TY - JOUR
T1 - 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
AU - Pfeiffer, Martin
AU - Wildberger, Patricia
AU - Nidetzky, Bernd
PY - 2014
Y1 - 2014
N2 - 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 (kcat/KM= 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.
AB - 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 (kcat/KM= 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.
KW - HAD superfamily
KW - Oriented immobilization
KW - Phosphatase
KW - Substrate selectivity
KW - Sugar phosphate
UR - http://www.scopus.com/inward/record.url?scp=84908006709&partnerID=8YFLogxK
U2 - 10.1016/j.molcatb.2014.09.004
DO - 10.1016/j.molcatb.2014.09.004
M3 - Article
AN - SCOPUS:84908006709
SN - 1381-1177
VL - 110
SP - 39
EP - 46
JO - Journal of Molecular Catalysis B
JF - Journal of Molecular Catalysis B
ER -