Ybiv from Escherichia coli K12 is a HAD phosphatase

Abstract

The protein YbiV from Escherichia coli K12 MG1655 is a hypothetical protein with sequence homology to the haloacid dehalogenase (HAD) superfamily of proteins. Although numerous members of this family have been identified, the functions of few are known. Using the crystal structure, sequence analysis, and biochemical assays, we have characterized YbiV as a HAD phosphatase. The crystal structure of YbiV reveals a two-domain protein, one with the characteristic HAD hydrolase fold, the other an inserted alpha/beta fold. In an effort to understand the mechanism, we also solved and report the structures of YbiV in complex with beryllofluoride (BeF3-) and aluminum trifluoride (AlF3), which have been shown to mimic the phosphorylated intermediate and transition state for hydrolysis, respectively, in analogy to other HAD phosphatases. Analysis of the structures reveals the substrate-binding cavity, which is hydrophilic in nature. Both structure and sequence homology indicate YbiV may be a sugar phosphatase, which is supported by biochemical assays that measured the release of free phosphate on a number of sugar-like substrates. We also investigated available genomic and functional data in an effort to determine the physiological substrate.

The α-Phosphoglucomutase of Lactococcus lactis Is Unrelated to the α-d-Phosphohexomutase Superfamily and Is Encoded by the Essential Gene pgmH

“…1 and 3), an unrelated group of proteins that, despite their phosphohexomutase activity, belong to the HAD superfamily. The pgmH product features the conserved sequence motifs characteristic of the HAD superfamily (38,39,42,43) and is similar in size to the eukaryotic PMMs (Fig. 1).…”

“…ac.uk/cgi-bin/Pfam). Despite the low overall identity, the ␣-PGM sequence contains the four conserved motifs that characterize the HAD superfamily: motif I (DXDX(T/V)), motif II ((S/T)XX), and motifs III and IV (KX 18 -30 (G/S)(D/S)) (38,39).…”

Genome-wide Analysis of Substrate Specificities of the Escherichia coli Haloacid Dehalogenase-like Phosphatase Family

“…The biochemically and structurally studied HADs include phosphoserine phosphatase SerB from Methanococcus jannaschii (5), phosphoglycolate phosphatase from Thermoplasma acidophilum (6), phosphonacetaldehyde hydrolase from Bacillus cereus (7), ␤-phosphoglucomutase from Lactococcus lactis (8), haloacid dehalogenases from Pseudomonas sp. YL (9), and Xanthobacter autotrophicus (10), and two E. coli phosphatases, YbiV and NagD (11,12). However, the vast majority of HADs remains uncharacterized.…”

“…None of the E. coli HADs is essential for bacterial growth (13). The physiological substrates have been experimentally identified for three soluble E. coli HADs, namely phosphoglycolate phosphatase Gph, 3-deoxy-D-manno-octulosonate 8-phosphate phosphatase YrbI, and trehalose 6-phosphatase OtsB (14 -16) Only three enzymes (Gph, YrbI, and NagD) have been characterized biochemically (12,14,15), and three-dimensional structures have been resolved for YbiV and NagD (11,12).…”

Structure-based inference of molecular functions of proteins of unknown function from Berkeley Structural Genomics Center

“…Thus, the crystal structure immediately suggests a phosphatase function of TM0651 with a carbohydrate molecule as a substrate. The molecular function was proved later by biochemical assays with a TM0651 homologue, YbiV from E. coli which hydrolyzes a phosphate from various sugar-like substrates [14]. (GI 4982034) from T. maritima [16], TM1717 from T. maritima (GI 4982294) [17], AF2373 (GI 2650718) from Archaeoglobus fulgidus [18], all of which are the homologues of M. pneumoniae and M. genitalium proteins.…”