Cellular localization of D-lactate dehydrogenase and NADH oxidase from Archaeoglobus fulgidus.
Members of the genus Archaeoglobus are hyperthermophilic sulfate reducers with an optimal growth temperature of 83 degrees C. Archaeoglobus fulgidus can utilize simple compounds including D-lactate, L-lactate and pyruvate as the sole substrate for carbon and electrons for dissimilatory sulfate reduction. Previously we showed that this organism makes a D-lactate dehydrogenase (Dld) that requires FAD and Zn2+ for activity. To determine the cellular location and topology of Dld and to identify proteins that interact with Dld, an antibody directed against Dld was prepared. Immunocytochemical studies using gold particle-coated secondary antibodies show that more than 85% of Dld is associated with the membrane. A truncated form of Dld was detected in immunoblots of whole cells treated with protease, showing that Dld is an integral membrane protein and that a significant portion of Dld, including part of the FAD-binding pocket, is outside the membrane facing the S-layer. The gene encoding Dld is part of an operon that includes noxA2, which encodes one of several NADH oxidases in A. fulgidus. Previous studies have shown that NoxA2 remains bound to Dld during purification. Thin sections of A. fulgidus probed simultaneously with antibodies against Dld and NoxA2 show that both proteins co-localized to the same sites in the membrane. Although these data show a tight interaction between NoxA2 and Dld, the role of NoxA2 in electron transport reactions is unknown. Rather, NoxA2 may protect proteins involved in electron transfer by reducing O2 to H2O2 or H2O.
Pagala
Vishwajeeth Reddy
VR
Department of Microbiology, Molecular Biology, and Biochemistry, University of Idaho, Moscow, ID 83844-3052, USA.
Park
Joohye
J
Reed
David W
DW
Hartzell
Patricia L
PL
D019843:Archaeal Proteins / Q000302:isolation & purification; D019611:Archaeoglobus fulgidus / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000502:physiology; D016764:Cell Polarity / Q000502:physiology; D003593:Cytoplasm / Q000201:enzymology; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000302:isolation & purification*; D009097:Multienzyme Complexes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D013104:Spheroplasts / Q000201:enzymology; D014305:Triose-Phosphate Isomerase / Q000378:metabolism
D019843:Archaeal Proteins; D009097:Multienzyme Complexes; D007770:L-Lactate Dehydrogenase; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D014305:Triose-Phosphate Isomerase