Structural and functional studies of Bacillus stearothermophilus serine hydroxymethyltransferase: the role of Asn341, Tyr60 and Phe351 in tetrahydrofolate binding

SHMT (serine hydoxymethyltransferase), a type I pyridoxal 5'-
phosphate-dependent enzyme, catalyses the conversion of L-serine
and THF (tetrahydrofolate) into glycine and 5,10-methylene THF.
SHMT also catalyses several THF-independent side reactions such
as cleavage of β-hydroxy amino acids, transamination,
racemization and decarboxylation. In the present study, the
residues Asn341, Tyr60 and Phe351, which are likely to influence
THF binding, were mutated to alanine, alanine and glycine
respectively, to elucidate the role of these residues in THFdependent
and -independent reactions catalysed by SHMT. The
N341A and Y60A bsSHMT (Bacillus stearothermophilus SHMT)
mutants were inactive for the THF-dependent activity, while the
mutations had no effect on THF-independent activity. However,
mutation of Phe351 to glycine did not have any effect on either of the
activities. The crystal structures of the glycine binary complexes of
the mutants showed that N341A bsSHMT
forms an external aldimine as in bsSHMT, whereas Y60A and
F351G bsSHMTs exist as a mixture of internal/external aldimine
and gem-diamine forms. Crystal structures of all of the three
mutants obtained in the presence of L-allo-threonine were similar
to the respective glycine binary complexes. The structure of the
ternary complex of F351G bsSHMT with glycine and FTHF (5-
formyl THF) showed that the monoglutamate side chain of FTHF
is ordered in both the subunits of the asymmetric unit, unlike in the
wild-type bsSHMT. The present studies demonstrate that the
residues Asn341 and Tyr60 are pivotal for the binding of THF/FTHF,
whereas Phe351 is responsible for the asymmetric binding of FTHF
in the two subunits of the dimer.