UDP-3-O-(R-3-Hydroxymyristoyl)-N-acetylglucosamine Deacetylase of Escherichia coli Is a Zinc Metalloenzyme

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• 作者：Jane E. Jackman ; Christian R. H. Raetz ; and Carol A. Fierke
• 刊名：Biochemistry
• 出版年：1999
• 出版时间：February 9, 1999
• 年：1999
• 卷：38
• 期：6
• 页码：1902 - 1911
• 全文大小：139K
• 年卷期：v.38,no.6(February 9, 1999)
• ISSN：1520-4995

文摘

The enzyme UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc deacetylase (LpxC) catalyzes thecommitted step in the biosynthesis of lipid A and is therefore a potential antibiotic target. Inhibition ofthis enzyme by hydroxamate compounds [Onishi, H. R.; Pelak, B. A.; Gerckens, L. S.; Silver, L. L.;Kahan, F. M.; Chen, M. H.; Patchett, A. A.; Stachula, S. S.; Anderson, M. S.; Raetz, C. R. H. (1996)Science 274, 980-982] suggested the presence of a metal ion cofactor. We have investigated the substratespecificity and metal dependence of the deacetylase using spectroscopic and kinetic analyses. Comparisonof the steady-state kinetic parameters for the physiological substrate UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc and an alternative substrate, UDP-GlcNAc, demonstrates that the ester-linked R-3-hydroxymyristoylchain increases k_cat/K_M (5 × 10⁶)-fold. Metal-chelating reagents, such as dipicolinic acid (DPA) andethylenediaminetetraacetic acid, completely inhibit LpxC activity, implicating an essential metal ion. Plasmaemission spectroscopy and colorimetric assays directly demonstrate that purified LpxC contains boundZn²⁺. This Zn²⁺ can be removed by incubation with DPA, causing a decrease in the LpxC activity thatcan be restored by subsequent addition of Zn²⁺. However, high concentrations of Zn²⁺ also inhibit LpxC.Addition of Co²⁺, Ni²⁺, or Mn²⁺ to apo-LpxC also activates the enzyme to varying degrees while noadditional activity is observed upon the addition of Cd²⁺, Ca²⁺, Mg²⁺, or Cu²⁺. This is consistent withthe profile of metals that substitute for catalytic zinc ions in metalloproteinases. Co²⁺ ions stimulate LpxCactivity maximally at a stoichiometry of 1:1. These data demonstrate that E. coli LpxC is a metalloenzymethat requires bound Zn²⁺ for optimal activity.