Crystal Structure and Functional Assignment of YfaU, a Metal Ion Dependent Class II Aldolase from Escherichia coli K12
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- 作者：Dean Rea ; Rebecca Hovington ; John F. Rakus ; John A. Gerlt ; Vilmos Fü ; lö ; p ; Timothy D. H. Bugg ; David I. Roper
- 刊名：Biochemistry
- 出版年：2008
- 出版时间：September 23, 2008
- 年：2008
- 卷：47
- 期：38
- 页码：9955-9965
- 全文大小：549K
- 年卷期：v.47,no.38(September 23, 2008)
- ISSN：1520-4995
文摘
One of the major challenges in the postgenomic era is the functional assignment of proteins using sequence- and structure-based predictive methods coupled with experimental validation. We have used these approaches to investigate the structure and function of the Escherichia coli K-12 protein YfaU, annotated as a putative 4-hydroxy-2-ketoheptane-1,7-dioate aldolase (HpcH) in the sequence databases. HpcH is the final enzyme in the degradation pathway of the aromatic compound homoprotocatechuate. We have determined the crystal structure of apo-YfaU and the Mg2+−pyruvate product complex. Despite greater sequence and structural similarity to HpcH, genomic context suggests YfaU is instead a 2-keto-3-deoxy sugar aldolase like the homologous 2-dehydro-3-deoxygalactarate aldolase (DDGA). Enzyme kinetic measurements show activity with the probable physiological substrate 2-keto-3-deoxy-l-rhamnonate, supporting the functional assignment, as well as the structurally similar 2-keto-3-deoxy-l-mannonate and 2-keto-3-deoxy-l-lyxonate (see accompanying paper: Rakus, J. F., Fedorov, A. A., Fedorov, E. V., Glasner, M. E., Hubbard, B. K., Delli, J. D., Babbitt, P. C., Almo, S. C., and Gerlt, J. A. (2008) Biochemistry 47, 9944−9954). YfaU has similar activity toward the HpcH substrate 4-hydroxy-2-ketoheptane-1,7-dioate and synthetic substrates 4-hydroxy-2-ketopentanoic acid and 4-hydroxy-2-ketohexanoic acid. This indicates a relaxed substrate specificity that complicates the functional assignment of members of this enzyme superfamily. Crystal structures suggest these enzymes use an Asp-His intersubunit dyad to activate a metal-bound water or hydroxide for proton transfer during catalysis.