Protein Science, Vol 7, Issue 5 1147-1155, Copyright © 1998 by Cold Spring Harbor Laboratory Press

ARTICLE

A thioredoxin fusion protein of VanH, a D-lactate dehydrogenase from Enterococcus faecium: Cloning, expression, purification, kinetic analysis, and crystallization

V. S. STOLL, A. V. MANOHAR, W. GILLON, ELA. MACFARLANE, R. C. HYNES and E. F. PAI
Department of Biochemistry, University of Toronto, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada Division of Molecular and Structural Biology, Ontario Cancer Institute, 610 University Avenue, Toronto, Ontario M5G 2M9, Canada Current address: Abbott Laboratories, Department 46Y, 100 Abbott Park Road, Abbott Park, Illinois 60064-3500.

The gene encoding the vancomycin resistance protein VanH from Enterococcus faecium, a D-lactate dehydrogenase, has been cloned into a thioredoxin expression system (pTRxFus) and expressed as a fusion protein. The use of several other expression systems yielded only inclusion bodies from which no functional protein could be recovered. Experiments to remove the thioredoxin moiety by enterokinase cleavage at the engineered recognition site under a variety of conditions resulted in nonspecific proteolysis and inactivation of the protein. The intact fusion protein was, therefore, used for kinetic studies and crystallization trials. It has been purified to greater than 90% homogeneity by ammonium sulfate precipitation followed by phenyl Sepharose chromatography. Based on k(cat)/K(M) for pyruvate, it is 20% as active as native VanH. Michaelis constants for NADPH, NADH, and pyruvate, of ~3.5 {mu}M, 19.0 {mu}M, and 1.5 mM, respectively, were comparable to those reported for the native VanH (Bugg TDH et al., 1991, Biochemistry 30:10408-10415). Like native VanH, maximum activity of the fusion protein requires the presence of an anion (phosphate or acetate), however, in addition, a strongly reducing environment is needed for optimal efficacy. Competitive inhibition constants for ADP-ribose, NAD(+), and oxamate have also been determined. Crystallization by hanging drop vapor diffusion produced two different crystal forms, one hexagonal and the other tetragonal. Flash-frozen crystals of the tetragonal form diffracted to 3.0 A resolution at a synchrotron radiation source.
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