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PDBsum entry 1xql
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References listed in PDB file
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Key reference
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Title
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Effect of a y265f mutant on the transamination-Based cycloserine inactivation of alanine racemase.
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Authors
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T.D.Fenn,
T.Holyoak,
G.F.Stamper,
D.Ringe.
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Ref.
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Biochemistry, 2005,
44,
5317-5327.
[DOI no: ]
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PubMed id
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Abstract
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The requirement for d-alanine in the peptidoglycan layer of bacterial cell walls
is fulfilled in part by alanine racemase (EC 5.1.1.1), a pyridoxal 5'-phosphate
(PLP)-assisted enzyme. The enzyme utilizes two antiparallel bases focused at the
C(alpha) position and oriented perpendicular to the PLP ring to facilitate the
equilibration of alanine enantiomers. Understanding how this two-base system is
utilized and controlled to yield reaction specificity is therefore a potential
means for designing antibiotics. Cycloserine is a known alanine racemase suicide
substrate, although its mechanism of inactivation is based on transaminase
chemistry. Here we characterize the effects of a Y265F mutant (Tyr265 acts as
the catalytic base in the l-isomer case) of Bacillus stearothermophilus alanine
racemase on cycloserine inactivation. The Y265F mutant reduces racemization
activity 1600-fold [Watanabe, A., Yoshimura, T., Mikami, B., and Esaki, N.
(1999) J. Biochem. 126, 781-786] and only leads to formation of the isoxazole
end product (the result of the transaminase pathway) in the case of
d-cycloserine, in contrast to results obtained using the wild-type enzyme.
l-Cycloserine, on the other hand, utilizes a number of alternative pathways in
the absence of Y265, emphasizing the importance of Y265 in both the inactivation
and racemization pathway. In combination with the kinetics of inactivation,
these results suggest roles for each of the two catalytic bases in racemization
and inactivation, as well as the importance of Y265 in "steering" the chemistry
to favor one pathway over another.
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