Multiple states of the Tyr318Leu mutant of dihydroorotate dehydrogenase revealed by single-molecule kinetics.
Dihydroorotate dehydrogenase (DHOD) from Escherichia coli is a monomeric membrane-associated flavoprotein that catalyzes the oxidation of dihydroorotate to orotate. By using confocal fluorescence spectroscopy on the highly fluorescent Tyr318Leu DHOD mutant, we studied the catalytic turnover of single enzyme molecules through the characteristic on-off fluorescence signal, which corresponds to flavin mononucleotide (FMN) interconverting between the oxidized and reduced states during turnover. Our single-molecule data provide evidence of a distinct static heterogeneity in the enzymatic activity, with some molecules going through the on-off cycles 5-fold faster than others, however, there is no detectable dynamic disorder in DHOD turnover. When 0.1% reduced Triton X-100, a detergent that more closely simulates the natural membrane environment, is added, our data suggest the degree of static molecular heterogeneity is reduced. The observation of static heterogeneity suggests that the enzyme, which associates with the membrane in vivo, is present in distinct conformations that result in different catalytic efficiencies. The alternate conformations are most likely the result of the loss of van der Waals or other interactions between tyrosine 318 and FMN in the catalytic site with the mutation of Tyr318Leu, which disrupts the native structure of wild-type DHOD.
Shi
Jue
J
Biophysics Research Division, University of Michigan, Ann Arbor, Michigan 48105, USA.
Palfey
Bruce A
BA
Dertouzos
Joe
J
Jensen
Kaj Frank
KF
Gafni
Ari
A
Steel
Duncan
D
D001665:Binding Sites; D002384:Catalysis; D000090722:Dihydroorotate Dehydrogenase; D004926:Escherichia coli; D005486:Flavin Mononucleotide / Q000378:metabolism; D007700:Kinetics; D007930:Leucine / Q000235:genetics*; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation / Q000235:genetics*; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014443:Tyrosine / Q000235:genetics*
D000090722:Dihydroorotate Dehydrogenase; D014443:Tyrosine; D005486:Flavin Mononucleotide; D044925:Oxidoreductases Acting on CH-CH Group Donors; D007930:Leucine