Cleavage of the moaX-encoded fused molybdopterin synthase from Mycobacterium tuberculosis is necessary for activity

BMC Microbiology

Bavesh D Kana¹  Valerie Mizrahi²  Edith Erika Machowski¹  Nicole C Narrandes¹ 

[1]DST/NRF Centre of Excellence for Biomedical TB Research, Faculty of Health Sciences, University of the Witwatersrand, National Health Laboratory Service, Johannesburg 2000, South Africa

[2]MRC/NHLS/UCT Molecular Mycobacteriology Research Unit and DST/NRF Centre of Excellence for Biomedical TB Research, Institute of Infectious Disease & Molecular Medicine and Division of Medical Microbiology, University of Cape Town, Cape Town, South Africa

关键词: Nitrate reductase;    MoaE;    MoaD;    MoaX;    mycobacteria;    Molybdopterin synthase;    MoCo biosynthesis;   

Others  :  1137440
DOI  :  10.1186/s12866-015-0355-2

 received in 2014-11-14, accepted in 2015-01-19,  发布年份 2015

【 摘 要 】

Background

Molybdopterin cofactor (MoCo) biosynthesis in Mycobacterium tuberculosis is associated with a multiplicity of genes encoding several enzymes in the pathway, including the molybdopterin (MPT) synthase, a hetero tetramer comprising two MoaD and two MoaE subunits. In addition to moaD1, moaD2, moaE1, moaE2, the M. tuberculosis genome also contains a moaX gene which encodes an MPT-synthase in which the MoaD and MoaE domains are located on a single polypeptide. In this study, we assessed the requirement for post-translational cleavage of MoaX for functionality of this novel, fused MPT synthase and attempted to establish a functional hierarchy for the various MPT-synthase encoding genes in M. tuberculosis.

Results

Using a heterologous Mycobacterium smegmatis host and the activity of the MoCo-dependent nitrate reductase, we confirmed that moaD2 and moaE2 from M. tuberculosis together encode a functional MPT synthase. In contrast, moaD1 displayed no functionality in this system, even in the presence of the MoeBR sulphurtransferase, which contains the rhodansese-like domain, predicted to activate MoaD subunits. We demonstrated that cleavage of MoaX into its constituent MoaD and MoaE subunits was required for MPT synthase activity and confirmed that cleavage occurs between the Gly82 and Ser83 residues in MoaX. Further analysis of the Gly81-Gly82 motif confirmed that both of these residues are necessary for catalysis and that the Gly81 was required for recognition/cleavage of MoaX by an as yet unidentified protease. In addition, the MoaE component of MoaX was able to function in conjunction with M. smegmatis MoaD2 suggesting that cleavage of MoaX renders functionally interchangeable subunits. Expression of MoaX in E. coli revealed that incorrect post-translational processing is responsible for the lack of activity of MoaX in this heterologous host.

Conclusions

There is a degree of functional interchangeability between the MPT synthase subunits of M. tuberculosis. In the case of MoaX, post-translational cleavage at the Gly82 residue is required for function.

【 授权许可】

   
2015 Narrandes et al.; licensee BioMed Central.

【 图 表 】

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【 参考文献 】

• [1]Dutta NK, Karakousis PC: Latent Tuberculosis Infection: Myths, Models, and Molecular Mechanisms. Microbiol Mol Biol Rev 2014, 78(3):343-71.
• [2]Berney M, Cook GM: Unique flexibility in energy metabolism allows mycobacteria to combat starvation and hypoxia. PLoS One 2010, 5(1):e8614.
• [3]Wang J, Behr MA: Building a better bacillus: the emergence of Mycobacterium tuberculosis. Front Microbiol 2014, 5:139.
• [4]Williams M, Mizrahi V, Kana BD: Molybdenum cofactor: A key component of Mycobacterium tuberculosis pathogenesis? Crit Rev Microbiol 2014, 40(1):18-29.
• [5]Pitterle DM, Rajagopalan KV: The biosynthesis of molybdopterin in Escherichia coli. Purification and characterization of the converting factor. J Biol Chem 1993, 268(18):13499-505.
• [6]Mendel RR: The molybdenum cofactor. J Biol Chem 2013, 288(19):13165-72.
• [7]Schwarz G, Mendel RR, Ribbe MW: Molybdenum cofactors, enzymes and pathways. Nature 2009, 460(7257):839-47.
• [8]Iobbi-Nivol C, Leimkuhler S: Molybdenum enzymes, their maturation and molybdenum cofactor biosynthesis in Escherichia coli. Biochim Biophys Acta 2013, 1827(8–9):1086-101.
• [9]McGuire AM, Weiner B, Park ST, Wapinski I, Raman S, Dolganov G, et al.: Comparative analysis of Mycobacterium and related Actinomycetes yields insight into the evolution of Mycobacterium tuberculosis pathogenesis. BMC Genomics 2012, 13(1):120. BioMed Central Full Text
• [10]Shi T, Xie J: Molybdenum enzymes and molybdenum cofactor in mycobacteria. J Cell Biochem 2012, 112(10):2721-8.
• [11]Rudolph MJ, Wuebbens MM, Rajagopalan KV, Schindelin H: Crystal structure of molybdopterin synthase and its evolutionary relationship to ubiquitin activation. Nat Struct Biol 2001, 8(1):42-6.
• [12]Mendel RR, Bittner F: Cell biology of molybdenum. Biochim Biophys Acta 2006, 1763(7):621-35.
• [13]Gn S, Mendel RR: Molybdenum cofactor biosynthesis and molybdenum enzymes. Annu Rev Plant Biol 2006, 57:623-47.
• [14]Leimkuhler S, Wuebbens MM, Rajagopalan KV: Characterization of Escherichia coli MoeB and its involvement in the activation of molybdopterin synthase for the biosynthesis of the molybdenum cofactor. J Biol Chem 2001, 276(37):34695-701.
• [15]Gutzke G, Fischer B, Mendel RR, Schwarz G: Thiocarboxylation of molybdopterin synthase provides evidence for the mechanism of dithiolene formation in metal-binding pterins. J Biol Chem 2001, 276(39):36268-74.
• [16]Lake MW, Wuebbens MM, Rajagopalan KV, Schindelin H: Mechanism of ubiquitin activation revealed by the structure of a bacterial MoeB-MoaD complex. Nature 2001, 414(6861):325-9.
• [17]Zhang W, Urban A, Mihara H, Leimkuhler S, Kurihara T, Esaki N: IscS functions as a primary sulfur-donating enzyme by interacting specifically with MoeB and MoaD in the biosynthesis of molybdopterin in Escherichia coli. J Biol Chem 2010, 285(4):2302-8.
• [18]Marquet A: Enzymology of carbon-sulfur bond formation. Curr Opin Chem Biol 2001, 5(5):541-9.
• [19]Dahl JU, Urban A, Bolte A, Sriyabhaya P, Donahue JL, Nimtz M, et al.: The identification of a novel protein involved in molybdenum cofactor biosynthesis in Escherichia coli. J Biol Chem 2011, 286(41):35801-12.
• [20]Cole ST, Brosch R, Parkhill J, Garnier T, Churcher C, Harris D, et al.: Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 1998, 393(6685):537-44.
• [21]Williams MJ, Kana BD, Mizrahi V: Functional analysis of molybdopterin biosynthesis in mycobacteria identifies a fused molybdopterin synthase in Mycobacterium tuberculosis. J Bacteriol 2011, 193(1):98-106.
• [22]Rudolph MJ, Wuebbens MM, Turque O, Rajagopalan KV, Schindelin H: Structural studies of molybdopterin synthase provide insights into its catalytic mechanism. J Biol Chem 2003, 278(16):14514-22.
• [23]Schmitz J, Wuebbens MM, Rajagopalan KV, Leimkuhler S: Role of the C-terminal Gly-Gly motif of Escherichia coli MoaD, a molybdenum cofactor biosynthesis protein with a ubiquitin fold. Biochemistry 2007, 46(3):909-16.
• [24]Voss M, Nimtz M, Leimkuhler S: Elucidation of the dual role of Mycobacterial MoeZR in molybdenum cofactor biosynthesis and cysteine biosynthesis. PLoS One 2011, 6(11):e28170.
• [25]Malm S, Tiffert Y, Micklinghoff J, Schultze S, Joost I, Weber I, et al.: The roles of the nitrate reductase NarGHJI, the nitrite reductase NirBD and the response regulator GlnR in nitrate assimilation of Mycobacterium tuberculosis. Microbiology 2009, 155(Pt 4):1332-9.
• [26]Leimkuhler S, Hodson R, George GN, Rajagopalan KV: Recombinant Rhodobacter capsulatus xanthine dehydrogenase, a useful model system for the characterization of protein variants leading to xanthinuria I in humans. J Biol Chem 2003, 278(23):20802-11.
• [27]Stinear TP, Seemann T, Harrison PF, Jenkin GA, Davies JK, Johnson PDR, et al.: Insights from the complete genome sequence of Mycobacterium marinum on the evolution of Mycobacterium tuberculosis. Genome Res 2008, 18(5):729-41.
• [28]Senaratne RH, De Silva AD, Williams SJ, Mougous JD, Reader JR, Zhang T, et al.: 5′-Adenosinephosphosulphate reductase (CysH) protects Mycobacterium tuberculosis against free radicals during chronic infection phase in mice. Mol Microbiol 2006, 59(6):1744-53.
• [29]Mehra S, Kaushal D: Functional genomics reveals extended roles of the Mycobacterium tuberculosis stress response factor sigmaH. J Bacteriol 2009, 191(12):3965-80.
• [30]Leimkuhler S, Rajagopalan KV: A Sulfurtransferase Is Required in the Transfer of Cysteine Sulfur in the in Vitro Synthesis of Molybdopterin from Precursor Z in Escherichia coli. J Biol Chem 2001, 276(25):22024-31.
• [31]Giel JL, Nesbit AD, Mettert EL, Fleischhacker AS, Wanta BT, Kiley PJ: Regulation of iron-sulphur cluster homeostasis through transcriptional control of the Isc pathway by [2Fe-2S]-IscR in Escherichia coli. Mol Microbiol 2012, 87(3):478-92.
• [32]Brodin P, Poquet Y, Levillain F, Peguillet I, Larrouy-Maumus G, Gilleron M, et al.: High content phenotypic cell-based visual screen identifies Mycobacterium tuberculosis acyltrehalose-containing glycolipids involved in phagosome remodeling. PLoS Pathog 2010, 6(9):e1001100.
• [33]Camacho LR, Ensergueix D, Perez E, Gicquel B, Guilhot C: Identification of a virulence gene cluster of Mycobacterium tuberculosis by signature-tagged transposon mutagenesis. Mol Microbiol 1999, 34(2):257-67.
• [34]Dutta NK, Mehra S, Didier PJ, Roy CJ, Doyle LA, Alvarez X, et al.: Genetic requirements for the survival of tubercle bacilli in primates. J Infect Dis 2010, 201(11):1743-52.
• [35]MacGurn JA, Cox JS: A genetic screen for Mycobacterium tuberculosis mutants defective for phagosome maturation arrest identifies components of the ESX-1 secretion system. Infect Immun 2007, 75(6):2668-78.
• [36]Rosas-Magallanes V, Stadthagen-Gomez G, Rauzier J, Barreiro LB, Tailleux L, Boudou F, et al.: Signature-tagged transposon mutagenesis identifies novel Mycobacterium tuberculosis genes involved in the parasitism of human macrophages. Infect Immun 2007, 75(1):504-7.
• [37]Sassetti CM, Boyd DH, Rubin EJ: Genes required for mycobacterial growth defined by high density mutagenesis. Mol Microbiol 2003, 48(1):77-84.
• [38]Hopp TP, Prickett KS, Price VL, Libby RT, March CJ, Cerretti DP, et al.: A short polypeptide marker sequence useful for recombinant protein identification and purification. Biotechnology 1988, 6:7.
• [39]Smith AM, Klugman KP: “Megaprimer” method of PCR-based mutagenesis: the concentration of megaprimer is a critical factor. Biotechniques 1997, 22(3):438. 442