title: Yihx-encoded haloacid dehalogenase-like phosphatase HAD4 fromEscherichia coliis a specific α-d-glucose 1-phosphate hydrolase useful for substrate-selective sugar phosphate transformations.
abstract: 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-P), we selected haloacid dehalogenase-like phosphatase 4 (HAD4) fromEscherichia coliand obtained highly active recombinant enzyme through a fusion protein (Zbasic2_HAD4) that contained Zbasic2, a strongly positively charged three α-helical bundle module, at its N-terminus. Highly pure Zbasic2_HAD4 was prepared directly fromE. colicell extract using capture and polishing combined in a single step of cation exchange chromatography. Kinetic studies showed Zbasic2_HAD4 to exhibit 565-fold preference for hydrolyzing αGlc 1-P(kcat/KM= 1.87 ± 0.03 mM-1s-1; 37 °C, pH 7.0) as compared to d-glucose 6-phosphate (Glc 6-P). Also among other sugar phosphates, αGlc 1-Pwas clearly preferred. Using different mixtures of αGlc 1-Pand Glc 6-P(e.g. 180 mM each) as the substrate, Zbasic2_HAD4 could be used to selectively convert the αGlc 1-Ppresent, leaving back all of the Glc 6-Pfor recovery. Zbasic2_HAD4 was immobilized conveniently using direct loading ofE. colicell 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 Zbasic2module. Selective removal of αGlc 1-Pfrom sugar phosphate preparations could be an interesting application of Zbasic2_HAD4 for which readily available broad-spectrum phosphatases are unsuitable.
authors.affiliation: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/1, A-8010 Graz, Austria.
authors.forename: Martin
authors.initials: M
authors.lastname: Pfeiffer
authors.affiliation: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/1, A-8010 Graz, Austria.
authors.forename: Patricia
authors.initials: P
authors.lastname: Wildberger
authors.affiliation: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/1, A-8010 Graz, Austria ; ACIB - Austrian Centre of Industrial Biotechnology, Petersgasse 14, A-8010 Graz, Austria.
authors.forename: Bernd
authors.initials: B
authors.lastname: Nidetzky
keywords: HAD superfamily; Oriented immobilization; Phosphatase; Substrate selectivity; Sugar phosphate