Structure determination and biochemical studies on Bacillus stearothermophilus E53Q serine hydroxymethyltransferase and its complexes provide insights on function and enzyme memory

Serine hydroxymethyltransferase (SHMT) belongs to the a-family of
pyridoxal 5¢-phosphate-dependent enzymes and catalyzes the reversible
conversion of l-Ser and tetrahydrofolate to Gly and 5,10-methylene
tetrahydrofolate. 5,10-Methylene tetrahydrofolate serves as a source of
one-carbon fragment in many biological processes. SHMT also catalyzes
the tetrahydrofolate-independent conversion of l-allo-Thr to Gly and
acetaldehyde. The crystal structure of Bacillus stearothermophilus SHMT
(bsSHMT) suggested that E53 interacts with the substrate, l-Ser and tetrahydrofolate.
To elucidate the role of E53, it was mutated to Q and structural
and biochemical studies were carried out with the mutant enzyme.
The internal aldimine structure of E53QbsSHMT was similar to that of the
wild-type enzyme, except for significant changes at Q53, Y60 and Y61. The
carboxyl of Gly and side chain of l-Ser were in two conformations in
the respective external aldimine structures. The mutant enzyme was completely
inactive for tetrahydrofolate-dependent cleavage of l-Ser, whereas
there was a 1.5-fold increase in the rate of tetrahydrofolate-independent
reaction with l-allo-Thr. The results obtained from these studies suggest
that E53 plays an essential role in tetrahydrofolate ⁄ 5-formyl tetrahydrofolate
binding and in the proper positioning of Cb of l-Ser for direct
attack by N5 of tetrahydrofolate. Most interestingly, the structure of the
complex obtained by cocrystallization of E53QbsSHMT with Gly and
5-formyl tetrahydrofolate revealed the gem-diamine form of pyridoxal
5¢-phosphate bound to Gly and active site Lys. However, density for
5-formyl tetrahydrofolate was not observed. Gly carboxylate was in a single
conformation, whereas pyridoxal 5¢-phosphate had two distinct conformations.
The differences between the structures of this complex and Gly
external aldimine suggest that the changes induced by initial binding of
5-formyl tetrahydrofolate are retained even though 5-formyl tetrahydrofolate
is absent in the final structure. Spectral studies carried out with this
mutant enzyme also suggest that 5-formyl tetrahydrofolate binds to the
E53QbsSHMT-Gly complex forming a quinonoid intermediate and falls off within 4 h of dialysis, leaving behind the mutant enzyme in the gemdiamine
form. This is the first report to provide direct evidence for enzyme
memory based on the crystal structure of enzyme complexes.