Dataset Information

Roles of cysteine in the structure and metabolic function of Mycobacterium tuberculosis CYP142A1.

ABSTRACT: CYP142A1 is a cytochrome P450 (CYP) enzyme expressed in Mycobacterium tuberculosis (Mtb), which supports the growth of Mtb H37Rv relying on cholesterol, in the absence of CYP125A1. Since cysteine residues usually play a fundamental role in maintaining the structure and function of CYP enzymes, in this study, we aimed to determine the potential biochemical functions of six cysteine residues except for the heme-binding cysteine in the amino acid sequence of recombinant Mtb CYP142A1 by replacing each one using site-directed mutagenesis. Recombinant CYP142A1 mutants were heterologously expressed, purified, and analyzed using ESI-MS, far-UV CD spectroscopy, UV-vis spectrophotometric titration, and metabolic function assays. Substitution of the cysteine residues caused various effects on the structure and function of CYP142A1. Separate substitution of the six cysteine residues resulted in numerous changes in the secondary structure, expression level, substrate-binding ability, inhibitor-binding ability, thermal stability and oxidation efficiency of the enzyme. These results contribute to our understanding of the biochemical roles of cysteine residues in the structure and function of Mtb CYP enzymes, especially their effects on the structure and function of CYP142A1.

SUBMITTER: Lu Y

PROVIDER: S-EPMC9425443 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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Roles of cysteine in the structure and metabolic function of Mycobacterium tuberculosis CYP142A1.

Lu Yun Y Sun Lilan L Pang Jing J Li Congran C Wang Xiukun X Hu Xinxin X Li Guoqing G Li Xue X Zhang Youwen Y Wang Hao H Yang Xinyi X You Xuefu X

RSC advances 20220830 38

CYP142A1 is a cytochrome P450 (CYP) enzyme expressed in Mycobacterium tuberculosis (Mtb), which supports the growth of Mtb H37Rv relying on cholesterol, in the absence of CYP125A1. Since cysteine residues usually play a fundamental role in maintaining the structure and function of CYP enzymes, in this study, we aimed to determine the potential biochemical functions of six cysteine residues except for the heme-binding cysteine in the amino acid sequence of recombinant Mtb< ...[more]

PMID: 36128375

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Project description:Accurate and timely functional genome annotation is essential for translating basic pathogen research into clinically impactful advances. Here, through literature curation and structure-function inference, we systematically update the functional genome annotation of Mycobacterium tuberculosis virulent type strain H37Rv. First, we systematically curated annotations for 589 genes from 662 publications, including 282 gene products absent from leading databases. Second, we modeled 1,711 underannotated proteins and developed a semiautomated pipeline that captured shared function between 400 protein models and structural matches of known function on Protein Data Bank, including drug efflux proteins, metabolic enzymes, and virulence factors. In aggregate, these structure- and literature-derived annotations update 940/1,725 underannotated H37Rv genes and generate hundreds of functional hypotheses. Retrospectively applying the annotation to a recent whole-genome transposon mutant screen provided missing function for 48% (13/27) of underannotated genes altering antibiotic efficacy and 33% (23/69) required for persistence during mouse tuberculosis (TB) infection. Prospective application of the protein models enabled us to functionally interpret novel laboratory generated pyrazinamide (PZA)-resistant mutants of unknown function, which implicated the emerging coenzyme A depletion model of PZA action in the mutants' PZA resistance. Our findings demonstrate the functional insight gained by integrating structural modeling and systematic literature curation, even for widely studied microorganisms. Functional annotations and protein structure models are available at https://tuberculosis.sdsu.edu/H37Rv in human- and machine-readable formats. IMPORTANCE Mycobacterium tuberculosis, the primary causative agent of tuberculosis, kills more humans than any other infectious bacterium. Yet 40% of its genome is functionally uncharacterized, leaving much about the genetic basis of its resistance to antibiotics, capacity to withstand host immunity, and basic metabolism yet undiscovered. Irregular literature curation for functional annotation contributes to this gap. We systematically curated functions from literature and structural similarity for over half of poorly characterized genes, expanding the functionally annotated Mycobacterium tuberculosis proteome. Applying this updated annotation to recent in vivo functional screens added functional information to dozens of clinically pertinent proteins described as having unknown function. Integrating the annotations with a prospective functional screen identified new mutants resistant to a first-line TB drug, supporting an emerging hypothesis for its mode of action. These improvements in functional interpretation of clinically informative studies underscore the translational value of this functional knowledge. Structure-derived annotations identify hundreds of high-confidence candidates for mechanisms of antibiotic resistance, virulence factors, and basic metabolism and other functions key in clinical and basic tuberculosis research. More broadly, they provide a systematic framework for improving prokaryotic reference annotations.

Dataset Information

Roles of cysteine in the structure and metabolic function of Mycobacterium tuberculosis CYP142A1.

Publications

Roles of cysteine in the structure and metabolic function of <i>Mycobacterium tuberculosis</i> CYP142A1.

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