Structural and functional studies of Bacillus stearothermophilus serine hydroxymethyltransferase: the role of Asn(341), Tyr(60) and Phe(351) 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 beta-hydroxy amino acids, transamination, racemization and decarboxylation. In the present study, the residues Asn(341), Tyr(60) and Phe(351), which are likely to influence THF binding, were mutated to alanine, alanine and glycine respectively, to elucidate the role of these residues in THF-dependent 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 Phe(351) 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 Asn(341) and Tyr(60) are pivotal for the binding of THF/FTHF, whereas Phe(351) is responsible for the asymmetric binding of FTHF in the two subunits of the dimer.
Pai
Vinitha R
VR
Department of Biochemistry, Indian Institute of Science, Bangalore, India.
Rajaram
V
V
Bisht
Shveta
S
Bhavani
B S
BS
Rao
N Appaji
NA
Murthy
M R N
MR
Savithri
H S
HS
D000595:Amino Acid Sequence; D001216:Asparagine / Q000378:metabolism*; D001411:Geobacillus stearothermophilus / Q000201:enzymology / Q000235:genetics; D012696:Glycine Hydroxymethyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D002955:Leucovorin / Q000378:metabolism; D010649:Phenylalanine / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D013763:Tetrahydrofolates / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*
D013763:Tetrahydrofolates; D014443:Tyrosine; C030371:5,6,7,8-tetrahydrofolic acid; D010649:Phenylalanine; D001216:Asparagine; D012696:Glycine Hydroxymethyltransferase; D002955:Leucovorin