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PDBsum entry 1yjs
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References listed in PDB file
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Key reference
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Title
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Role of lys-226 in the catalytic mechanism of bacillus stearothermophilus serine hydroxymethyltransferase--Crystal structure and kinetic studies.
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Authors
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S.Bhavani,
V.Trivedi,
V.R.Jala,
H.S.Subramanya,
P.Kaul,
V.Prakash,
N.Appaji rao,
H.S.Savithri.
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Ref.
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Biochemistry, 2005,
44,
6929-6937.
[DOI no: ]
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PubMed id
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Abstract
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Serine hydroxymethyltransferase (SHMT), a pyridoxal 5'-phosphate (PLP)-dependent
enzyme catalyzes the reversible conversion of l-Ser and
tetrahydropteroylglutamate (H(4)PteGlu) to Gly and 5,10-methylene
tetrahydropteroylglutamate (CH(2)-H(4)PteGlu). Biochemical and structural
studies on this enzyme have implicated several residues in the catalytic
mechanism, one of them being the active site lysine, which anchors PLP. It has
been proposed that this residue is crucial for product expulsion. However, in
other PLP-dependent enzymes, the corresponding residue has been implicated in
the proton abstraction step of catalysis. In the present investigation, Lys-226
of Bacillus stearothermophilus SHMT (bsSHMT) was mutated to Met and Gln to
evaluate the role of this residue in catalysis. The mutant enzymes contained 1
mol of PLP per mol of subunit suggesting that Schiff base formation with lysine
is not essential for PLP binding. The 3D structure of the mutant enzymes
revealed that PLP was bound at the active site in an orientation different from
that of the wild-type enzyme. In the presence of substrate, the PLP ring was in
an orientation superimposable with that of the external aldimine complex of
wild-type enzyme. However, the mutant enzymes were inactive, and the kinetic
analysis of the different steps of catalysis revealed that there was a drastic
reduction in the rate of formation of the quinonoid intermediate. Analysis of
these results along with the crystal structures suggested that K-226 is
responsible for flipping of PLP from one orientation to another which is crucial
for H(4)PteGlu-dependent Calpha-Cbeta bond cleavage of l-Ser.
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