Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1993 Jun;175(11):3511–3519. doi: 10.1128/jb.175.11.3511-3519.1993

Cloning, sequencing, and expression of the gene encoding methylmalonyl-coenzyme A mutase from Streptomyces cinnamonensis.

A Birch 1, A Leiser 1, J A Robinson 1
PMCID: PMC204751  PMID: 8099072

Abstract

In streptomycetes, the conversion of succinyl-coenzyme A (CoA) into methylmalonyl-CoA, catalyzed by methylmalonyl-CoA mutase, most likely represents an important source of building blocks for polyketide antibiotic biosynthesis. In this work, the structural gene for methylmalonyl-CoA mutase from Streptomyces cinnamonensis was cloned by using a heterologous gene probe encoding the mutase from Propionibacterium shermanii. A 5,732-bp fragment was sequenced, within which four open reading frames were identified on one DNA strand. The two largest (mutA and mutB) overlap by 1 nucleotide and encode proteins of 616 and 733 residues showing high amino acid sequence similarities to each other and to methylmalonyl-CoA mutases from P. shermanii and mammalian sources. The transcriptional start of the mutA-mutB message, determined by S1 mapping, coincides with the first nucleotide of the translational start codon. Evidence that these two open reading frames encode a functional mutase in S. cinnamonensis was obtained by subcloning and expression in Streptomyces lividans TK64. The mutA and mutB gene products were detected in Western blots (immunoblots) with mutase-specific antibodies and by direct detection of mutase activity with a newly developed assay method. The methylmalonyl-CoA mutase was unable to catalyze the conversion of isobutyryl-CoA into n-butyryl-CoA, another closely related adenosylcobalamin-dependent rearrangement known to occur in S. cinnamonensis.

Full text

PDF
3511

Images in this article

3517Image
on p.3517
3517Image
on p.3517

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arrowsmith T. J., Malpartida F., Sherman D. H., Birch A., Hopwood D. A., Robinson J. A. Characterisation of actI-homologous DNA encoding polyketide synthase genes from the monensin producer Streptomyces cinnamonensis. Mol Gen Genet. 1992 Aug;234(2):254–264. doi: 10.1007/BF00283846. [DOI] [PubMed] [Google Scholar]
  2. Ashworth D. M., Holmes D. S., Robinson J. A., Oikawa H., Cane D. E. Selection of a specifically blocked mutant of Streptomyces cinnamonensis: isolation and synthesis of 26-deoxymonensin A. J Antibiot (Tokyo) 1989 Jul;42(7):1088–1099. doi: 10.7164/antibiotics.42.1088. [DOI] [PubMed] [Google Scholar]
  3. Bibb M. J., Cohen S. N. Gene expression in Streptomyces: construction and application of promoter-probe plasmid vectors in Streptomyces lividans. Mol Gen Genet. 1982;187(2):265–277. doi: 10.1007/BF00331128. [DOI] [PubMed] [Google Scholar]
  4. Bibb M. J., Findlay P. R., Johnson M. W. The relationship between base composition and codon usage in bacterial genes and its use for the simple and reliable identification of protein-coding sequences. Gene. 1984 Oct;30(1-3):157–166. doi: 10.1016/0378-1119(84)90116-1. [DOI] [PubMed] [Google Scholar]
  5. Birch A., Häusler A., Vögtli M., Krek W., Hütter R. Extremely large chromosomal deletions are intimately involved in genetic instability and genomic rearrangements in Streptomyces glaucescens. Mol Gen Genet. 1989 Jun;217(2-3):447–458. doi: 10.1007/BF02464916. [DOI] [PubMed] [Google Scholar]
  6. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  7. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Donadio S., Staver M. J., McAlpine J. B., Swanson S. J., Katz L. Modular organization of genes required for complex polyketide biosynthesis. Science. 1991 May 3;252(5006):675–679. doi: 10.1126/science.2024119. [DOI] [PubMed] [Google Scholar]
  9. Feinberg A. P., Vogelstein B. "A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity". Addendum. Anal Biochem. 1984 Feb;137(1):266–267. doi: 10.1016/0003-2697(84)90381-6. [DOI] [PubMed] [Google Scholar]
  10. Geistlich M., Irniger S., Hütter R. Localization and functional analysis of the regulated promoter from the Streptomyces glaucescens mel operon. Mol Microbiol. 1989 Aug;3(8):1061–1069. doi: 10.1111/j.1365-2958.1989.tb00256.x. [DOI] [PubMed] [Google Scholar]
  11. Harms E., Higgins E., Chen J. W., Umbarger H. E. Translational coupling between the ilvD and ilvA genes of Escherichia coli. J Bacteriol. 1988 Oct;170(10):4798–4807. doi: 10.1128/jb.170.10.4798-4807.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hopwood D. A., Sherman D. H. Molecular genetics of polyketides and its comparison to fatty acid biosynthesis. Annu Rev Genet. 1990;24:37–66. doi: 10.1146/annurev.ge.24.120190.000345. [DOI] [PubMed] [Google Scholar]
  13. Horinouchi S., Furuya K., Nishiyama M., Suzuki H., Beppu T. Nucleotide sequence of the streptothricin acetyltransferase gene from Streptomyces lavendulae and its expression in heterologous hosts. J Bacteriol. 1987 May;169(5):1929–1937. doi: 10.1128/jb.169.5.1929-1937.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hunaiti A. A., Kolattukudy P. E. Source of methylmalonyl-coenzyme A for erythromycin synthesis: methylmalonyl-coenzyme A mutase from Streptomyces erythreus. Antimicrob Agents Chemother. 1984 Feb;25(2):173–178. doi: 10.1128/aac.25.2.173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jansen R., Kalousek F., Fenton W. A., Rosenberg L. E., Ledley F. D. Cloning of full-length methylmalonyl-CoA mutase from a cDNA library using the polymerase chain reaction. Genomics. 1989 Feb;4(2):198–205. doi: 10.1016/0888-7543(89)90300-5. [DOI] [PubMed] [Google Scholar]
  16. Kamoun P. Valine is a precursor of propionyl-CoA. Trends Biochem Sci. 1992 May;17(5):175–176. doi: 10.1016/0968-0004(92)90258-b. [DOI] [PubMed] [Google Scholar]
  17. Katz E., Thompson C. J., Hopwood D. A. Cloning and expression of the tyrosinase gene from Streptomyces antibioticus in Streptomyces lividans. J Gen Microbiol. 1983 Sep;129(9):2703–2714. doi: 10.1099/00221287-129-9-2703. [DOI] [PubMed] [Google Scholar]
  18. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  19. Marsh E. N., Leadlay P. F. Methylmalonyl-CoA mutase from Propionibacterium shermanii. Evidence for the presence of two masked cysteine residues. Biochem J. 1989 Jun 1;260(2):339–343. doi: 10.1042/bj2600339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Marsh E. N., McKie N., Davis N. K., Leadlay P. F. Cloning and structural characterization of the genes coding for adenosylcobalamin-dependent methylmalonyl-CoA mutase from Propionibacterium shermanii. Biochem J. 1989 Jun 1;260(2):345–352. doi: 10.1042/bj2600345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Massey L. K., Sokatch J. R., Conrad R. S. Branched-chain amino acid catabolism in bacteria. Bacteriol Rev. 1976 Mar;40(1):42–54. doi: 10.1128/br.40.1.42-54.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Matthies C., Schink B. Reciprocal isomerization of butyrate and isobutyrate by the strictly anaerobic bacterium strain WoG13 and methanogenic isobutyrate degradation by a defined triculture. Appl Environ Microbiol. 1992 May;58(5):1435–1439. doi: 10.1128/aem.58.5.1435-1439.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  24. Messing J., Crea R., Seeburg P. H. A system for shotgun DNA sequencing. Nucleic Acids Res. 1981 Jan 24;9(2):309–321. doi: 10.1093/nar/9.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Murray N. E., Brammar W. J., Murray K. Lambdoid phages that simplify the recovery of in vitro recombinants. Mol Gen Genet. 1977 Jan 7;150(1):53–61. doi: 10.1007/BF02425325. [DOI] [PubMed] [Google Scholar]
  26. Muto A., Osawa S. The guanine and cytosine content of genomic DNA and bacterial evolution. Proc Natl Acad Sci U S A. 1987 Jan;84(1):166–169. doi: 10.1073/pnas.84.1.166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Norrander J., Kempe T., Messing J. Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene. 1983 Dec;26(1):101–106. doi: 10.1016/0378-1119(83)90040-9. [DOI] [PubMed] [Google Scholar]
  28. Omura S., Tsuzuki K., Tanaka Y., Sakakibara H., Aizawa M., Lukacs G. Valine as a precursor of n-butyrate unit in the biosynthesis of macrolide aglycone. J Antibiot (Tokyo) 1983 May;36(5):614–616. doi: 10.7164/antibiotics.36.614. [DOI] [PubMed] [Google Scholar]
  29. Pospísil S., Sedmera P., Havránek M., Krumphanzl V., Vanek Z. Biosynthesis of monensins A and B. J Antibiot (Tokyo) 1983 May;36(5):617–619. doi: 10.7164/antibiotics.36.617. [DOI] [PubMed] [Google Scholar]
  30. Priestley N. D., Robinson J. A. Purification and catalytic properties of L-valine dehydrogenase from Streptomyces cinnamonensis. Biochem J. 1989 Aug 1;261(3):853–861. doi: 10.1042/bj2610853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Robinson J. A. Polyketide synthase complexes: their structure and function in antibiotic biosynthesis. Philos Trans R Soc Lond B Biol Sci. 1991 May 29;332(1263):107–114. doi: 10.1098/rstb.1991.0038. [DOI] [PubMed] [Google Scholar]
  32. Roy I., Leadlay P. F. Physical map location of the new Escherichia coli gene sbm. J Bacteriol. 1992 Sep;174(17):5763–5764. doi: 10.1128/jb.174.17.5763-5764.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sherman M. M., Yue S., Hutchinson C. R. Biosynthesis of lasalocid A. Metabolic interrelationships of carboxylic acid precursors and polyether antibiotics. J Antibiot (Tokyo) 1986 Aug;39(8):1135–1143. doi: 10.7164/antibiotics.39.1135. [DOI] [PubMed] [Google Scholar]
  35. Strohl W. R. Compilation and analysis of DNA sequences associated with apparent streptomycete promoters. Nucleic Acids Res. 1992 Mar 11;20(5):961–974. doi: 10.1093/nar/20.5.961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Stubbe J. Radicals in biological catalysis. Biochemistry. 1988 May 31;27(11):3893–3900. doi: 10.1021/bi00411a001. [DOI] [PubMed] [Google Scholar]
  37. Ward J. M., Janssen G. R., Kieser T., Bibb M. J., Buttner M. J., Bibb M. J. Construction and characterisation of a series of multi-copy promoter-probe plasmid vectors for Streptomyces using the aminoglycoside phosphotransferase gene from Tn5 as indicator. Mol Gen Genet. 1986 Jun;203(3):468–478. doi: 10.1007/BF00422072. [DOI] [PubMed] [Google Scholar]
  38. Wilkemeyer M. F., Crane A. M., Ledley F. D. Primary structure and activity of mouse methylmalonyl-CoA mutase. Biochem J. 1990 Oct 15;271(2):449–455. doi: 10.1042/bj2710449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Woese C. R. Bacterial evolution. Microbiol Rev. 1987 Jun;51(2):221–271. doi: 10.1128/mr.51.2.221-271.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  41. Zagalak B., Rétey J. Studies on methylmalonyl-CoA mutase from Propionibacterium shermanii. Eur J Biochem. 1974 May 15;44(2):529–535. doi: 10.1111/j.1432-1033.1974.tb03512.x. [DOI] [PubMed] [Google Scholar]
  42. Zalkin H., Ebbole D. J. Organization and regulation of genes encoding biosynthetic enzymes in Bacillus subtilis. J Biol Chem. 1988 Feb 5;263(4):1595–1598. [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES