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

Martin Pfeiffer, Patricia Wildberger, Bernd Nidetzky

Research output: Contribution to journalArticleResearchpeer-review

Abstract

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.

Original languageEnglish
Pages (from-to)39-46
Number of pages8
JournalJournal of molecular catalysis / B
Volume110
DOIs
Publication statusPublished - 2014

Fingerprint

Sugar Phosphates
Hydrolases
Phosphatases
Phosphoric Monoester Hydrolases
Escherichia coli
Glucose
Phosphates
Glucose-6-Phosphate
Substrates
Cell Extracts
Sulfonic Acids
Enzymes
Biotransformation
Polishing
Cations
Fusion reactions
Biocatalysts
glucose-1-phosphate
2-haloacid dehalogenase
Recovery

Keywords

  • HAD superfamily
  • Oriented immobilization
  • Phosphatase
  • Substrate selectivity
  • Sugar phosphate

ASJC Scopus subject areas

  • Biochemistry
  • Bioengineering
  • Catalysis
  • Process Chemistry and Technology

Cite this

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title = "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 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.",
keywords = "HAD superfamily, Oriented immobilization, Phosphatase, Substrate selectivity, Sugar phosphate",
author = "Martin Pfeiffer and Patricia Wildberger and Bernd Nidetzky",
year = "2014",
doi = "10.1016/j.molcatb.2014.09.004",
language = "English",
volume = "110",
pages = "39--46",
journal = "Journal of molecular catalysis / B",
issn = "1381-1177",
publisher = "Elsevier B.V.",

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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

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U2 - 10.1016/j.molcatb.2014.09.004

DO - 10.1016/j.molcatb.2014.09.004

M3 - Article

VL - 110

SP - 39

EP - 46

JO - Journal of molecular catalysis / B

JF - Journal of molecular catalysis / B

SN - 1381-1177

ER -