CRYSTALLIZATION AND CRYSTALLOGRAPHY OF THE MAJOR FLAVIN REDUCTASE OF Vibrio fischeri. Hideaki Koike1, Hiroshi Sasaki1, Shuhei Zenno2, Kaoru Saigo2, Michael E. P. Murphy3, Elinor T. Adman3, Masaru Tanokura1, 1Biotechnology Research Center, 2Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan, 3Department of Biological Structure, Box 357420, School of Medicine, University of Washington, Seattle, WA 98195-7420, USA
We crystallized and characterized the major NAD(P)H:FMN oxidoreductase (flavin reductase) of a bioluminescent bacterium V. Fischeri. It diffracted X-ray to further than 2.2 Å. We have found a good derivative of mercury and are now searching other derivatives to get better isomorphous phasing.
The enzyme catalyzes the reduction of FMN to FMNH2 using NAD(P)H as an electron donor and supplies the FMNH2 to bacterial luciferase. The enzyme was purified from an expression system of E. coli and characterized on the biochemical properties. The enzyme works as a dimer and has FMN as a cofactor. From the steady state kinetics and anaerobic reductive titration by dithionite and NADH, we have proposed the reaction mechanism of the enzyme as follows: 2 electrons flow from NADH to the cofactor FMN, then NAD+ leaves from the enzyme. Next, a substrate FMN binds to the enzyme and is given 2 electrons from the reduced cofactor FMN.
Crystals were obtained using PEG 4000 as a precipitant. Precession photographs revealed that the crystals belong to monoclinic, space group C2, and cell parameters were a = 101.6, b = 63.3, c = 74.4 Å, and [[beta]] = 100.0deg. and have a dimer in an asymmetric unit. We collected the native intensity data using synchrotron radiation in the Photon Factory at National Laboratory for High Energy Physics. The data sets had the statistics of 79.9% completeness with 23,163 independent reflections (2.2 Å resolution). The data of the crystals soaked in solutions containing heavy atom compounds indicated a good isomorphous derivative of a mercury compound, EMTS. We also observed anomalous scattering data of the derivative, and calculated isomorphous phase with the program MLPHARE of CCP4. Figure of merits and phasing power for centric reflections at 2.7 Å resolution were 0.75 and 1.35, respectively. To improve the phase, we are searching another good derivative.