Abstract
The Escherichia coli periplasmic glucose-1-phosphatase is a member of the histidine acid phosphatase family and acts primarily as a glucose scavenger. Previous substrate profiling studies have demonstrated some of the intriguing properties of the enzyme, including its unique and highly selective inositol phosphatase activity. The enzyme is also potentially involved in pathogenic inositol phosphate signal transduction pathways via type III secretion into the host cell. We have determined the crystal structure of E. coli glucose-1-phosphatase in an effort to unveil the structural mechanism underlying such unique substrate specificity. The structure was determined by the method of multiwavelength anomalous dispersion using a tungstate derivative together with the H18A inactive mutant complex structure with glucose 1-phosphate at 2.4-A resolution. In the active site of glucose-1-phosphatase, there are two unique gating residues, Glu-196 and Leu-24, in addition to the conserved features of histidine acid phosphatases. Together they create steric and electrostatic constraints responsible for the unique selectivity of the enzyme toward phytate and glucose-1-phosphate as well as its unusually high pH optimum for the latter. Based on the structural characterization, we were able to derive simple structural principles that not only precisely explains the substrate specificity of glucose-1-phosphatase and the hydrolysis products of various inositol phosphate substrates but also rationalizes similar general characteristics across the histidine acid phosphatase family.
Highlights
Encoded by the agp gene, Escherichia coli glucose-1-phosphatase (EC 3.1.3.10, D-glucose-1phosphate phosphohydrolase, hereafter abbreviated as G1Pase1) belongs to the high molecular weight acid phosphatases family, which is considered to be functionally conserved from prokaryotes to higher eukaryotes (1)
The Escherichia coli periplasmic glucose-1-phosphatase is a member of the histidine acid phosphatase family and acts primarily as a glucose scavenger
Despite the possession of the same active site residues (the RH(G/N)XRXP and downstream R and HD) found in other histidine acid phosphatases, a recent analysis of G1Pase by Cottrill et al (10) indicates the enzyme specificity toward inositol phosphates. This specific inositol phosphate activity is intriguing because several inositol phosphatases of the related Gram-negative Enterobacteriaceae family members, Salmonella and Shigella spp., were reported to be translocated directly into the eukaryotic host cell via type III secretion systems to interfere with a variety of host cell signaling pathways (16)
Summary
Wavelength (Å) Space group Cell axes a, b, c (Å) Resolution range (Å) No of observed reflections No of unique reflections Rsym (%)d,e Overall completeness (%)e Anomalous completeness (%)e No of non-H atoms (protein/water/ligand) R-factor/free R-factor f Root mean square deviations (bond lengths (Å)/angles (°)). 24.60/31.80 0.0075/1.43 a MAD phasing was calculated using 3 as a native and 1 and 2 as derivatives. C Phasing power ϭ͗FH(CALC)͉/͉E͘, where FH(CALC) is the calculated anomalous difference and E is the lack of closure. D Rsym ϭ ⌺͉I(k) ϪI͘/⌺I(k), where I(k) andIrepresent the diffraction intensities of the individual measurements and the corresponding mean values, respectively. In this paper we report structural studies of E. coli glucose-1-phosphatase along with its H18A inactive mutant structure in a complex with the natural substrate glucose 1-phosphate
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