependent reactions have been linked to several virulence factors. F420carries multiple glutamate residues in the side chain, forming F420-nspecies (n, number of glutamate residues), and the length of this side chain impacts cellular physiology.M. tuberculosisstrains with F420species carrying shorter side chains exhibit resistance to delamanid and pretomanid, two new tuberculosis (TB) drugs. Thus, the process of polyglutamylation of F420is of great interest. It has been known from genetic analysis that in mycobacteria an F420-0 γ-glutamyl ligase (FbiB) introduces up to seven glutamate residues into F420. However, purified FbiB ofM. tuberculosis(MtbFbiB) is either inefficient or incapable of incorporating more than two glutamates. We found that,in vitro,MtbFbiB synthesized side chains containing up to seven glutamate residues if F420was presented to the enzyme in a two-electron reduced state (F420H2). Our genetic analysis inMycobacterium bovisBCG andMycobacterium smegmatisand an analysis of literature data onM. tuberculosisrevealed that in these mycobacteria the polyglutamylation process requires the assistance of F420-dependent glucose-6-phosphate dehydrogenase (Fgd) which reduces F420to F420H2. We hypothesize that, starting with F420-0H2, the amino-terminal domain of FbiB builds F420-2H2, which is then transferred to the carboxy-terminal domain for further glutamylation; F420-2H2modifies the carboxy-terminal domain structurally to accommodate longer glutamyl chains. This system is analogous to folylpolyglutamate synthase, which introduces more than one glutamate residue into folate only after this vitamin is reduced to tetrahydrofolate.IMPORTANCECoenzyme F420-dependent reactions ofMycobacterium tuberculosis, which causes tuberculosis, potentially contributes to the virulence of this bacterium. The coenzyme carries a glutamic acid-derived tail, the length of which influences the metabolism ofM. tuberculosis. Mutations that eliminate the production of F420with longer tails makeM. tuberculosisresistant to two new tuberculosis drugs. This report describes that the synthesis of longer glutamyl tails of F420requires concerted actions of two enzymes, one of which reduces the coenzyme prior to the action of the other, which catalyzes polyglutamylation. This knowledge will help to develop more effective tuberculosis (TB) drugs. Remarkably, the introduction of multiple glutamate residues into the sidechain of folate (vitamin B9) requires similar concerted actions, where one enzyme reduces the vitamin to tetrahydrofolate and the other catalyzes polyglutamylation; folate is required for DNA and amino acid synthesis. Thus, the reported research has also revealed a key similarity between two important cellular systems.

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Backbone and side-chain 1H, 13C, and 15N NMR assignments of the N-terminal domain of Escherichia coli LpoA

Biomolecular NMR Assignments ◽

10.1007/s12104-014-9546-2 ◽

2014 ◽

Vol 9 (1) ◽

pp. 65-69 ◽

Cited By ~ 1

Author(s):

Nicolas L. Jean ◽

Catherine Bougault ◽

Adeline Derouaux ◽

Gilles Callens ◽

Waldemar Vollmer ◽

...

Keyword(s):

Escherichia Coli ◽

Nmr Assignments ◽

Side Chain ◽

15N Nmr ◽

Terminal Domain

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Structural Basis for the C-Terminal Domain of Mycobacterium tuberculosis Ribosome Maturation Factor RimM to Bind Ribosomal Protein S19

Biomolecules ◽

10.3390/biom11040597 ◽

2021 ◽

Vol 11 (4) ◽

pp. 597

Author(s):

Haoran Zhang ◽

Qiuxiang Zhou ◽

Chenyun Guo ◽

Liubin Feng ◽

Huilin Wang ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Ribosomal Protein ◽

Solution Structure ◽

Molecular Mechanisms ◽

Structural Model ◽

Ribosomal Subunit ◽

Structural Basis ◽

Hydrophobic Core ◽

Terminal Domain ◽

Ribosomal Protein S19

Multidrug-resistant tuberculosis (TB) is a serious threat to public health, calling for the development of new anti-TB drugs. Chaperon protein RimM, involved in the assembly of ribosomal protein S19 into 30S ribosomal subunit during ribosome maturation, is a potential drug target for TB treatment. The C-terminal domain (CTD) of RimM is primarily responsible for binding S19. However, both the CTD structure of RimM from Mycobacterium tuberculosis (MtbRimMCTD) and the molecular mechanisms underlying MtbRimMCTD binding S19 remain elusive. Here, we report the solution structure, dynamics features of MtbRimMCTD, and its interaction with S19. MtbRimMCTD has a rigid hydrophobic core comprised of a relatively conservative six-strand β-barrel, tailed with a short α-helix and interspersed with flexible loops. Using several biophysical techniques including surface plasmon resonance (SPR) affinity assays, nuclear magnetic resonance (NMR) assays, and molecular docking, we established a structural model of the MtbRimMCTD–S19 complex and indicated that the β4-β5 loop and two nonconserved key residues (D105 and H129) significantly contributed to the unique pattern of MtbRimMCTD binding S19, which might be implicated in a form of orthogonality for species-dependent RimM–S19 interaction. Our study provides the structural basis for MtbRimMCTD binding S19 and is beneficial to the further exploration of MtbRimM as a potential target for the development of new anti-TB drugs.

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Structural insights into the interaction of helicase and primase in Mycobacterium tuberculosis

Biochemical Journal ◽

10.1042/bcj20180673 ◽

2018 ◽

Vol 475 (21) ◽

pp. 3493-3509 ◽

Cited By ~ 2

Author(s):

Dhakaram Pangeni Sharma ◽

Ramachandran Vijayan ◽

Syed Arif Abdul Rehman ◽

Samudrala Gourinath

Keyword(s):

Mycobacterium Tuberculosis ◽

Replication Initiation ◽

Binding Studies ◽

Functional Conservation ◽

Terminal Domain ◽

Binding Domains ◽

Initiation System ◽

Helical Hairpin ◽

Helicase Dnab ◽

Species Specific

The helicase–primase interaction is an essential event in DNA replication and is mediated by the highly variable C-terminal domain of primase (DnaG) and N-terminal domain of helicase (DnaB). To understand the functional conservation despite the low sequence homology of the DnaB-binding domains of DnaGs of eubacteria, we determined the crystal structure of the helicase-binding domain of DnaG from Mycobacterium tuberculosis (MtDnaG-CTD) and did so to a resolution of 1.58 Å. We observed the overall structure of MtDnaG-CTD to consist of two subdomains, the N-terminal globular region (GR) and the C-terminal helical hairpin region (HHR), connected by a small loop. Despite differences in some of its helices, the globular region was found to have broadly similar arrangements across the species, whereas the helical hairpins showed different orientations. To gain insights into the crucial helicase–primase interaction in M. tuberculosis, a complex was modeled using the MtDnaG-CTD and MtDnaB-NTD crystal structures. Two nonconserved hydrophobic residues (Ile605 and Phe615) of MtDnaG were identified as potential key residues interacting with MtDnaB. Biosensor-binding studies showed a significant decrease in the binding affinity of MtDnaB-NTD with the Ile605Ala mutant of MtDnaG-CTD compared with native MtDnaG-CTD. The loop, connecting the two helices of the HHR, was concluded to be largely responsible for the stability of the DnaB–DnaG complex. Also, MtDnaB-NTD showed micromolar affinity with DnaG-CTDs from Escherichia coli and Helicobacter pylori and unstable binding with DnaG-CTD from Vibrio cholerae. The interacting domains of both DnaG and DnaB demonstrate the species-specific evolution of the replication initiation system.

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Enzymatic β-Oxidation of the Cholesterol Side Chain in Mycobacterium Tuberculosis Bifurcates Stereospecifically at Hydration of 3-Oxo-Cholest-4,22- Dien-24-Oyl-CoA

10.26434/chemrxiv.13728112 ◽

2021 ◽

Author(s):

Tianao Yuan ◽

Joshua Werman ◽

Xingyu Yin ◽

Meng Yang ◽

Miguel Garcia-Diaz ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Fatty Acid ◽

Alcohol Dehydrogenase ◽

Side Chain ◽

Crystallographic Structure ◽

X Ray ◽

Steroid Substrate ◽

Unique Ability ◽

Therapeutics Development ◽

Chain Type

The unique ability of Mycobacterium tuberculosis (Mtb) to utilize host lipids such as cholesterol for survival, persistence, and virulence has made the metabolic pathway of cholesterol an area of great interest for therapeutics development, and bioproduction of valuable sterol intermediates. Herein, we identify and characterize two genes from the <a></a><a>Cho-region of the Mtb genome</a>, chsH3 (Rv3538) and chsB1 (Rv3502c). Their protein products catalyze <a></a><a>two sequential stereospecific</a>hydration and dehydrogenation steps in the b-oxidation of the cholesterol side chain. ChsH3 favors the 22S hydration of 3-oxo-cholest-4,22-dien-24-oyl-CoA in contrast to the previously reported EchA19 (Rv3516) which catalyzes formation of the (22R)-hydroxy-3-oxo-cholest-4-en-24-oyl-CoA from the same enoyl-CoA substrate. ChsB1 is stereospecific and catalyzes dehydrogenation of the ChsH3 product, but not the EchA19 product. The X-ray crystallographic structure of the ChsB1 apo-protein was determined at a resolution of 2.03 Å and the holo-enzyme with bound NAD+ cofactor at 2.21 Å.The homodimeric structure is representative of a classical NAD+ utilizing short-chain type alcohol dehydrogenase/reductase, including a Rossmann-fold motif, but exhibits a unique substrate binding site architecture that is of greater length and width than its homologous counterparts, likely to accommodate the bulky steroid substrate. Intriguingly, Mtb utilizes MaoC-like hydratases in sterol side-chain catabolism in contrast to fatty acid b-oxidation in other species that utilize the evolutionarily distinct crotonase family of hydratases.

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