Abstract
UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) catalyzes the deacetylation of UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine to form UDP-3-O-myristoylglucosamine and acetate in Gram-negative bacteria. This second, and committed, step in lipid A biosynthesis is a target for antibiotic development. LpxC was previously identified as a mononuclear Zn(II) metalloenzyme; however, LpxC is 6-8-fold more active with the oxygen-sensitive Fe(II) cofactor (Hernick, M., Gattis, S. G., Penner-Hahn, J. E., and Fierke, C. A. (2010) Biochemistry 49, 2246-2255). To analyze the native metal cofactor bound to LpxC, we developed a pulldown method to rapidly purify tagged LpxC under anaerobic conditions. The metal bound to LpxC purified from Escherichia coli grown in minimal medium is mainly Fe(II). However, the ratio of iron/zinc bound to LpxC varies with the metal content of the medium. Furthermore, the iron/zinc ratio bound to native LpxC, determined by activity assays, has a similar dependence on the growth conditions. LpxC has significantly higher affinity for Zn(II) compared with Fe(II) with K(D) values of 60 ± 20 pM and 110 ± 40 nM, respectively. However, in vivo concentrations of readily exchangeable iron are significantly higher than zinc, suggesting that Fe(II) is the thermodynamically favored metal cofactor for LpxC under cellular conditions. These data indicate that LpxC expressed in E. coli grown in standard medium predominantly exists as the Fe(II)-enzyme. However, the metal cofactor in LpxC can switch between iron and zinc in response to perturbations in available metal ions. This alteration may be important for regulating the LpxC activity upon changes in environmental conditions and may be a general mechanism of regulating the activity of metalloenzymes.
Highlights
Lipid A biosynthetic pathway (Fig. 1A) [1, 2], an essential building block of lipopolysaccharides (LPS) that make up the outer leaflet surrounding the cell wall in Gram-negative bacteria [2,3,4]
LpxC Purified from E. coli Contains Mainly Bound Fe(II)— LpxC purified from E. coli was previously shown to contain bound Zn(II); this protein was purified under aerobic conditions with 2 mM dithiothreitol as the reducing agent [15], which is not sufficient to prevent oxidation of bound Fe(II) [18]
To clarify the identity of the biologically relevant metal ion cofactor, we purified LpxC under anaerobic conditions to reexamine the identity of the native metal cofactor
Summary
Lipid A biosynthetic pathway (Fig. 1A) [1, 2], an essential building block of lipopolysaccharides (LPS) that make up the outer leaflet surrounding the cell wall in Gram-negative bacteria [2,3,4]. The combination of the higher concentration of readily exchangeable Fe(II) and the enhanced reactivity of Fe(II)-bound LpxC [18] suggest that the native cofactor bound to LpxC in E. coli might be Fe(II) rather than Zn(II) To test this hypothesis, we developed rapid purification methods and activity assays to analyze the metal ion status of LpxC in vivo. We developed rapid purification methods and activity assays to analyze the metal ion status of LpxC in vivo Using these methods, we demonstrate that under normal growth conditions iron-bound LpxC predominates. LpxC binds Zn(II) with much higher affinity than Fe(II), the higher cellular concentration of Fe(II)free contributes to the formation of Fe(II)-bound LpxC in vivo These data indicate that Fe(II) is the biologically relevant cofactor for LpxC under common growth conditions, metal switching occurs in response to metal availability. Metal switching may be important for regulating the LpxC activity upon changes in cellular conditions
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