Purification and properties of an enzyme involved in the ATP-dependent activation of the methanol:2-mercaptoethanesulfonic acid methyltransferase reaction in Methanosarcina barkeri.
In Methanosarcina barkeri the transfer of the methyl group from methanol to 2-mercaptoethanesulfonic acid is catalyzed by the concerted action of two methyltransferases. The first one is the corrinoid-containing methanol:5-hydroxybenzimidazolylcobamide methyltransferase (MT1), which binds the methyl group of methanol to its corrinoid prosthetic group. MT1 is only catalytically active when the cobalt atom of the corrinoid is present in the highly reduced Co(I) state. In the course of its purification and even during catalysis, MT1 becomes oxidatively inactivated. The enzyme, however, may be reductively reactivated by a suitable reducing system (hydrogen and hydrogenase), ATP, and an enzyme called methyltransferase activation protein (MAP). In order to elucidate its role in the reactivation process, MAP was purified to apparent homogeneity. The protein had an Mr = 60,000. Preincubation of the enzymic components involved with 8-azido-ATP or with ATP demonstrated MAP to be the primary site of action of ATP. In agreement herewith, the protein was autophosphorylated by [gamma-32P]ATP in a 1:1 stoichiometry. Phosphorylated MAP substituted for ATP in the activation of MT1, and the addition of increasing amounts of MAP phosphate resulted in a corresponding increase of active MT1. However, in the presence of limiting amounts of MAP, maximal activation of MT1 could be achieved during a lag phase provided ATP was present, indicating that MAP acts as a catalyst. This paper is the first to report on the presence, isolation, and function of a phosphorylated protein in a methanogenic archaeon.
Daas
P J
PJ
Department of Microbiology, Faculty of Science, University of Nijmegen, Toernooiveld, NL-6525 ED Nijmegen, The Netherlands.
Wassenaar
R W
RW
Willemsen
P
P
Theunissen
R J
RJ
Keltjens
J T
JT
van der Drift
C
C
Vogels
G D
GD
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D019843:Archaeal Proteins; D001386:Azides / Q000378:metabolism; D002793:Cholic Acids; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D017021:Methanosarcina barkeri / Q000201:enzymology*; D008780:Methyltransferases / Q000378:metabolism*; D008970:Molecular Weight; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism; D014466:Ultraviolet Rays
D019843:Archaeal Proteins; D001386:Azides; D002793:Cholic Acids; C017040:8-azidoadenosine 5'-triphosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D008780:Methyltransferases; C036890:methanol-2-mercaptoethanesulfonic acid methyltransferase; C040528:methanol-5-hydroxybenzimidazolylcobamide methyltransferase; D011494:Protein Kinases; C101667:methyltransferase activation protein, Methanosarcina barkeri; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate