Abstract
Methanol:5-hydroxybenzimidazolylcobamide methyltransferase (MT1) is the first of two enzymes involved in the transmethylation reaction from methanol to 2-mercaptoethanesulfonic acid in Methanosarcina barkeri. MT1 only binds the methyl group of methanol when the cobalt atom of its corrinoid prosthetic groups is present in the highly reduced Co(I) state. Formation of this redox state requires H2, hydrogenase, methyltransferase activation protein, and ATP. Optical and electron paramagnetic resonance spectroscopy studies were employed to determine the oxidation states and coordinating ligands of the corrinoids of MT1 during the activation process. Purified MT1 contained 1.7 corrinoids per enzyme with cobalt in the fully oxidized Co(III) state. Water and N-3 of the 5-hydroxybenzimidazolyl base served as the upper and lower ligands, respectively. Reduction to the Co(II) level was accomplished by H2 and hydrogenase. The cob(II)amide of MT1 had the base coordinated at this stage. Subsequent addition of methyltransferase activation protein and ATP resulted in the formation of base-uncoordinated Co(II) MT1. The activation mechanism is discussed within the context of a proposed model and compared to those described for other corrinoid-containing methyl group transferring proteins.
Highlights
The corrinoid prosthetic group of MT1 can only be methylated by methanol when the central cobalt atom of the cobamide
The activation of MT1 proceeds by a novel mechanism, which is presented in a model and compared to those described for other corrinoid-containing methyl group-transferring proteins
MT1 activity was determined by measuring the methanol-dependent HS-CoM conversion to CH3-S-CoM when added to a reaction mixture containing MT2/hydrogenase, methyltransferase activation protein (MAP), and ferredoxin fractions obtained after DEAE-Sepharose fractionation of cell extract of M. barkeri
Summary
MT1 activity was determined by measuring the methanol-dependent HS-CoM conversion to CH3-S-CoM when added to a reaction mixture containing MT2/hydrogenase, MAP, and ferredoxin fractions obtained after DEAE-Sepharose fractionation of cell extract of M. barkeri [6]. Protein Purification—Because several of the enzymes involved in the methanol:HS-CoM methyltransferase reaction are oxygen-labile [1, 4, 6, 8], all handlings were performed in an anaerobic glove box (97.5% N2, 2.5% H2) at room temperature. The purification procedure started by applying 10 ml of cell extract to a DEAE-Sepharose-CL-6B column and separating the proteins involved in the methyltransferase reaction as zimidazolyl; B12-HBI, 5-hydroxybenzimidazolylcobamide; CAPS, 3- described previously [6]. MT2 and hydrogenase activity eluted between (cyclohexylamino)-1-propanesulfonic acid; kPa, kilopascal(s). Enzyme assays were performed as described under “Experimental Procedures.” Units are expressed as micromoles of 2-mercaptoethanesulfonic acid methylated per min
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