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. 1992 Feb;89(2):385–391. doi: 10.1172/JCI115597

Cloning and expression of a mutant methylmalonyl coenzyme A mutase with altered cobalamin affinity that causes mut- methylmalonic aciduria.

A M Crane 1, R Jansen 1, E R Andrews 1, F D Ledley 1
PMCID: PMC442864  PMID: 1346616

Abstract

Distinct genotypic and phenotypic forms of methylmalonyl CoA mutase (MCM) apoenzyme deficiency can be delineated by biochemical analysis of mutant fibroblasts. One form, designated mut-, expresses a phenotype in which residual enzyme activity is evident in cultured cells exposed to high concentrations of hydroxycobalamin. We describe cloning of an MCM cDNA from cells exhibiting a mut- phenotype and characterization of the mutant gene product overexpressed in primary muto human fibroblasts and Saccharomyces cerevisiae. Three novel base changes were observed. Recombinant clones containing one of these base changes (G717V) express four characteristics of the mut- phenotype: failure to constitute [14C]propionate incorporation activity in fibroblasts assayed under basal cell culture conditions, constitution of [14C]propionate incorporation activity in fibroblasts stimulated with 0.1-1.0 micrograms/ml hydroxycobalamin, interallelic complementation with alleles bearing an R93H mutation, and an apparent Km (adenosylcobalamin) 1,000-fold higher than normal. These results demonstrate that the G717V mutation produces the mut- phenotype and localizes determinants for adenosylcobalamin binding near the carboxyl terminus of MCM.

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Selected References

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

  1. CANNATA J. J., FOCESI A., Jr, MAZUMDER R., WARNER R. C., OCHOA S. METABOLISM OF PROPIONIC ACID IN ANIMAL TISSUES. XII. PROPERTIES OF MAMMALIAN METHYLMALONYL COENZYME A MUTASE. J Biol Chem. 1965 Aug;240:3249–3257. [PubMed] [Google Scholar]
  2. Chou P. Y., Fasman G. D. Conformational parameters for amino acids in helical, beta-sheet, and random coil regions calculated from proteins. Biochemistry. 1974 Jan 15;13(2):211–222. doi: 10.1021/bi00699a001. [DOI] [PubMed] [Google Scholar]
  3. Chou P. Y., Fasman G. D. Prediction of protein conformation. Biochemistry. 1974 Jan 15;13(2):222–245. doi: 10.1021/bi00699a002. [DOI] [PubMed] [Google Scholar]
  4. Fenton W. A., Hack A. M., Willard H. F., Gertler A., Rosenberg L. E. Purification and properties of methylmalonyl coenzyme A mutase from human liver. Arch Biochem Biophys. 1982 Apr 1;214(2):815–823. doi: 10.1016/0003-9861(82)90088-1. [DOI] [PubMed] [Google Scholar]
  5. Francalanci F., Davis N. K., Fuller J. Q., Murfitt D., Leadlay P. F. The subunit structure of methylmalonyl-CoA mutase from Propionibacterium shermanii. Biochem J. 1986 Jun 1;236(2):489–494. doi: 10.1042/bj2360489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Jansen R., Ledley F. D. Heterozygous mutations at the mut locus in fibroblasts with mut0 methylmalonic acidemia identified by polymerase-chain-reaction cDNA cloning. Am J Hum Genet. 1990 Nov;47(5):808–814. [PMC free article] [PubMed] [Google Scholar]
  8. Kolhouse J. F., Stabler S. P., Allen R. H. L-methylmalonyl-CoA mutase from human placenta. Methods Enzymol. 1988;166:407–414. doi: 10.1016/s0076-6879(88)66053-8. [DOI] [PubMed] [Google Scholar]
  9. Kolhouse J. F., Utley C., Allen R. H. Isolation and characterization of methylmalonyl-CoA mutase from human placenta. J Biol Chem. 1980 Apr 10;255(7):2708–2712. [PubMed] [Google Scholar]
  10. Ledley F. D., Crane A. M., Klish K. T., May G. S. Is there methylmalonyl CoA mutase in Aspergillus nidulans? Biochem Biophys Res Commun. 1991 Jun 28;177(3):1076–1081. doi: 10.1016/0006-291x(91)90648-q. [DOI] [PubMed] [Google Scholar]
  11. Ledley F. D., Crane A. M., Lumetta M. Heterogeneous alleles and expression of methylmalonyl CoA mutase in mut methylmalonic acidemia. Am J Hum Genet. 1990 Mar;46(3):539–547. [PMC free article] [PubMed] [Google Scholar]
  12. Ledley F. D., Jansen R., Nham S. U., Fenton W. A., Rosenberg L. E. Mutation eliminating mitochondrial leader sequence of methylmalonyl-CoA mutase causes muto methylmalonic acidemia. Proc Natl Acad Sci U S A. 1990 Apr;87(8):3147–3150. doi: 10.1073/pnas.87.8.3147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ledley F. D., Levy H. L., Shih V. E., Benjamin R., Mahoney M. J. Benign methylmalonic aciduria. N Engl J Med. 1984 Oct 18;311(16):1015–1018. doi: 10.1056/NEJM198410183111604. [DOI] [PubMed] [Google Scholar]
  14. Ledley F. D., Lumetta M., Nguyen P. N., Kolhouse J. F., Allen R. H. Molecular cloning of L-methylmalonyl-CoA mutase: gene transfer and analysis of mut cell lines. Proc Natl Acad Sci U S A. 1988 May;85(10):3518–3521. doi: 10.1073/pnas.85.10.3518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ledley F. D. Perspectives on methylmalonic acidemia resulting from molecular cloning of methylmalonyl CoA mutase. Bioessays. 1990 Jul;12(7):335–340. doi: 10.1002/bies.950120706. [DOI] [PubMed] [Google Scholar]
  16. MacGregor G. R., Caskey C. T. Construction of plasmids that express E. coli beta-galactosidase in mammalian cells. Nucleic Acids Res. 1989 Mar 25;17(6):2365–2365. doi: 10.1093/nar/17.6.2365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Marsh N., Leadlay P. F., Evans P. R. Crystallization and preliminary diffraction data for adenosylcobalamin-dependent methylmalonyl-CoA mutase from Propionibacterium shermanii. J Mol Biol. 1988 Mar 20;200(2):421–422. doi: 10.1016/0022-2836(88)90252-5. [DOI] [PubMed] [Google Scholar]
  19. Matsui S. M., Mahoney M. J., Rosenberg L. E. The natural history of the inherited methylmalonic acidemias. N Engl J Med. 1983 Apr 14;308(15):857–861. doi: 10.1056/NEJM198304143081501. [DOI] [PubMed] [Google Scholar]
  20. McDonnell D. P., Pike J. W., Drutz D. J., Butt T. R., O'Malley B. W. Reconstitution of the vitamin D-responsive osteocalcin transcription unit in Saccharomyces cerevisiae. Mol Cell Biol. 1989 Aug;9(8):3517–3523. doi: 10.1128/mcb.9.8.3517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Raff M. L., Crane A. M., Jansen R., Ledley F. D., Rosenblatt D. S. Genetic characterization of a MUT locus mutation discriminating heterogeneity in mut0 and mut- methylmalonic aciduria by interallelic complementation. J Clin Invest. 1991 Jan;87(1):203–207. doi: 10.1172/JCI114972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rosenblatt D. S., Cooper B. A., Pottier A., Lue-Shing H., Matiaszuk N., Grauer K. Altered vitamin B12 metabolism in fibroblasts from a patient with megaloblastic anemia and homocystinuria due to a new defect in methionine biosynthesis. J Clin Invest. 1984 Dec;74(6):2149–2156. doi: 10.1172/JCI111641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Shigekawa K., Dower W. J. Electroporation of eukaryotes and prokaryotes: a general approach to the introduction of macromolecules into cells. Biotechniques. 1988 Sep;6(8):742–751. [PubMed] [Google Scholar]
  24. Wilkemeyer M. F., Crane A. M., Ledley F. D. Differential diagnosis of mut and cbl methylmalonic aciduria by DNA-mediated gene transfer in primary fibroblasts. J Clin Invest. 1991 Mar;87(3):915–918. doi: 10.1172/JCI115098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Willard H. F., Ambani L. M., Hart A. C., Mahoney M. J., Rosenberg L. E. Rapid prenatal and postnatal detection of inborn errors of propionate, methylmalonate, and cobalamin metabolism: a sensitive assay using cultured cells. Hum Genet. 1976 Dec 15;34(3):277–283. doi: 10.1007/BF00295291. [DOI] [PubMed] [Google Scholar]
  27. Willard H. F., Rosenberg L. E. Inherited deficiencies of human methylmalonyl CaA mutase activity: reduced affinity of mutant apoenzyme for adenosylcobalamin. Biochem Biophys Res Commun. 1977 Oct 10;78(3):927–934. doi: 10.1016/0006-291x(77)90511-3. [DOI] [PubMed] [Google Scholar]
  28. Willard H. F., Rosenberg L. E. Inherited methylmalonyl CoA mutase apoenzyme deficiency in human fibroblasts: evidence for allelic heterogeneity, genetic compounds, and codominant expression. J Clin Invest. 1980 Mar;65(3):690–698. doi: 10.1172/JCI109715. [DOI] [PMC free article] [PubMed] [Google Scholar]

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