TITLE:
Electron Donor Systems to Facilitate Development of Assays for Two Flavoproteins Involved in Tetrahydromethanopterin Biosynthesis
AUTHORS:
Chao Pang, Jose Moscaira, Jenny Gong, Madeline E. Rasche
KEYWORDS:
Methanopterin, Coenzyme Biosynthesis, Dihydromethanopterin Reductase, Flavoprotein, Archaea, Methanogenesis
JOURNAL NAME:
Advances in Microbiology,
Vol.10 No.6,
June
28,
2020
ABSTRACT: Methane production by archaea depends on tetrahydromethanopterin (H4MPT),
a pterin-containing cofactor that carries one-carbon units. Two redox reactions
within the nine steps of H4MPT side chain biosynthesis have been
hypothesized. Biochemical assays have demonstrated that the archaeal
iron-sulfur flavoprotein dihydromethanopterin reductase X (DmrX or MM1854)
catalyzes the final reaction of the pathway, the reduction of
dihydromethanopterin to H4MPT, using
dithiothreitol (DTT) as an artificial electron donor. The crystal structure of
DmrB, a bacterial DmrX homolog that lacks iron-sulfur clusters, has led to a
proposed ping-pong mechanism of electron transfer between FMNH2 and
the FMN prosthetic group of DmrB. However, an enzymatic assay to test the
hypothetical DmrB mechanism is lacking because a suitable electron donor has
not previously been identified. Furthermore, a second uncharacterized archaeal
flavoprotein (MM1853) has been hypothesized to function in H4MPT
side chain biosynthesis. In this work, to
facilitate the development of assays to elucidate the functions of DmrB and
MM1853, we tested a variety of electron donors, including dithiothreitol,
ferredoxin, and a system consisting of NADH and an NADH-dependent flavin-reducing enzyme (Fre). Reduction of the
DmrB prosthetic group (FMN) was measured as a decrease in absorbance at 460 nm.
NADPH, NADH, and DTT were unable to reduce
DmrB. However, NADH/Fre was able to reduce DmrB within 70 min (initial rate of 1.3 μM/min), providing the basis for
a future DmrB activity assay. Carbon monoxide (CO)/CO dehydrogenase/ferredoxin
reduced DmrB more rapidly within 6 min. Both electron transfer systems reduced
a second flavin-containing archaeal protein MM1853, which is predicted to
catalyze the third step of H4MPT biosynthesis. While NADH and NADPH
were incapable of directly reducing the FMN cofactor of MM1853, DTT or NADH/Fre
could eliminate the FMN peaks. These results establish the basis for new
oxidoreductase assays that will facilitate testing several proposed DmrB
mechanisms and defining the specific function of MM1853 in methanogen cofactor
biosynthesis.