Purification and characterization of the reduced-nicotinamide-dependent 2,2'-dithiodiethanesulfonate reductase from Methanobacterium thermoautotrophicum delta H.
A novel reduced nicotinamide-dependent disulfide reductase, the 2,2'-dithiodiethanesulfonate [(S-CoM)2] reductase (CoMDSR) of Methanobacterium thermoautotrophicum was purified 405-fold to electrophoretic homogeneity. Both NADPH and NADH functioned as electron donors, although rates with NADPH were three times higher. Reduced factor F420, the deazaflavin electron carrier characteristic of methanogenic bacteria, was not a substrate for the enzyme. The enzyme was most active with (S-CoM)2 but could also reduce L-cystine at 23% the (S-CoM)2 rate. Results of sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that the enzyme was monomeric with an Mr of about 64,000; spectral analysis showed that it was a flavoprotein with an estimated composition of one molecule of flavin per polypeptide. Maximal activity occurred at 64 degrees C, and the pH optimum was 8.5. The apparent Km for both NADPH and (S-CoM)2 was 80 microM. The enzyme was completely inactivated by oxygen in crude cell extracts but was oxygen stable in the homogeneous state. The low activity of the CoMDSR in cell extracts as well as its relatively low rate of reducing CoM-S-S-HTP (the heterodisulfide of the two thiol cofactors involved in the last step of methanogenesis) make it unlikely that it plays a role in the methylreductase system. It may be involved in the redox balance of the cell, such as the NADPH-dependent bis-gamma-glutamylcystine reductase with which it shows physical similarity in another archaebacterium, Halobacterium halobium (A. R. Sundquist and R. C. Fahey, J. Bacteriol. 170:3459-3467, 1988). The CoMDSR might also be involved in regenerating the coenzyme M trapped as its homodisulfide, a nonutilizable form of the cofactor.
Smith
S G
SG
Department of Microbiology, University of Illinois, Urbana 61801.
Rouvière
P E
PE
D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D004220:Disulfides / Q000378:metabolism; D019605:Euryarchaeota / Q000201:enzymology*; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D013053:Spectrophotometry; D013379:Substrate Specificity
D004220:Disulfides; D009243:NAD; D009249:NADP; D009247:NADH, NADPH Oxidoreductases; C066235:2,2'-dithiodiethanesulfonate reductase (NAD(P)H)