Biosynthesis of the phosphodiester bond in coenzyme F-420 in the methanoarchaea

The biochemical route for the formation of the phosphodiester bond in coenz yme F-420, one of the methanogenic coenzymes, has been established in the m ethanoarchaea Methanosarcina thermophila and Methanococcus jannaschii. The first step in the formation of this portion of the F-420 structure is the G TP-dependent phosphorylation Of L-lactate to 2-phospho-L-lactate and GDP. T he 2-phospho-L-lactate represents a new natural product that was chemically identified in Methanobacterium thermoautotrophicum, M. thermophila, and Mc . jannaschii. Incubation of cell extracts of both M. thermophila and Mc. ja nnaschii with [hydroxy-O-18, carboxyl-O-18(2)] lactate and GTP produced 2-p hospho-L-lactate with the same O-18, distribution as found in both the star ting lactate and the lactate recovered from the incubation. These results i ndicate that the carboxyl oxygens are not involved in the phosphorylation r eaction. Incubation of Sephadex G-25 purified cell extracts of M. thermophi la or Mc. jannaschii with 7,8-didemethyl-8-hydroxy-5-deazariboflavin (Fo), 2-phospho-L-lactate, and GTP or ATP lead to the formation of F-420-0 (F-420 with no glutamic acids). This transformation was shown to involve two step s: (i) the GTP- or ATP-dependent activation of 2-phospho-L-lactate to eithe r lactyl(2)diphospho-(5')guanosine (LPPG) or lactyl(2)diphospho(5')adenosin e (LPPA) and (ii) the reaction of the resulting LPPG or LPPA with Fo to for m F-420-0 with release of GMP or AMP. Attempts to identify LPPG or LPPA int ermediates by incubation of cell extracts with L-[U-C-14]lactate, [U-C-14]2 -phospho-L-lactate, or [8-H-3]GTP were not successful owing to the instabil ity of these compounds toward hydrolysis. Synthetically prepared LPPG and L PPA had half-lives of 10 min at 50 degreesC (at pH 7.0) and decomposed into GMP or AMP and 2-phospho-L-lactate via cyclic 2-phosphol-lactate. No evide nce for the functioning of the cyclic 2-phospho-L-lactate in the in vitro b iosynthesis could be demonstrated. Incubation of cell extracts of M. thermo phila or Mc. jannaschii with either LPPG or LPPA and Fo generated F-420-0. In summary, this study demonstrates that the formation of the phosphodieste r bond in coenzyme F-420 follows a reaction scheme like that found in one o f the steps of the DNA ligase reaction and in the biosynthesis of coenzyme B-12 and phospholipids.