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PMID:20709752

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Citation

Gattis, SG, Hernick, M and Fierke, CA (2010) Active site metal ion in UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) switches between Fe(II) and Zn(II) depending on cellular conditions. J. Biol. Chem. 285:33788-96

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.

Links

PubMed PMC2962478 Online version:10.1074/jbc.M110.147173

Keywords

Amidohydrolases/chemistry; Catalytic Domain; Escherichia coli/enzymology; Escherichia coli/metabolism; Immunoglobulin G/chemistry; Iron/chemistry; Kinetics; Metalloproteins/chemistry; Metals/chemistry; Models, Chemical; Models, Statistical; Plasmids/metabolism; Protein Binding; Thermodynamics; Zinc/chemistry

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

ECOLI:LPXC

enables

GO:0005506: iron ion binding

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

ECOBD:C6EAV9

GO:0008759: UDP-3-O-(3-hydroxymyristoyl) N-acetylglucosamine deacetylase activity

ECO:0000314:

F

Native LpxC activity varies with metal supplementation in the growth medium. E. coli BL21(DE3) cells (without LpxC expression plasmid) were grown in minimal medium with and without 20 μm metal supplementation, lysed, and assayed for deacetylase activity as described under “Materials and Methods” either anaerobically (black bars) or after exposure to room oxygen for 2.5 h (gray bars).

complete


See also

References

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