Biosynthesis of the Phosphodiester Bond in Coenzyme F₄₂₀in the Methanoarchaea

Abstract

The biochemical route for the formation of the phosphodiester bond in coenzyme F(420), one of the methanogenic coenzymes, has been established in the methanoarchaea Methanosarcina thermophila and Methanococcus jannaschii. The first step in the formation of this portion of the F(420) structure is the GTP-dependent phosphorylation of L-lactate to 2-phospho-L-lactate and GDP. The 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. jannaschii with [hydroxy-(18)O, carboxyl-(18)O(2)]lactate and GTP produced 2-phospho-L-lactate with the same (18)O distribution as found in both the starting lactate and the lactate recovered from the incubation. These results indicate that the carboxyl oxygens are not involved in the phosphorylation reaction. Incubation of Sephadex 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 F(420)-0 (F(420) with no glutamic acids). This transformation was shown to involve two steps: (i) the GTP- or ATP-dependent activation 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 F(420)-0 with release of GMP or AMP. Attempts to identify LPPG or LPPA intermediates by incubation of cell extracts with L-[U-(14)C]lactate, [U-(14)C]2-phospho-L-lactate, or [8-(3)H]GTP were not successful owing to the instability of these compounds toward hydrolysis. Synthetically prepared LPPG and LPPA had half-lives of 10 min at 50 degrees 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 biosynthesis could be demonstrated. Incubation of cell extracts of M. thermophila or Mc. jannaschii with either LPPG or LPPA and Fo generated F(420)-0. In summary, this study demonstrates that the formation of the phosphodiester bond in coenzyme F(420) follows a reaction scheme like that found in one of the steps of the DNA ligase reaction and in the biosynthesis of coenzyme B(12) and phospholipids.

Physiology, Biochemistry, and Applications of F420- and Fo-Dependent Redox Reactions

“…Though unconfirmed, it is assumed that lactate (synthesized from glycolytic pyruvate by L-lactate dehydrogenase) is also the precursor for LPPG in bacteria. It has been shown in methanogens that lactate can be phosphorylated to form 2-phospho-Llactate in a GTP-dependent manner (116); however, the enzyme responsible (to be named CofB) has remained elusive in the 15 years since the reaction was discovered. Finally, the 2-phospho-Llactate is converted to LPPG by the GTP-dependent enzyme 2-phospho-L-lactate guanylyltransferase (CofC) (PDB ID 2I5E) (116,131).…”

“…In the first, the lactate-derived intermediate L-lactyl-2-diphospho-5=-guanosine (LPPG) is condensed with F o (116) to form the phosphodiester F 420 -0 (i.e., F 420 containing no glutamate side chain). This reaction is catalyzed by a 2-phospho-L-lactate transferase (named CofD in archaea and FbiA in actinobacteria) (117,118).…”

The methanogenic redox cofactor F420 is widely synthesized by aerobic soil bacteria

“…In the first, a riboflavin precursor (5-amino-6-(D-ribitylamino)uracil) is condensed with tyrosine to form 8-hydroxy-5-deazaflavin, also known as F o ; this step is catalyzed by the radical S-adenosylmethionine enzymes CofG and CofH that are fused into a single protein in some bacteria (known as CofGH or FbiC) (Choi et al, 2002;Philmus et al, 2015). Subsequently, LPPG (L-lactyl-2-diphospho-5 0 -guanosine) is proposed to be synthesized from 2-phospho-L-lactate by CofC (Grochowski et al, 2008) and transferred to F o by CofD (also known as FbiA) (Choi et al, 2001;Graupner and White, 2001;Graupner et al, 2002). The resulting LPPG sidechain is finally elongated with glutamate residues by the F 420 :γ-L-glutamyl ligase CofE (also known as FbiB) (Choi et al, 2001;Li et al, 2003;Nocek et al, 2007) that is fused with an FMNdependent oxidoreductase in Actinobacteria (Bashiri et al, 2016).…”

Methanococcus jannaschii Coenzyme F 420 Analogs Contain a Terminal α-Linked Glutamate

“…As part of our work to establish the genes and pathway involved in F 420 biosynthesis in Methanococcus jannaschii (13)(14)(15) we examined the F 420 species present in this euryarchaeon. These analyses established that the F 420 s present in these cells are unique and consist of a series of ␥-linked F 420 s capped with a single terminal ␣-linked L-glutamate.…”