Importance of tyrosine residues of Bacillus stearothermophilus serine hydroxymethyltransferase in cofactor binding and L-allo-Thr cleavage Crystal structure and biochemical studies

Serine hydroxymethyltransferase (SHMT) from Bacillus stearothermophilus
(bsSHMT) is a pyridoxal 5¢-phosphate-dependent enzyme that catalyses the
conversion of l-serine and tetrahydrofolate to glycine and 5,10-methylene
tetrahydrofolate. In addition, the enzyme catalyses the tetrahydrofolateindependent
cleavage of 3-hydroxy amino acids and transamination. In this
article, we have examined the mechanism of the tetrahydrofolate-independent
cleavage of 3-hydroxy amino acids by SHMT. The three-dimensional
structure and biochemical properties of Y51F and Y61A bsSHMTs and
their complexes with substrates, especially l-allo-Thr, show that the cleavage
of 3-hydroxy amino acids could proceed via Ca proton abstraction
rather than hydroxyl proton removal. Both mutations result in a complete
loss of tetrahydrofolate-dependent and tetrahydrofolate-independent activities.
The mutation of Y51 to F strongly affects the binding of pyridoxal
5¢-phosphate, possibly as a consequence of a change in the orientation of
the phenyl ring in Y51F bsSHMT. The mutant enzyme could be completely
reconstituted with pyridoxal 5¢-phosphate. However, there was an
alteration in the kmax value of the internal aldimine (396 nm), a decrease in
the rate of reduction with NaCNBH3 and a loss of the intermediate in the
interaction with methoxyamine (MA). The mutation of Y61 to A results in
the loss of interaction with Ca and Cb of the substrates. X-Ray structure
and visible CD studies show that the mutant is capable of forming an
external aldimine. However, the formation of the quinonoid intermediate is
hindered. It is suggested that Y61 is involved in the abstraction of the Ca
proton from 3-hydroxy amino acids. A new mechanism for the cleavage of
3-hydroxy amino acids via Ca proton abstraction by SHMT is proposed.