Skip to content
1887

Microbiology Society logo Microbiology

Volume 140, Issue 11

Malate synthase from : sequence analysis of the gene and biochemical characterization of the enzyme Free

Abstract

Malate synthase is one of the key enzymes of the glyoxylate cycle and is essential for growth on acetate as sole carbon source. The gene from , encoding malate synthase, was isolated, subcloned and expressed in and . Sequencing of a 3024 bp DNA fragment containing the gene revealed that it is located close to the isocitrate lyase gene . The two genes are separated by 597 bp and are transcribed in divergent directions. The predicted gene product consists of 739 amino acids with an of 82362. Interestingly, this polypeptide shows only weak identity with malate synthase polypeptides from other organisms and possesses an extra N-terminal sequence of about 170 amino acid residues. Inactivation of the chromosomal gene led to the absence of malate synthase activity and to the inability to grow on acetate, suggesting that only one malate synthase is present in . The malate synthase was purified from an -overexpressing strain and biochemically characterized. The native enzyme was shown to be a monomer migrating at an of about 80000. By sequencing the N-terminus of malate synthase the predicted translational start site of the enzyme was confirmed. The enzyme displayed values of 30 μM and 12 μM for the substrates glyoxylate and acetyl CoA, respectively. Oxalate, glycolate and ATP were found to be inhibitors of malate synthase activity. The present study provides evidence that the malate synthase from is functionally similar to other malate synthase enzymes but is different both in size and primary structure.

  • Published Online:
Keyword(s): acetate metabolism , Corynebacterium glutamicum , glyoxylate cycle , isocitrate lyase and malate synthase
© Society for General Microbiology 1994
Loading

Article metrics loading...

/content/journal/micro/10.1099/13500872-140-11-3099
1994-11-01
2025-04-28
Loading full text...

Full text loading...

/deliver/fulltext/micro/140/11/mic-140-11-3099.html?itemId=/content/journal/micro/10.1099/13500872-140-11-3099&mimeType=html&fmt=ahah

References

  1. Akashi K., Shibai H., Hi rose Y. Comparison between acetic acid and glucose as a substrate in threonine fermentation. Agric Biol Chem 1979; 43:1563–1566
     [Google Scholar]
  2. Alter G.M., Casazza J.P., Zhi W., Nemeth P., Srere P.A., Evans C.T. Mutation of essential catalytic residues in pig citrate synthase. Biochemistry 1990; 29:7557–7563
     [Google Scholar]
  3. Armitt S., McCullough W., Roberts W. Analysis of acetate non-utilizing (acu) mutants in Aspergillus nidulans. J Gen Microbiol 1976; 92:263–282
     [Google Scholar]
  4. Birnboim H.C. A rapid alkaline extraction method for the isolation of plasmid DNA. Methods Enaymol 1983; 100:243–255
     [Google Scholar]
  5. Bormann E.R., Eikmanns B.J., Sahm H. Molecular analysis of the Corynebacterium glutamicum gdh gene encoding glutamate dehydrogenase. Mol Microbiol 1992; 6:317–326
     [Google Scholar]
  6. Brice C.B., Kornberg H.L. Genetic control of isocitrate lyase activity in Escherichia coli. J Bacteriol 1968; 96:2185–2186
     [Google Scholar]
  7. Byrne C., Stokes H.W., Ward K.A. Nucleotide sequence of the aceB gene encoding malate synthase A in Escherichia coli. Nucleic Acids Res 1988; 16:9342
     [Google Scholar]
  8. Chang A.C.Y., Cohen S.N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic plasmid. J Bacteriol 1978; 134:1141–1156
     [Google Scholar]
  9. Chell R.M., Sundaram T.K. Isolation and characterization of isocitrate lyase and malate synthase from Bacillus stearothermophilus. Biochem Soc Trans 1975; 3:303–306
     [Google Scholar]
  10. Chell R.M., Sundaram T.K. Structural basis of the thermostability of monomeric malate synthase from a thermophilic Bacillus. J Bacteriol 1978; 135:334–341
     [Google Scholar]
  11. Chung T., Klumpp D.J., LaPorte D.C. Glyoxylate bypass operon of Escherichia coli: cloning and determination of the functional map. J Bacteriol 1988; 170:386–392
     [Google Scholar]
  12. Cioni M., Pinzauti G., Vanni P. Comparative biochemistry of the glyoxylate cycle. Comp Biochem Physiol 1981; 70B:1–26
     [Google Scholar]
  13. Comai L., Baden C.S., Harada J.J. Deduced sequence of a malate synthase polypeptide encoded by a subclass of the gene family. J Biol Chem 1989; 264:2778–2782
     [Google Scholar]
  14. Cortay J.C., Bleicher F., Duclos B., Cenatiempo Y., Gautier C., Prato J.L., Cozzone A.J. Utilization of acetate in Escherichia coli: structural organization and differential expression of the ace operon. Biochimie 1989; 71:1043–1049
     [Google Scholar]
  15. Dixon G.H., Kornberg H.L. Assay methods for key enzymes of the glyoxylate cycle. Biochem J 1959; 72:3 P
    [Google Scholar]
  16. Dixon G.H., Kornberg H.L., Lund P. Purification and properties of malate synthetase. Biochim Biophys Acta 1960; 41:217–233
     [Google Scholar]
  17. Eikmanns B.J. Identification, sequence analysis, and expression of a Corynebacterium glutamicum gene cluster encoding the three glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, and triosephosphate isomerase. J Bacteriol 1992; 174:6076–6086
     [Google Scholar]
  18. Eikmanns B.J., Kleinertz E., Liebl W., Sahm H. A family of Corynebacterium glutamicum/ Escherichia coli shuttle vectors for cloning, controlled gene expression, and promoter probing. Gene 1991a; 102:93–98
     [Google Scholar]
  19. Eikmanns B.J., Metzger M., Reinscheid D.J., Sahm H. Amplification of three threonine biosynthesis genes in Corynebacterium glutamicum and its influence on carbon flux in different strains. Appl Microbiol Biotechnol 1991b; 34:617–622
     [Google Scholar]
  20. Falmagne P., Wiame J.-M. Purification et caracterisation partielle des deux malate synthases d’Escherichia coli. Eur J Biochem 1973; 37:415–424
     [Google Scholar]
  21. Fernandez E., Fernandez M., Rodicio R. Two structural genes are encoding malate synthase isoenzymes in Saccharomyces cerevisiae. FEBS Lett 1993; 320:271–275
     [Google Scholar]
  22. Fukawa H., Ejiri S., Katsumata T. Purification and some properties of malate synthase from the pollen of Pinus densiflora Sieb. et Zucc. Agric Biol Chem 1987; 51:1553–1560
     [Google Scholar]
  23. Gornall A.G., Bardawill C.J., David M.M. Determination of serum proteins by means of the Biuret reaction. J Biol Chem 1949; 177:751–766
     [Google Scholar]
  24. Graham I.A., Smith L.M., Brown J.W.S., Leaver C.J., Smith S.M. The malate synthase gene of cucumber. Plant Mol Biol 1989; 13:673–684
     [Google Scholar]
  25. Hartig M., Simon M.M., Schuster T., Daugherty J.R., Yoo H.S., Cooper T.G. Differentially regulated malate synthase genes participate in carbon and nitrogen metabolism of Saccharomyces cerevisiae. Nucleic Acids Res 1992; 20:5677–5686
     [Google Scholar]
  26. Hikida M., Atomi H., Fukuda Y., Aoki A., Hishida T., Teranishi Y., Ueda M., Tanaka A. Presence of two transcribed malate synthase genes in an «-alkane-utilizing yeast, Candida tropicalis. J Biochem 1991; 110:909–914
     [Google Scholar]
  27. Hohn B., Collins J. A small cosmid for efficient cloning of large DNA fragments. Gene 1980; 11:291–298
     [Google Scholar]
  28. Kinoshita S. Amino acids. In Biology of Industrial Organisms 1985 Edited by Demain A.L., Solomon N.A. London: Benjamin/Cummings Publishing; pp 115–142
     [Google Scholar]
  29. Kinoshita S., Tanaka K. Glutamic acid. In The Microbial Production of Amino Acids 1972 Edited by Amada K.Y. New York: John Wiley & Sons; pp 263–324
     [Google Scholar]
  30. Kornberg H.L. The role and control of the glyoxylate cycle in Escherichia coli. Biochem J 1966; 99:1–11
     [Google Scholar]
  31. Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227:680–685
     [Google Scholar]
  32. LaPorte D.C., Thorsness P.E., Koshland D.E. Jr Compensatory phosphorylation of isocitrate dehydrogenase, a mechanism for adaptation to the intracellular environment. J Biol Chem 1985; 260:10563–10568
     [Google Scholar]
  33. Liebl W. The genus Corynebacterium - nonmedical. In The Prokaryotes 1991 Edited by Balows A., Trüper H.G., Dworkin M., Harder W., Schleifer K.-H. New York: Springer Verlag; 2 pp 1157–1171
     [Google Scholar]
  34. Liebl W., Bayerl A., Schein B., Stillner U., Schleifer K.H. High efficiency electroporation of intact Corynebacterium glutamicum cells. FEMS Microbiol Lett 1989; 65:299–304
     [Google Scholar]
  35. Maloy S.R., Nunn W.D. Genetic regulation of the glyoxylate shunt in Escherichia coli K12. J Bacteriol 1982; 149:173–180
     [Google Scholar]
  36. Okada H., Ueda M., Tanaka A. Purification of peroxisomal malate synthase from alkane-grown Candida tropicalis and some properties of the purified enzyme. Arch Microbiol 1986; 144:137–144
     [Google Scholar]
  37. Reinscheid D., Eikmanns B.J., Sahm H. Characterization of the isocitrate lyase gene from Corynebacterium glutamicum and biochemical analysis of the enzyme. J Bacteriol 1994; 176:3474–3483
     [Google Scholar]
  38. Sambrook J., Fritsch E.F., Maniatis J. Molecular Cloning: A Laboratory Manual 1989 Cold Spring Harbor: Cold Spring Harbor Laboratory Press;
     [Google Scholar]
  39. Sandemann R.A., Hynes M.J. Isolation of the facA (acetyl-coenzyme A synthetase) and acuE (malate synthase) genes of Aspergillus nidulans. Mol & Gen Genet 1989; 218:87–92
     [Google Scholar]
  40. Sandemann R.A., Hynes M.J., Fincham J.R.S., Connerton I.F. Molecular organisation of the malate synthase genes of Aspergillus nidulans and Neurospora crassa. Mol & Gen Genet 1991; 228:445–452
     [Google Scholar]
  41. Sanger F., Nicklen S., Coulson A.R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 1977; 74:5463–5467
     [Google Scholar]
  42. Schäfer A., Kalinowski J., Simon R., Seep-Feldhaus A.H., Plihler A. High frequency conjugal plasmid transfer from gram-negative Escherichia coli to various gram-positive coryneform bacteria. J Bacteriol 1990; 172:1663–1666
     [Google Scholar]
  43. Schwarzer A., Pühler A. Manipulation of Corynebhcterium glutamicum by gene disruption and replacement. Bio/Technology 1991; 9:84–87
     [Google Scholar]
  44. Segel I.H. Enzyme Kinetics 1975 New York: Wiley Interscience;
     [Google Scholar]
  45. Simon R., Priefer U., Pühler A. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Bio/Technology 1983; 1:784–791
     [Google Scholar]
  46. Thomas G.H., Connerton I.F., Fincham J.R.S. Molecular cloning, identification and transcriptional analysis of genes involved in acetate utilization in Neurospora crassa. Mol Microbiol 1988; 2:599–606
     [Google Scholar]
  47. Tinoco I., Borer P.N., Dengler B., Levine M.D., Uhlenbeck O.C., Crothers D.M., Gralla J. Improved estimation of secondary structure in ribonucleic acid. Nature New Biol 1973; 246:40–41
     [Google Scholar]
  48. Vanderwinkel E., De Vlieghere M. Physiologie et genetique de l’isocitritase et des malate synthases chez Escherichia coli. Eur J Biochem 1968; 5:81–90
     [Google Scholar]
  49. Vieira J., Messing J. The pUC plasmids, an M13mp7- derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 1982; 19:259–268
     [Google Scholar]
  50. Wiegand G., Remington S.J. Citrate synthesis: structure, control, and mechanism. Annu Rep Biophys Biophys Chem 1986; 15:97–117
     [Google Scholar]
  51. Wilson R.B., Maloy S.R. Isolation and characterization of Salmonella typhimurium glyoxylate shunt mutants. J Bacteriol 1987; 169:3029–3034
     [Google Scholar]
/content/journal/micro/10.1099/13500872-140-11-3099
Loading
Malate synthase from Corynebacterium glutamicum: sequence analysis of the gene and biochemical characterization of the enzyme
Microbiology 140, 3099 (1994); https://doi.org/10.1099/13500872-140-11-3099
/content/journal/micro/10.1099/13500872-140-11-3099
/content/journal/micro/10.1099/13500872-140-11-3099
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error