Biochemical and structural characterization of a novel cold-active esterase-like protein from the psychrophilic yeast Glaciozyma antarctica

Noor Haza Fazlin Hashim, Nor Muhammad Mahadi, Rosli Md Illias, Shevin Rizal Feroz, Farah Diba Abu Bakar, Abdul Munir Abd. Murad

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Dienelactone hydrolase, an α/β hydrolase enzyme, catalyzes the hydrolysis of dienelactone to maleylacetate, an intermediate for the Krebs cycle. Genome sequencing of the psychrophilic yeast, Glaciozyma antarctica predicted a putative open reading frame (ORF) for dienelactone hydrolase (GaDlh) with 52% sequence similarity to that from Coniophora puteana. Phylogenetic tree analysis showed that GaDlh is closely related to other reported dienelactone hydrolases, and distantly related to other α/β hydrolases. Structural prediction using MODELLER 9.14 showed that GaDlh has the same α/β hydrolase fold as other dienelactone hydrolases and esterase/lipase enzymes, with a catalytic triad consisting of Cys–His–Asp and a G–x–C–x–G–G motif. Based on the predicted structure, GaDlh exhibits several characteristics of cold-adapted proteins such as glycine clustering in the binding pocket, reduced protein core hydrophobicity, and the absence of proline residues in loops. The putative ORF was amplified, cloned, and overexpressed in an Escherichia coli expression system. The recombinant protein was overexpressed as soluble proteins and was purified via Ni–NTA affinity chromatography. Biochemical characterization of GaDlh revealed that it has an optimal temperature at 10 °C and that it retained almost 90% of its residual activity when incubated for 90 min at 10 °C. The optimal pH was at pH 8.0 and it was stable between pH 5–9 when incubated for 60 min (more than 50% residual activity). Its Km value was 256 μM and its catalytic efficiency was 81.7 s−1. To our knowledge, this is the first report describing a novel cold-active dienelactone hydrolase-like protein.

Original languageEnglish
Pages (from-to)1-10
Number of pages10
JournalExtremophiles
DOIs
Publication statusAccepted/In press - 20 Mar 2018

Fingerprint

carboxymethylenebutenolidase
Fungal Proteins
Esterases
Hydrolases
Open Reading Frames
Proteins
Citric Acid Cycle
Enzymes
Lipase
Hydrophobic and Hydrophilic Interactions
Affinity Chromatography
Recombinant Proteins
Proline
Glycine
Cluster Analysis
Hydrolysis
Yeasts
Genome
Escherichia coli
Temperature

Keywords

  • Antarctic microbiology
  • Dienelactone hydrolase
  • Esterase
  • Recombinant proteins
  • Thermolabile

ASJC Scopus subject areas

  • Microbiology
  • Molecular Medicine

Cite this

Biochemical and structural characterization of a novel cold-active esterase-like protein from the psychrophilic yeast Glaciozyma antarctica. / Hashim, Noor Haza Fazlin; Mahadi, Nor Muhammad; Illias, Rosli Md; Feroz, Shevin Rizal; Abu Bakar, Farah Diba; Abd. Murad, Abdul Munir.

In: Extremophiles, 20.03.2018, p. 1-10.

Research output: Contribution to journalArticle

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abstract = "Dienelactone hydrolase, an α/β hydrolase enzyme, catalyzes the hydrolysis of dienelactone to maleylacetate, an intermediate for the Krebs cycle. Genome sequencing of the psychrophilic yeast, Glaciozyma antarctica predicted a putative open reading frame (ORF) for dienelactone hydrolase (GaDlh) with 52{\%} sequence similarity to that from Coniophora puteana. Phylogenetic tree analysis showed that GaDlh is closely related to other reported dienelactone hydrolases, and distantly related to other α/β hydrolases. Structural prediction using MODELLER 9.14 showed that GaDlh has the same α/β hydrolase fold as other dienelactone hydrolases and esterase/lipase enzymes, with a catalytic triad consisting of Cys–His–Asp and a G–x–C–x–G–G motif. Based on the predicted structure, GaDlh exhibits several characteristics of cold-adapted proteins such as glycine clustering in the binding pocket, reduced protein core hydrophobicity, and the absence of proline residues in loops. The putative ORF was amplified, cloned, and overexpressed in an Escherichia coli expression system. The recombinant protein was overexpressed as soluble proteins and was purified via Ni–NTA affinity chromatography. Biochemical characterization of GaDlh revealed that it has an optimal temperature at 10 °C and that it retained almost 90{\%} of its residual activity when incubated for 90 min at 10 °C. The optimal pH was at pH 8.0 and it was stable between pH 5–9 when incubated for 60 min (more than 50{\%} residual activity). Its Km value was 256 μM and its catalytic efficiency was 81.7 s−1. To our knowledge, this is the first report describing a novel cold-active dienelactone hydrolase-like protein.",
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AU - Illias, Rosli Md

AU - Feroz, Shevin Rizal

AU - Abu Bakar, Farah Diba

AU - Abd. Murad, Abdul Munir

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