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CRYSTAL STRUCTURE OF KETOPANTOATE REDUCTASE SOLVED BY SeMet MAD

Escherichia coli ketopantoate reductase (KPR), encoded by the gene panE, is an NADPH dependent oxidoreductase which catalyses the reduction of ketopantoate to pantoate, a step in the biosynthetic pathway of pantothenate (vitamin B5). KPR is the least well-characterised enzyme of the pathway because of the presence of another enzyme which can carry out the same reaction,1 acetohydroxyisomeroreductase, an enzyme from valine biosynthesis. In order to understand the catalytic mechanism of KPR we overexpressed and purified it and solved the crystal structure. The structure of KPR was solved by a selenomethionine MAD experiment at beamline BW7A at the EMBL outstation in DESY, Hamburg. Data were collected to 2.4 A at 3 wavelengths, selected from an X-ray fluorescence spectrum to optimise the anomalous signal. Selenium sites were found with the program SHELXS96 and data phased with SHARP using all three wavelength data sets. Experimental phases were improved by solvent flattening using Solomon, with a solvent content of 43%. The final electron-density map was easily interpretable. Native data were collected to 1.7 A at Daresbury SRS beamline 9.6. KPR crystals are tetragonal, space group P42212 (a = 104.2 and c = 55.8 A), with one molecule in the asymmetric unit. The enzyme is monomeric and has two domains. The N-terminal domain has an alpha-beta fold of Rossman type, while the C-terminal domain is all alpha helical. There is no evidence of bound cofactor (NADPH). Comparison of the fold against all structures in the Protein Data Bank using the DALI server revealed that it is similar to norvaline dehydrogenase.2 _____________ [1] M. E. Frodyma and D. Downs, J. Biol. Chem 273 (1998) 5572. [2] K. L. Britton, Y. Asano and D. W. Rice, Nature Struct. Biol. 5 (1998) 593.

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