CRYSTALLOGRAPHIC STUDES OF LACTATE MONOOXYGENASE (LMO) AND OLD YELLOW ENZYME2 (OYE2). Zhan Deng, P. Andrew Karplus, Section of Biochemistry, Cell and Molecular Biology, Cornell University, Ithaca, NY 14853, Vincent Massey, Department of Biological Chemistry, Medical School, University of Michigan, Ann Arbor, MI48109

The flavoprotein L-lactate 2-monooxygenase (LMO) catalyzes the oxidative decarboxylation of L-lactate to acetate, carbon dioxide and water. Although LMO is closely related to glycollate oxidase (GOX) and flavocytochrome b2 (FCB), it has notably different kinetic properties compared to GOX and FCB. In particular, GOX and FCB have keto acids as their final oxidative products, whereas in LMO, the intermediate pyruvate is tightly bound to the enzyme and is subsequently decarboxylated. Crystallographic study of LMO will provide a good opportunity to see how subtle structural change can lead to large change in function.

Crystals of LMO from Mycobacterium smegmatis have been grown in a primitive tetragonal space group with a=b=143Å, c=269Å. A 2.6Å data set has just been collected from a frozen LMO crystal at CHESS. We plan to solve the structure by molecular replacement method using GOX as a search model, and gain insights into the structural cause of their functional differences. An update of the structure determination will be presented.

Old yellow enzyme (OYE) (E.C. 1.6.99.1) was the first identified flavoprotein and has been used as a model protein in the studies of flavoprotein and flavin chemistry. However its physiological function still remains elusive. Recent discoveries that OYE interacts with actin protein and deletion of OYE genes in S. cerevisiae leads to perturbed actin cytoskeleton indicate that OYE plays an important role in yeast. The structure of old yellow enzyme from brewer's yeast (OYE 1) was solved to 2.0Å resolution, showing a TIM-barrel domain structure.

We have grown crystals of OYE2, an OYE isoform from S. Cerevisiae. The crystals belong to space group P2_l2₁2_l (a=70Å, b=96Å, c= 151Å) and diffract to 2.5Å resolution. The structure determination is in progress by molecular replacement and we plan to report a comparison between the structures of OYE1 and OYE2 isoforms.