-encoded haloacid dehalogenase-like phosphatase HAD4 from is a specific α-d-glucose 1-phosphate hydrolase useful for substrate-selective sugar phosphate transformations.

Phosphomonoester hydrolases (phosphatases; EC 3.1.3.) often exhibit extremely relaxed substrate specificity which limits their application to substrate-selective biotransformations. In search of a phosphatase catalyst specific for hydrolyzing α-d-glucose 1-phosphate (αGlc 1-), we selected haloacid dehalogenase-like phosphatase 4 (HAD4) from and obtained highly active recombinant enzyme through a fusion protein (Z_HAD4) that contained Z, a strongly positively charged three α-helical bundle module, at its N-terminus. Highly pure Z_HAD4 was prepared directly from cell extract using capture and polishing combined in a single step of cation exchange chromatography. Kinetic studies showed Z_HAD4 to exhibit 565-fold preference for hydrolyzing αGlc 1- (/ = 1.87 ± 0.03 mM s; 37 °C, pH 7.0) as compared to d-glucose 6-phosphate (Glc 6-). Also among other sugar phosphates, αGlc 1- was clearly preferred. Using different mixtures of αGlc 1- and Glc 6- (e.g. 180 mM each) as the substrate, Z_HAD4 could be used to selectively convert the αGlc 1- present, leaving back all of the Glc 6- for recovery. Z_HAD4 was immobilized conveniently using direct loading of cell extract on sulfonic acid group-containing porous carriers, yielding a recyclable heterogeneous biocatalyst that was nearly as effective as the soluble enzyme, probably because protein attachment to the anionic surface occurred in a preferred orientation via the cationic Z module. Selective removal of αGlc 1- from sugar phosphate preparations could be an interesting application of Z_HAD4 for which readily available broad-spectrum phosphatases are unsuitable.