Biosynthesis of Isonitrile Lipopeptide Metallophores from Pathogenic Mycobacteria

Overview

Journal Biochemistry

Specialty Biochemistry

Date 2023 Jan 13

PMID 36638317

Authors

Antonio Del Rio Flores

Maanasa Narayanamoorthy

Wenlong Cai

Rui Zhai

Siyue Yang

Yuanbo Shen

Kaushik Seshadri

Kyle De Matias

Zhaoqiang Xue

Wenjun Zhang

Affiliations

Soon will be listed here.

Abstract

Isonitrile lipopeptides (INLPs) are known to be related to the virulence of pathogenic mycobacteria by mediating metal transport, but their biosynthesis remains obscure. In this work, we use in vitro biochemical assays, site-directed mutagenesis, chemical synthesis, and spectroscopy techniques to scrutinize the activity of core enzymes required for INLP biosynthesis in mycobacteria. Compared to environmental , pathogenic employ a similar chemical logic and enzymatic machinery in INLP biosynthesis, differing mainly in the fatty-acyl chain length, which is controlled by multiple enzymes in the pathway. Our in-depth study on the non-heme iron(II) and α-ketoglutarate-dependent dioxygenase for isonitrile generation, including Rv0097 from (), demonstrates that it recognizes a free-standing small molecule substrate, different from the recent hypothesis that a carrier protein is required for Rv0097 in . A key residue in Rv0097 is further identified to dictate the varied fatty-acyl chain length specificity between and .

Citing Articles

Bioinformatic, structural, and biochemical analysis leads to the discovery of novel isonitrilases and decodes their substrate selectivity.

Hostetler T, Chen T, Chang W RSC Chem Biol. 2025; .

PMID: 39944535 PMC: 11811631. DOI: 10.1039/d4cb00304g.

Characterization of the Flavin-Dependent Monooxygenase Involved in the Biosynthesis of the Nocardiosis-Associated Polyketide†.

Del Rio Flores A, Khosla C Biochemistry. 2024; 63(21):2868-2877.

PMID: 39433512 PMC: 11872153. DOI: 10.1021/acs.biochem.4c00480.

Isonitrile biosynthesis by non-heme iron(II)-dependent oxidases/decarboxylases.

Del Rio Flores A, Zhai R, Zhang W Methods Enzymol. 2024; 704:143-172.

PMID: 39300646 PMC: 11424024. DOI: 10.1016/bs.mie.2024.06.002.

Variation in biosynthesis and metal-binding properties of isonitrile-containing peptides produced by Mycobacteria versus Streptomyces.

Chen T, Chen J, Ruszczycky M, Hilovsky D, Hostetler T, Liu X ACS Catal. 2024; 14(7):4975-4983.

PMID: 38895101 PMC: 11185824. DOI: 10.1021/acscatal.4c00645.

Biosynthesis of isonitrile lipopeptides.

Jia K, Sun H, Zhou Y, Zhang W Curr Opin Chem Biol. 2024; 81:102470.

PMID: 38788523 PMC: 11323250. DOI: 10.1016/j.cbpa.2024.102470.

References

Giovannini D, Cappelli G, Jiang L, Castilletti C, Colone A, Serafino A . A new Mycobacterium tuberculosis smooth colony reduces growth inside human macrophages and represses PDIM Operon gene expression. Does an heterogeneous population exist in intracellular mycobacteria?. Microb Pathog. 2012; 53(3-4):135-46. DOI: 10.1016/j.micpath.2012.06.002. View

Martinez S, Hausinger R . Catalytic Mechanisms of Fe(II)- and 2-Oxoglutarate-dependent Oxygenases. J Biol Chem. 2015; 290(34):20702-20711. PMC: 4543632. DOI: 10.1074/jbc.R115.648691. View

Sassetti C, Rubin E . Genetic requirements for mycobacterial survival during infection. Proc Natl Acad Sci U S A. 2003; 100(22):12989-94. PMC: 240732. DOI: 10.1073/pnas.2134250100. View

Chisuga T, Miyanaga A, Kudo F, Eguchi T . Structural analysis of the dual-function thioesterase SAV606 unravels the mechanism of Michael addition of glycine to an α,β-unsaturated thioester. J Biol Chem. 2017; 292(26):10926-10937. PMC: 5491777. DOI: 10.1074/jbc.M117.792549. View

Hotter G, Wards B, Mouat P, Besra G, Gomes J, Singh M . Transposon mutagenesis of Mb0100 at the ppe1-nrp locus in Mycobacterium bovis disrupts phthiocerol dimycocerosate (PDIM) and glycosylphenol-PDIM biosynthesis, producing an avirulent strain with vaccine properties at least equal to those of M. bovis.... J Bacteriol. 2005; 187(7):2267-77. PMC: 1065232. DOI: 10.1128/JB.187.7.2267-2277.2005. View

Rengarajan J, Bloom B, Rubin E . Genome-wide requirements for Mycobacterium tuberculosis adaptation and survival in macrophages. Proc Natl Acad Sci U S A. 2005; 102(23):8327-32. PMC: 1142121. DOI: 10.1073/pnas.0503272102. View

Matthews B, Nicholson H, Becktel W . Enhanced protein thermostability from site-directed mutations that decrease the entropy of unfolding. Proc Natl Acad Sci U S A. 1987; 84(19):6663-7. PMC: 299143. DOI: 10.1073/pnas.84.19.6663. View

OBrien J, Wright G . An ecological perspective of microbial secondary metabolism. Curr Opin Biotechnol. 2011; 22(4):552-8. DOI: 10.1016/j.copbio.2011.03.010. View

Amagai K, Takaku R, Kudo F, Eguchi T . A unique amino transfer mechanism for constructing the β-amino fatty acid starter unit in the biosynthesis of the macrolactam antibiotic cremimycin. Chembiochem. 2013; 14(15):1998-2006. DOI: 10.1002/cbic.201300370. View

10.

Chen T, Chen J, Tang Y, Zhou J, Guo Y, Chang W . Pathway from N-Alkylglycine to Alkylisonitrile Catalyzed by Iron(II) and 2-Oxoglutarate-Dependent Oxygenases. Angew Chem Int Ed Engl. 2020; 59(19):7367-7371. PMC: 7180123. DOI: 10.1002/anie.201914896. View

11.

Barber C, Zhang W . Small molecule natural products in human nasal/oral microbiota. J Ind Microbiol Biotechnol. 2021; 48(3-4). PMC: 8210680. DOI: 10.1093/jimb/kuab010. View

12.

Cappelli G, Volpe E, Grassi M, Liseo B, Colizzi V, Mariani F . Profiling of Mycobacterium tuberculosis gene expression during human macrophage infection: upregulation of the alternative sigma factor G, a group of transcriptional regulators, and proteins with unknown function. Res Microbiol. 2006; 157(5):445-55. DOI: 10.1016/j.resmic.2005.10.007. View

13.

Pakula A, Sauer R . Genetic analysis of protein stability and function. Annu Rev Genet. 1989; 23:289-310. DOI: 10.1146/annurev.ge.23.120189.001445. View

14.

Chhabra A, Haque A, Pal R, Goyal A, Rai R, Joshi S . Nonprocessive [2 + 2]e- off-loading reductase domains from mycobacterial nonribosomal peptide synthetases. Proc Natl Acad Sci U S A. 2012; 109(15):5681-6. PMC: 3326462. DOI: 10.1073/pnas.1118680109. View

15.

Jonnalagadda R, Del Rio Flores A, Cai W, Mehmood R, Narayanamoorthy M, Ren C . Biochemical and crystallographic investigations into isonitrile formation by a nonheme iron-dependent oxidase/decarboxylase. J Biol Chem. 2020; 296:100231. PMC: 7949033. DOI: 10.1074/jbc.RA120.015932. View

16.

Wang F, Langley R, Gulten G, Wang L, Sacchettini J . Identification of a type III thioesterase reveals the function of an operon crucial for Mtb virulence. Chem Biol. 2007; 14(5):543-51. DOI: 10.1016/j.chembiol.2007.04.005. View

17.

Sassetti C, Boyd D, Rubin E . Genes required for mycobacterial growth defined by high density mutagenesis. Mol Microbiol. 2003; 48(1):77-84. DOI: 10.1046/j.1365-2958.2003.03425.x. View

18.

Winkler R . ESIprot: a universal tool for charge state determination and molecular weight calculation of proteins from electrospray ionization mass spectrometry data. Rapid Commun Mass Spectrom. 2010; 24(3):285-94. DOI: 10.1002/rcm.4384. View

19.

Del Rio Flores A, Kastner D, Du Y, Narayanamoorthy M, Shen Y, Cai W . Probing the Mechanism of Isonitrile Formation by a Non-Heme Iron(II)-Dependent Oxidase/Decarboxylase. J Am Chem Soc. 2022; 144(13):5893-5901. PMC: 8986608. DOI: 10.1021/jacs.1c12891. View

20.

Chen T, Zheng Z, Zhang X, Chen J, Cha L, Tang Y . Deciphering the Reaction Pathway of Mononuclear Iron Enzyme-Catalyzed N≡C Triple Bond Formation in Isocyanide Lipopeptide and Polyketide Biosynthesis. ACS Catal. 2022; 12(4):2270-2279. PMC: 9390461. DOI: 10.1021/acscatal.1c04869. View