新金分枝杆菌3β-脱氢酶(3β-HSD)的多基因控制机制
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摘要
目的分枝杆菌中类固醇3β位脱氢酶(3β-HSD)及其同工酶是植物甾醇降解过程中的关键限速步骤,本文作者在基因水平上确证其组成和生物学功能。方法以结核分枝杆菌中功能明确的Rv1106c基因序列为模板在新金分枝杆菌基因组中进行序列比对,对基因片段进行体外表达验证其活性,再通过基因敲除验证其在类固醇代谢中的生理功能。结果在新金分枝杆菌基因组中筛选获得4条类固醇3位脱氢酶基因同源序列,具有3β羟基氧化功能,敲除突变菌株对植物甾醇代谢只减慢约10%。结论证实了新金分枝杆菌中有多个3β位脱氢酶基因参与类固醇的代谢。
Objective 3β-hydroxyl steroid dehydrogenases and isoenzymes(3β-HSD/isomerases)are the key rate-limiting steps in the degradation of phytosterols in mycobacteria.To confirm their compositions and biological functions at the genetic level.Methods The function of Rv1106c gene in Mycobacterium tuberculosis H37 Rv had been verified and was used as reference sequence to identify putative 3β-hsd genes in Mycobacterium neoaurumDSM44074 genome.Four putative genes were identified and expressed heterologously for 3β-hsd activity.Their physiological roles as 3β-hydroxyl dehydrogenasein phytosterol metabolism were verified by gene knockout.Results Four functional 3β-hydroxyl dehydrogenase genes were discovered in Mycobacterium neoaurumDSM44074.The metabolism level of phytosterols in the knockout mutant of all these four genes was reduced by 10%.Conclusion Besides choM1,choM2 and the four hsd genes,there are more genes involved in 3β-hydroxyl steroid dehydrogenation in Mycobacterium neoaurumDSM44074.
Objective 3β-hydroxyl steroid dehydrogenases and isoenzymes(3β-HSD/isomerases)are the key rate-limiting steps in the degradation of phytosterols in mycobacteria.To confirm their compositions and biological functions at the genetic level.Methods The function of Rv1106c gene in Mycobacterium tuberculosis H37 Rv had been verified and was used as reference sequence to identify putative 3β-hsd genes in Mycobacterium neoaurumDSM44074 genome.Four putative genes were identified and expressed heterologously for 3β-hsd activity.Their physiological roles as 3β-hydroxyl dehydrogenasein phytosterol metabolism were verified by gene knockout.Results Four functional 3β-hydroxyl dehydrogenase genes were discovered in Mycobacterium neoaurumDSM44074.The metabolism level of phytosterols in the knockout mutant of all these four genes was reduced by 10%.Conclusion Besides choM1,choM2 and the four hsd genes,there are more genes involved in 3β-hydroxyl steroid dehydrogenation in Mycobacterium neoaurumDSM44074.
引文
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[11] HORINOUCHI S,ISHIZUKA H,BEPPU T.Cloning,nucleotide sequence,and transcriptional analysis of the NAD(P)-dependent cholesterol dehydrogenase gene from a Nocardia sp.and its hyperexpression in Streptomyces spp.[J].Applide & Environmental Microbiology,1991,57(5):1386-1393.
[12] YANG X,DUBNAU E,SMITH I,et al.Rv1106c from Mycobacterium tuberculosis is a 3β-hydroxysteroid dehydrogenase [J].Biochemistry,2007,46(31):9058-9067.
[13] UHIA I,GALAN B,MORALES V,et al.Initial step in the catabolism of cholesterol by mycobacterium smegmatis mc2155 [J].Environmental Microbiology,2011,13(4):943-959.
[14] YAO K,WANG F Q,ZHANG H C,et al.Identification and engineering of cholesterol oxidases involved in the initial step of sterols catabolism in Mycobacterium neoaurum.[J].Metabolic Engineering,2013,15(1):75-87.
[15] KISIELA M,SKARKA A,EBERT B,et al.Hydroxysteroid dehydrogenases(HSDs)in bacteria-A bioinformatic perspective [J].Journal of Steroid Biochemistry & Molecular Biology,2012,129(1/2):1-46.
[16] WEI W,FAN S,WANG F,et al.A new steroid-transforming strain of mycobacterium neoaurum and cloning of 3-ketosteroid 9α-hydroxylase in NwIB-01[J].Applied Biochemistry and Biotechnology ABAB,2010,162(5):1446-1456.
[2] HANNICH J T,UMEBAYASHI K,RIEZMAN H.Distribution and functions of sterols and sphingolipids [J].Cold Spring Harb Perspect Biol,2011,3(5):725-738.
[3] MERZENDORFER H,ZIMOCH L.Chitin metabolism in insects:structure,function and regulation of chitin synthases and chitinases [J].Journal of Experimental Biology,2003,206(24):4393.
[4] SZENTIRMAI A.Microbial physiology of sidechain degradation of sterols [J].Journal of Industrial Microbiology and Biotechnology,1990,6(2):101-115.
[5] GEIZE R V D,YAM K,HEUSER T,et al.A gene cluster encoding cholesterol catabolism in a soil actinomycete provides insight into Mycobacterium tuberculosis survival in macrophages [J].Proceedings of the National Academy of Sciences,2007,104(6):1947-1952.
[6] KENDALL S L,WITHERS M,SOFFAIR C N,et al.A highly conserved transcriptional repressor controls a large regulon involved in lipid degradation in Mycobacterium smegmatis and Mycobacterium tuberculosis [J].Molecular Microbiology,2007,65(3):684-699.
[7] PETRASMA M,DIJKHUIZEN L,VANDER G R.Rhodococcus rhodochrous DSM 43269 3-ketosteroid 9a-hydroxylase,a two-component iron-sulfur-containing monooxygenase with subtle steroid substrate specificity[J].Applied and Environmental Microbiology,2009,75(16):5300-5307.
[8]ROSLONIEC K Z,WILBRINK M H,CAPYK J K,et al.Cytochrome P450 125(CYP125)catalyses C26-hydroxylation to initiate sterol side-chain degradation in Rhodococcusjostii RHAL [J].Mol Microbiol,2009,74(5):1031-1043
[9] WILBRINK M H,PETMSMA M,DIJKHUIZEN L,et al.FadD19 of Rhodococcus rhodochrous DSM43269,a steroid-coenzyme a ligase essential for degradation of C-24 branched sterol side Chains[J].Applied and Environmental Microbiology,2011,77(13):4455-4464.
[10] CAPYK J K,ISRAL C,ROBERT G,et al.Activity of 3-ketosteroid 9α-hydroxylase(KshAB)indicates cholesterol side chain and ring degradation occur simultaneously in Mycobacterium tuberculosis[J].Journal of Biological Chemistry,2011,286(47):40717-40724.
[11] HORINOUCHI S,ISHIZUKA H,BEPPU T.Cloning,nucleotide sequence,and transcriptional analysis of the NAD(P)-dependent cholesterol dehydrogenase gene from a Nocardia sp.and its hyperexpression in Streptomyces spp.[J].Applide & Environmental Microbiology,1991,57(5):1386-1393.
[12] YANG X,DUBNAU E,SMITH I,et al.Rv1106c from Mycobacterium tuberculosis is a 3β-hydroxysteroid dehydrogenase [J].Biochemistry,2007,46(31):9058-9067.
[13] UHIA I,GALAN B,MORALES V,et al.Initial step in the catabolism of cholesterol by mycobacterium smegmatis mc2155 [J].Environmental Microbiology,2011,13(4):943-959.
[14] YAO K,WANG F Q,ZHANG H C,et al.Identification and engineering of cholesterol oxidases involved in the initial step of sterols catabolism in Mycobacterium neoaurum.[J].Metabolic Engineering,2013,15(1):75-87.
[15] KISIELA M,SKARKA A,EBERT B,et al.Hydroxysteroid dehydrogenases(HSDs)in bacteria-A bioinformatic perspective [J].Journal of Steroid Biochemistry & Molecular Biology,2012,129(1/2):1-46.
[16] WEI W,FAN S,WANG F,et al.A new steroid-transforming strain of mycobacterium neoaurum and cloning of 3-ketosteroid 9α-hydroxylase in NwIB-01[J].Applied Biochemistry and Biotechnology ABAB,2010,162(5):1446-1456.