From structure to function: YrbI from Haemophilus influenzae (HI1679) is a phosphatase

Abstract

The crystal structure of the YrbI protein from Haemophilus influenzae (HI1679) was determined at a 1.67-A resolution. The function of the protein had not been assigned previously, and it is annotated as hypothetical in sequence databases. The protein exhibits the alpha/beta-hydrolase fold (also termed the Rossmann fold) and resembles most closely the fold of the L-2-haloacid dehalogenase (HAD) superfamily. Following this observation, a detailed sequence analysis revealed remote homology to two members of the HAD superfamily, the P-domain of Ca(2+) ATPase and phosphoserine phosphatase. The 19-kDa chains of HI1679 form a tetramer both in solution and in the crystalline form. The four monomers are arranged in a ring such that four beta-hairpin loops, each inserted after the first beta-strand of the core alpha/beta-fold, form an eight-stranded barrel at the center of the assembly. Four active sites are located at the subunit interfaces. Each active site is occupied by a cobalt ion, a metal used for crystallization. The cobalt is octahedrally coordinated to two aspartate side-chains, a backbone oxygen, and three solvent molecules, indicating that the physiological metal may be magnesium. HI1679 hydrolyzes a number of phosphates, including 6-phosphogluconate and phosphotyrosine, suggesting that it functions as a phosphatase in vivo. The physiological substrate is yet to be identified; however the location of the gene on the yrb operon suggests involvement in sugar metabolism.

Fig. 1

Electron-density maps (1.67-Å resolution) together with the final model. The active-site Co²⁺ region is shown. The coefficients (2F_o − F_c) and calculated phases are used. The map is contoured at the 1σ level.

Fig. 2

Crystal structure of HI1679 and ribbon diagram of the tetramer. The cobalt atoms are shown as blue spheres. The figure was generated with Molscript and Raster3D.

Fig. 3

(A) Schematic diagram of the secondary structure topology of the HI1679 monomer. Triangles represent β-strands, and circles represent α-helices; The location of the key catalytic residue, Asp14, is indicated by an arrow. (B) Stereoscopic view of the Cα-trace. Every 20th residue is labeled.

Fig. 4

Stereoscopic view of the monomer–monomer interface, highlighting the network of charged residues, and some hydrophobic interactions. Atomic colors are as follows: red, oxygen atoms; blue, nitrogen atoms; and gray or green, carbon atoms of the two neighboring molecules.

Fig. 5

Stereoscopic view of key residues in the active site. The coloring is the same used in Figure 4.

Fig. 6

Molecular surface of the active site, together with key residues, and modeled 6-phosphogluconate, a substrate of HI1679. The transparent blue surface delineates the second molecule associated with the active-site formation. Atomic colors are as follows: red, oxygen atoms; blue, nitrogen atoms; gray, protein carbon atoms; green, substrate carbon atoms; and yellow, phosphorus. The molecular surface was generated by the program MSMS and was incorporated into Molscript.. Raster3D was used to render the figure.

Fig. 7

Multiple sequence alignment of HI1679 with sequences obtained from the first iteration of a PSI-BLAST search. The alignment was constructed with ClustalW. Aligned sequences are identified by their designation in the Comprehensive Microbial Resource (www.tigr.org/CMR) with the following exceptions: TaroF4, the F4 protein from T. aromatica, and ScNeuAc, HsNeuAc, OmNeuAc, and MmNeuAc, the CMP–NeuAcS enzymes from S. coelicolor, H. sapiens, O. mykiss, and M. musculus, respectively. The secondary structural elements of HI1679 are shown as arrows (β-strands) and corkscrews (α-helices). Invariant residues are shaded in red. The catalytic residue (D14 of HI1679) and the metal ligands (D107 of HI1679) are shaded in yellow for those sequences in which they are conserved. Conserved HAD family sequence motifs 1–3 are indicated by circles, triangles, and inverted triangles, respectively.

Fig. 8

Ribbon representation of a HI1679 monomer (sky blue) superimposed on PSPase (yellow). The Co²⁺ ion bound to HI1679 and the Mg²⁺ ion bound to PSPase are shown as blue and red spheres, respectively.