Poster abstracts

Poster number 11 submitted by Lance Rider

Understanding the Dihydrouridine Synthase from T. maritima

Lance W. Rider (University of Michigan), Maria Nelson (Univeristy of Michigan), Bruce Palfey (University of Michigan)

Abstract:
Dihydrouridine is a modified nucleoside present in the tRNAs of eubacteria, eukaryotes, and some archea. This modified nucleoside is formed by the reduction of the double bond in uridine at specific sites in tRNAs, most commonly the D-loop region. The family of proteins catalyzing this reaction, dihydrouridine synthases, have only recently been identified in E.coli and S. cerivesiae. We have cloned, expressed, and partially characterized a member of this family of proteins from the thermophilic bacterium Thermotoga maritima. This protein is a 38kD monomeric protein which utilizes an FMN prosthetic group. Catalysis is comprised of a reductive half reaction, in which NADPH reduces the flavin, and an oxidative half-reaction whereby the flavin reduces the double bond of uridine. The reductive half-reaction at 25 degrees Celcius shows an apparent limiting rate constant of 2.3 x 10-4 s-1 with a KD of 600 μM. Stereochemistry of the hydride trandsfer shows the enzyme prefers the pro-R face of NADPH, however no kinetic isotope effect is seen for this reaction, indicating that chemistry is not rate limiting. Oxidation of the enzyme by a non-native pre-tRNAtyr reoxidizes the flavin with a rate of 3.2 x 10-5 s-1.

Keywords: dihydrouridine, thermotoga, kinetics