Biosynthesis of the Phosphodiester Bond in Coenzyme F420 in the Methanoarchaea

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• 作者：Marion Graupner and Robert H. White
• 刊名：Biochemistry
• 出版年：2001
• 出版时间：September 11, 2001
• 年：2001
• 卷：40
• 期：36
• 页码：10859 - 10872
• 全文大小：142K
• 年卷期：v.40,no.36(September 11, 2001)
• ISSN：1520-4995

文摘

The biochemical route for the formation of the phosphodiester bond in coenzyme F₄₂₀, one ofthe methanogenic coenzymes, has been established in the methanoarchaea Methanosarcina thermophilaand Methanococcus jannaschii. The first step in the formation of this portion of the F₄₂₀ structure is theGTP-dependent phosphorylation of L-lactate to 2-phospho-L-lactate and GDP. The 2-phospho-L-lactaterepresents 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. jannaschiiwith [hydroxy-¹⁸O, carboxyl-¹⁸O₂]lactate and GTP produced 2-phospho-L-lactate with the same ¹⁸Odistribution as found in both the starting lactate and the lactate recovered from the incubation. Theseresults indicate that the carboxyl oxygens are not involved in the phosphorylation reaction. Incubation ofSephadex G-25 purified cell extracts of M. thermophila 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 F₄₂₀-0 (F₄₂₀ withno glutamic acids). This transformation was shown to involve two steps: (i) the GTP- or ATP-dependentactivation of 2-phospho-L-lactate to either lactyl(2)diphospho-(5')guanosine (LPPG) or lactyl(2)diphospho-(5')adenosine (LPPA) and (ii) the reaction of the resulting LPPG or LPPA with Fo to form F₄₂₀-0 withrelease of GMP or AMP. Attempts to identify LPPG or LPPA intermediates by incubation of cell extractswith L-[U-¹⁴C]lactate, [U-¹⁴C]2-phospho-L-lactate, or [8-³H]GTP were not successful owing to the instabilityof these compounds toward hydrolysis. Synthetically prepared LPPG and LPPA had half-lives of 10 minat 50 tities/deg.gif">C (at pH 7.0) and decomposed into GMP or AMP and 2-phospho-L-lactate via cyclic 2-phospho-L-lactate. No evidence for the functioning of the cyclic 2-phospho-L-lactate in the in vitro biosynthesiscould be demonstrated. Incubation of cell extracts of M. thermophila or Mc. jannaschii with either LPPGor LPPA and Fo generated F₄₂₀-0. In summary, this study demonstrates that the formation of thephosphodiester bond in coenzyme F₄₂₀ follows a reaction scheme like that found in one of the steps of theDNA ligase reaction and in the biosynthesis of coenzyme B₁₂ and phospholipids.