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. 1995 May;4(5):955–959. doi: 10.1002/pro.5560040515

Purification and crystallization of benzoylformate decarboxylase.

M S Hasson 1, A Muscate 1, G T Henehan 1, P F Guidinger 1, G A Petsko 1, D Ringe 1, G L Kenyon 1
PMCID: PMC2143116  PMID: 7663351

Abstract

A new large-scale purification method for benzoylformate decarboxylase from Pseudomonas putida has allowed us to undertake an X-ray crystallographic study of the enzyme. The previously observed instability of the enzyme was overcome by addition of 100 microM thiamine pyrophosphate to buffers used in the purification. The final enzyme preparation was more than 97% pure, as determined by denaturing gel electrophoresis and densitometry. The mobility of the enzyme on a gel filtration column indicates that it is a tetramer of 57-kDa subunits. Large, single crystals of benzoylformate decarboxylase were grown from solutions of buffered polyethylene glycol 400, pH 8.5. The crystals diffract to beyond 1.6 A resolution and are stable for days to X-ray radiation. Analysis of X-ray data from the crystals, along with the newly determined quaternary structure, identifies the space group as I222. The unit cell dimensions are a = 82 A, b = 97 A, c = 138 A. An average Vm value for the crystals is consistent with one subunit per asymmetric unit. The subunits of the tetramer must be arranged with tetrahedral 222 symmetry.

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Selected References

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  1. Diefenbach R. J., Duggleby R. G. Pyruvate decarboxylase from Zymomonas mobilis. Structure and re-activation of apoenzyme by the cofactors thiamin diphosphate and magnesium ion. Biochem J. 1991 Jun 1;276(Pt 2):439–445. doi: 10.1042/bj2760439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Dyda F., Furey W., Swaminathan S., Sax M., Farrenkopf B., Jordan F. Catalytic centers in the thiamin diphosphate dependent enzyme pyruvate decarboxylase at 2.4-A resolution. Biochemistry. 1993 Jun 22;32(24):6165–6170. doi: 10.1021/bi00075a008. [DOI] [PubMed] [Google Scholar]
  3. Hübner G., König S., Schnackerz K. D. Correlation of cofactor binding and the quaternary structure of pyruvate decarboxylase as revealed by 31P NMR spectroscopy. FEBS Lett. 1992 Dec 7;314(1):101–103. doi: 10.1016/0014-5793(92)81471-w. [DOI] [PubMed] [Google Scholar]
  4. Lindqvist Y., Schneider G., Ermler U., Sundström M. Three-dimensional structure of transketolase, a thiamine diphosphate dependent enzyme, at 2.5 A resolution. EMBO J. 1992 Jul;11(7):2373–2379. doi: 10.1002/j.1460-2075.1992.tb05301.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Matthews B. W. Solvent content of protein crystals. J Mol Biol. 1968 Apr 28;33(2):491–497. doi: 10.1016/0022-2836(68)90205-2. [DOI] [PubMed] [Google Scholar]
  6. Mitra B., Gerlt J. A., Babbitt P. C., Koo C. W., Kenyon G. L., Joseph D., Petsko G. A. A novel structural basis for membrane association of a protein: construction of a chimeric soluble mutant of (S)-mandelate dehydrogenase from Pseudomonas putida. Biochemistry. 1993 Dec 7;32(48):12959–12967. doi: 10.1021/bi00211a003. [DOI] [PubMed] [Google Scholar]
  7. Neidhart D. J., Kenyon G. L., Gerlt J. A., Petsko G. A. Mandelate racemase and muconate lactonizing enzyme are mechanistically distinct and structurally homologous. Nature. 1990 Oct 18;347(6294):692–694. doi: 10.1038/347692a0. [DOI] [PubMed] [Google Scholar]
  8. Nikkola M., Lindqvist Y., Schneider G. Refined structure of transketolase from Saccharomyces cerevisiae at 2.0 A resolution. J Mol Biol. 1994 May 6;238(3):387–404. doi: 10.1006/jmbi.1994.1299. [DOI] [PubMed] [Google Scholar]
  9. Nilsson U., Lindqvist Y., Kluger R., Schneider G. Crystal structure of transketolase in complex with thiamine thiazolone diphosphate, an analogue of the reaction intermediate, at 2.3 A resolution. FEBS Lett. 1993 Jul 12;326(1-3):145–148. doi: 10.1016/0014-5793(93)81779-y. [DOI] [PubMed] [Google Scholar]
  10. Reynolds L. J., Garcia G. A., Kozarich J. W., Kenyon G. L. Differential reactivity in the processing of [p-(halomethyl)benzoyl] formates by benzoylformate decarboxylase, a thiamin pyrophosphate dependent enzyme. Biochemistry. 1988 Jul 26;27(15):5530–5538. doi: 10.1021/bi00415a022. [DOI] [PubMed] [Google Scholar]
  11. Robinson B. H., Chun K. The relationships between transketolase, yeast pyruvate decarboxylase and pyruvate dehydrogenase of the pyruvate dehydrogenase complex. FEBS Lett. 1993 Aug 9;328(1-2):99–102. doi: 10.1016/0014-5793(93)80973-x. [DOI] [PubMed] [Google Scholar]
  12. Shigeoka S., Nakano Y. Characterization and molecular properties of 2-oxoglutarate decarboxylase from Euglena gracilis. Arch Biochem Biophys. 1991 Jul;288(1):22–28. doi: 10.1016/0003-9861(91)90160-k. [DOI] [PubMed] [Google Scholar]
  13. Stanier R. Y. Simultaneous Adaptation: A New Technique for the Study of Metabolic Pathways. J Bacteriol. 1947 Sep;54(3):339–348. doi: 10.1128/jb.54.3.339-348.1947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Tsou A. Y., Ransom S. C., Gerlt J. A., Buechter D. D., Babbitt P. C., Kenyon G. L. Mandelate pathway of Pseudomonas putida: sequence relationships involving mandelate racemase, (S)-mandelate dehydrogenase, and benzoylformate decarboxylase and expression of benzoylformate decarboxylase in Escherichia coli. Biochemistry. 1990 Oct 23;29(42):9856–9862. doi: 10.1021/bi00494a015. [DOI] [PubMed] [Google Scholar]
  15. Weiss P. M., Garcia G. A., Kenyon G. L., Cleland W. W., Cook P. F. Kinetics and mechanism of benzoylformate decarboxylase using 13C and solvent deuterium isotope effects on benzoylformate and benzoylformate analogues. Biochemistry. 1988 Mar 22;27(6):2197–2205. doi: 10.1021/bi00406a058. [DOI] [PubMed] [Google Scholar]
  16. Wilcocks R., Ward O. P., Collins S., Dewdney N. J., Hong Y., Prosen E. Acyloin formation by benzoylformate decarboxylase from Pseudomonas putida. Appl Environ Microbiol. 1992 May;58(5):1699–1704. doi: 10.1128/aem.58.5.1699-1704.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

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