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Biochemistry. 2007 Jan 9;46(1):129-36.

Acyl carrier protein phosphodiesterase (AcpH) of Escherichia coli is a non-canonical member of the HD phosphatase/phosphodiesterase family.

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1
Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.

Abstract

The Escherichia coli AcpH acyl carrier protein phosphodiesterase (also called ACP hydrolyase) is the only enzyme known to cleave a phosphodiester-linked post-translational protein modification. AcpH hydrolyzes the link between 4'-phosphopanthetheine and the serine-36 side chain of acyl carrier protein (ACP). Although the existence of this enzyme activity has long been known, study of the enzyme was hampered by its recalcitrant properties and scarcity. We recently isolated the gene encoding AcpH and have produced the recombinant enzyme in quantity (Thomas, J., and Cronan, J. E., (2005) J. Biol. Chem. 280, 34675-34683), thus allowing the first studies of its reaction mechanism. AcpH requires Mn2+ for activity, and thus, we focused on the metal binding ligands in order to locate the active site. Bioinformatic investigations indicated that AcpH and its homologues were weakly related to a phosphodiesterase of known structure, the hydrolyase domain of the bifunctional bacterial protein, SpoT, suggesting that AcpH is a member of the HD family of phosphatases/ phosphodiesterases despite lacking the characteristic histidine of the motif. Indeed, we found that AcpH could be convincingly modeled on the SpoT structure with acceptable parameters, which allowed the identification of putative metal binding ligands. These were then tested by site-directed mutagenesis. Mutagenic removal of any of the putative ligands resulted in a severe or total loss of phosphodiesterase activity. In two cases, the H6Q and D24N proteins, the residual activities could be markedly stimulated by addition of high Mn2+ concentrations, thereby demonstrating a role for these residues in metal binding. We conclude that AcpH is a member of the HD protein family despite the lack of the signature histidine residue.

PMID:
17198382
DOI:
10.1021/bi061789e
[Indexed for MEDLINE]
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