Sodium Ion Translocation by N₅‐Methyltetrahydromethanopterin: Coenzyme M Methyltransferase from Methanosarcina mazei Gö1 Reconstituted in Ether Lipid Liposomes

Abstract

The N'-methyltetrahydomethanopterin (H,MPT) :coenzyme M methyltransferase is a membrane associated, corrinoid-containing protein that uses the methylation of coenzyme M (HS-CoM) by methyltetrahydromethanopterin to drive an energy-conserving sodium ion pump. The enzyme was purified from acetate-grown Methanosarcina mazei GO1 by a two-step solubilization with s-octyl-P-glucoside, chromatography on hydroxyapatite, and by gel filtration on Superdex 200 or Sepharose CL-6B. The highly purified protein was apparently composed of six different subunits of 34, 28, 20, 13, 12, and 9 kDa. The N-terminal amino acid sequences of these polypeptides were determined. The native enzyme exhibited an apparent molecular mass of about 380 kDa. During purification, the enzyme was stabilized with 10 pM hydroxocobalamin. The highest specific activity reached during purification was 10.4 U/mg. The purified enzyme was reconstituted in monolayer liposomes prepared from ether lipids of M. mazei GO1. In experiments with radioactive sodium ions, it was shown that the methyltransferase catalyzes the vectorial translocation of sodium ions across the membrane. Methyltransferase activity was stimulated by sodium ions. 1.7 mol Na+/mol methyl groups transferred were translocated. Methyltetrahydrofolate and methylcobalamin could substitute for methyl-H,MPT.

Biochemistry of methanogenesis: a tribute to Marjory Stephenson:1998 Marjory Stephenson Prize Lecture

“…As indicated, methyl transfer from N5-methyltetrahydromethanopterin to coenzyme M in the oxidative part of the energy metabolism is coupled with the buildup of an electrochemical Na+ gradient (Becher et al, 1992a, b ;Lienard et al, 1996). In turn, the reduction of the heterodisulphide in the reductive part is coupled with electrogenic H+ translocation (Peinemann et al, 1990;Deppenmeier et al, 1990aDeppenmeier et al, , b, 1991Sparling et al, 1993).…”

“…The methyl group of CH,-H,MPT is transferred to coenzyme M in an exergonic reaction (AGO' = -30 kJ mol-l) coupled with energy conservation via an electrochemical sodium potential across the cytoplasmic membrane (Becher et al, 1992a, b;Becher & Miiller, 1994;Muller & Gottschalk, 1994;Lienard et al, 1996;Lienard & Gottschalk, 1998).…”

Energy Conservation in Acetogenic Bacteria

“…With the discovery that methanogenic archaea that also use in part the Wood-Ljungdahl pathway are strictly dependend on Na ϩ for CO 2 reduction (53, 54) and that they couple the methyltetrahydromethanopterin:coenzyme M methyltransferase to vectorial Na ϩ transport across the cytoplasmic membrane (37,45,49), it was obvious to look for Na ϩ dependence of acetogenesis. These studies revealed a Na ϩ dependence for growth and acetate formation in A. woodii (26), Thermoanaerobacter kivui (formerly Acetogenium kivui) (78), and Ruminococcus productus (formerly Peptostreptococcus productus) (20).…”

Cloning, sequencing and expression of the genes encoding the sodium translocating N5‐methyltetrahydromethanopterin:coenzyme M methyltransferase of the methylotrophic archaeon Methanosarcina mazei Gö1

“…The protein has been reported to be composed of at least six di¡erent polypeptides with apparent molecular masses of 34, 28, 20, 13, 12, and 9 kDa. The Nterminal amino acid sequences have been determined [3].…”

“…The enzyme was puri¢ed from acetate-grown Ms. mazei Go ë 1, reconstituted in ether lipid liposomes of the same organism, and thereby identi¢ed as being responsible for the translocation of sodium ions across the membrane [3]. The protein has been reported to be composed of at least six di¡erent polypeptides with apparent molecular masses of 34, 28, 20, 13, 12, and 9 kDa.…”