Yihx-encoded haloacid dehalogenase-like phosphatase HAD4 from Escherichia coli 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 substratespecificity which limits their application to substrate-selective biotransformations. In search of a phos-phatase catalyst specific for hydrolyzing α-d-glucose 1-phosphate (αGlc 1-P), we selected haloaciddehalogenase-like phosphatase 4 (HAD4) from Escherichia coli and obtained highly active recombinantenzyme through a fusion protein (Zbasic₂HAD4) that contained Zbasic₂, a strongly positively chargedthree α-helical bundle module, at its N-terminus. Highly pure Zbasic₂HAD4 was prepared directly fromE. coli cell extract using capture and polishing combined in a single step of cation exchange chro-matography. Kinetic studies showed Zbasic₂HAD4 to exhibit 565-fold preference for hydrolyzing αGlc 1-P (k_cat/K_M= 1.87 ± 0.03 mM^?1s^?1; 37°C, pH 7.0) as compared to d-glucose 6-phosphate (Glc 6-P). Alsoamong other sugar phosphates, αGlc 1-P was clearly preferred. Using different mixtures of αGlc 1-P and Glc 6-P (e.g. 180 mM each) as the substrate, Zbasic₂HAD4 could be used to selectively convert the αGlc1-P present, leaving back all of the Glc 6-P for recovery. Zbasic₂HAD4 was immobilized conveniently usingdirect loading of E. coli cell extract on sulfonic acid group-containing porous carriers, yielding a recyclableheterogeneous biocatalyst that was nearly as effective as the soluble enzyme, probably because proteinattachment to the anionic surface occurred in a preferred orientation via the cationic Zbasic₂module.Selective removal of αGlc 1-P from sugar phosphate preparations could be an interesting application ofZbasic₂HAD4 for which readily available broad-spectrum phosphatases are unsuitable.