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.

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) from Escherichia coli and obtained highly active recombinant enzyme through a fusion protein (Z_basic2_HAD4) that contained Z_basic2, a strongly positively charged three α-helical bundle module, at its N-terminus. Highly pure Z_basic2_HAD4 was prepared directly from E. coli cell extract using capture and polishing combined in a single step of cation exchange chromatography. Kinetic studies showed Z_basic2_HAD4 to exhibit 565-fold preference for hydrolyzing αGlc 1-P (k_cat/K_M = 1.87 ± 0.03 mM^-1 s^-1; 37 °C, pH 7.0) as compared to d-glucose 6-phosphate (Glc 6-P). Also among 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, Z_basic2_HAD4 could be used to selectively convert the αGlc 1-P present, leaving back all of the Glc 6-P for recovery. Z_basic2_HAD4 was immobilized conveniently using direct loading of E. coli 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_basic2 module. Selective removal of αGlc 1-P from sugar phosphate preparations could be an interesting application of Z_basic2_HAD4 for which readily available broad-spectrum phosphatases are unsuitable.

SUBMITTER: Pfeiffer M 

PROVIDER: S-EPMC4251788 | biostudies-literature |

REPOSITORIES: biostudies-literature