Effect of a Y265F Mutant on the Transamination-Based Cycloserine Inactivation of Alanine Racemase
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- 作者：Timothy D. Fenn ; Todd Holyoak ; Geoffrey F. Stamper ; and Dagmar Ringe
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：April 12, 2005
- 年：2005
- 卷：44
- 期：14
- 页码：5317 - 5327
- 全文大小：787K
- 年卷期：v.44,no.14(April 12, 2005)
- ISSN：1520-4995
文摘
The requirement for D-alanine in the peptidoglycan layer of bacterial cell walls is fulfilled inpart by alanine racemase (EC 5.1.1.1), a pyridoxal 5'-phosphate (PLP)-assisted enzyme. The enzymeutilizes two antiparallel bases focused at the C
position and oriented perpendicular to the PLP ring tofacilitate the equilibration of alanine enantiomers. Understanding how this two-base system is utilizedand 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 isbased on transaminase chemistry. Here we characterize the effects of a Y265F mutant (Tyr265 acts as thecatalytic base in the L-isomer case) of Bacillus stearothermophilus alanine racemase on cycloserineinactivation. 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 isoxazoleend product (the result of the transaminase pathway) in the case of D-cycloserine, in contrast to resultsobtained using the wild-type enzyme. L-Cycloserine, on the other hand, utilizes a number of alternativepathways in the absence of Y265, emphasizing the importance of Y265 in both the inactivation andracemization pathway. In combination with the kinetics of inactivation, these results suggest roles foreach 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.
position and oriented perpendicular to the PLP ring tofacilitate the equilibration of alanine enantiomers. Understanding how this two-base system is utilizedand 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 isbased on transaminase chemistry. Here we characterize the effects of a Y265F mutant (Tyr265 acts as thecatalytic base in the L-isomer case) of Bacillus stearothermophilus alanine racemase on cycloserineinactivation. 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 isoxazoleend product (the result of the transaminase pathway) in the case of D-cycloserine, in contrast to resultsobtained using the wild-type enzyme. L-Cycloserine, on the other hand, utilizes a number of alternativepathways in the absence of Y265, emphasizing the importance of Y265 in both the inactivation andracemization pathway. In combination with the kinetics of inactivation, these results suggest roles foreach 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.