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Abstract

Methylotrophy, the ability of microorganisms to grow on reduced
one-carbon substrates such as methane or methanol, is a feature of
various bacterial species. The prevailing oxidation pathway depends on
tetrahydromethanopterin (H4MPT) and methylofuran (MYFR), an analog of
methanofuran from methanogenic archaea. Formyltransferase/hydrolase
complex (Fhc) generates formate from formyl-H4MPT in two consecutive
reactions where MYFR acts as a carrier of one-carbon units. Recently, we
chemically characterized MYFR from the model methylotroph Methylorubrum
extorquens and identified an unusually long polyglutamate side chain of
up to 24 glutamates. Here, we report on the crystal structure of Fhc to
investigate the function of the polyglutamate side chain in MYFR and the
relatedness of the enzyme complex with the orthologous enzymes in
archaea. We identified MYFR as a prosthetic group that is tightly, but
noncovalently, bound to Fhc. Surprisingly, the structure of Fhc together
with MYFR revealed that the polyglutamate side chain of MYFR is branched
and contains glutamates with amide bonds at both their alpha- and
gamma-carboxyl groups. This negatively charged and branched
polyglutamate side chain interacts with a cluster of conserved
positively charged residues of Fhc, allowing for strong interactions.
The MYFR binding site is located equidistantly from the active site of
the formyltransferase (FhcD) and metallo-hydrolase (FhcA). The
polyglutamate serves therefore an additional function as a swinging
linker to shuttle the one-carbon carrying amine between the two active
sites, thereby likely increasing overall catalysis while decreasing the
need for high intracellular MYFR concentrations.