Molecular cloning, characterization, and expression of a redox-responsive cutinase from Monilinia fructicola (Wint.) Honey

Guang Yi Wang, Themis J. Michailides, Bruce D. Hammock, Young Moo Lee, Richard M. Bostock

Research output: Contribution to journalArticle

38 Citations (Scopus)

Abstract

cDNA clones encoding a cutinase expressed in cutin-induced cultures of the plant pathogen Monilinia fructicola were isolated using a protein-based strategy. The largest cDNA (Mfcut1) was found to contain an open reading frame of 603 bp that predicted a 20.2-kDa protein of 201 amino acids with a 20-amino-acid secretory signal peptide and a pl of 8.4. The predicted protein contained cutinase/lipase consensus sequences with active site serines and potential protein kinase phosphorylation sites. Comparison of the deduced amino sequence from Mfcut1 with other fungal cutinase sequences revealed new features, which include conserved cysteines, C-terminal aromatic residues, and a novel histidine substitution in the D-H active site motif. The presence in the growth medium of antioxidants, such as caffeic acid, suppressed mRNA accumulation and enzyme activity of a cutinase from M. fructicola. MFCUT1 was expressed at high levels as a His-tagged fusion protein in Pichia pastoris and purified to apparent homogeneity in a single step by Ni2+-nitrilotriacetic acid affinity chromatography. Analysis of variant MFCUT1 mutants in which the novel serine and histidine residues were replaced by site-directed mutagenesis indicated that these residues had an important effect on enzyme activity.

Original languageEnglish (US)
Pages (from-to)261-276
Number of pages16
JournalFungal Genetics and Biology
Volume35
Issue number3
DOIs
StatePublished - 2002

Fingerprint

Honey
Molecular Cloning
Oxidation-Reduction
Histidine
Catalytic Domain
Proteins
Complementary DNA
Nitrilotriacetic Acid
Amino Acids
Pichia
Protein-Serine-Threonine Kinases
Consensus Sequence
Enzymes
Protein Sorting Signals
Site-Directed Mutagenesis
Lipase
Affinity Chromatography
Serine
Open Reading Frames
Cysteine

Keywords

  • Brown rot
  • Cutinase
  • Monilinia fructicola
  • MOPAC-PCR
  • Redox regulation

ASJC Scopus subject areas

  • Genetics
  • Microbiology

Cite this

Molecular cloning, characterization, and expression of a redox-responsive cutinase from Monilinia fructicola (Wint.) Honey. / Wang, Guang Yi; Michailides, Themis J.; Hammock, Bruce D.; Lee, Young Moo; Bostock, Richard M.

In: Fungal Genetics and Biology, Vol. 35, No. 3, 2002, p. 261-276.

Research output: Contribution to journalArticle

Wang, Guang Yi ; Michailides, Themis J. ; Hammock, Bruce D. ; Lee, Young Moo ; Bostock, Richard M. / Molecular cloning, characterization, and expression of a redox-responsive cutinase from Monilinia fructicola (Wint.) Honey. In: Fungal Genetics and Biology. 2002 ; Vol. 35, No. 3. pp. 261-276.
@article{73a85120ea7c4429a5b064e78c6851a0,
title = "Molecular cloning, characterization, and expression of a redox-responsive cutinase from Monilinia fructicola (Wint.) Honey",
abstract = "cDNA clones encoding a cutinase expressed in cutin-induced cultures of the plant pathogen Monilinia fructicola were isolated using a protein-based strategy. The largest cDNA (Mfcut1) was found to contain an open reading frame of 603 bp that predicted a 20.2-kDa protein of 201 amino acids with a 20-amino-acid secretory signal peptide and a pl of 8.4. The predicted protein contained cutinase/lipase consensus sequences with active site serines and potential protein kinase phosphorylation sites. Comparison of the deduced amino sequence from Mfcut1 with other fungal cutinase sequences revealed new features, which include conserved cysteines, C-terminal aromatic residues, and a novel histidine substitution in the D-H active site motif. The presence in the growth medium of antioxidants, such as caffeic acid, suppressed mRNA accumulation and enzyme activity of a cutinase from M. fructicola. MFCUT1 was expressed at high levels as a His-tagged fusion protein in Pichia pastoris and purified to apparent homogeneity in a single step by Ni2+-nitrilotriacetic acid affinity chromatography. Analysis of variant MFCUT1 mutants in which the novel serine and histidine residues were replaced by site-directed mutagenesis indicated that these residues had an important effect on enzyme activity.",
keywords = "Brown rot, Cutinase, Monilinia fructicola, MOPAC-PCR, Redox regulation",
author = "Wang, {Guang Yi} and Michailides, {Themis J.} and Hammock, {Bruce D.} and Lee, {Young Moo} and Bostock, {Richard M.}",
year = "2002",
doi = "10.1006/fgbi.2001.1320",
language = "English (US)",
volume = "35",
pages = "261--276",
journal = "Fungal Genetics and Biology",
issn = "1087-1845",
publisher = "Academic Press Inc.",
number = "3",

}

TY - JOUR

T1 - Molecular cloning, characterization, and expression of a redox-responsive cutinase from Monilinia fructicola (Wint.) Honey

AU - Wang, Guang Yi

AU - Michailides, Themis J.

AU - Hammock, Bruce D.

AU - Lee, Young Moo

AU - Bostock, Richard M.

PY - 2002

Y1 - 2002

N2 - cDNA clones encoding a cutinase expressed in cutin-induced cultures of the plant pathogen Monilinia fructicola were isolated using a protein-based strategy. The largest cDNA (Mfcut1) was found to contain an open reading frame of 603 bp that predicted a 20.2-kDa protein of 201 amino acids with a 20-amino-acid secretory signal peptide and a pl of 8.4. The predicted protein contained cutinase/lipase consensus sequences with active site serines and potential protein kinase phosphorylation sites. Comparison of the deduced amino sequence from Mfcut1 with other fungal cutinase sequences revealed new features, which include conserved cysteines, C-terminal aromatic residues, and a novel histidine substitution in the D-H active site motif. The presence in the growth medium of antioxidants, such as caffeic acid, suppressed mRNA accumulation and enzyme activity of a cutinase from M. fructicola. MFCUT1 was expressed at high levels as a His-tagged fusion protein in Pichia pastoris and purified to apparent homogeneity in a single step by Ni2+-nitrilotriacetic acid affinity chromatography. Analysis of variant MFCUT1 mutants in which the novel serine and histidine residues were replaced by site-directed mutagenesis indicated that these residues had an important effect on enzyme activity.

AB - cDNA clones encoding a cutinase expressed in cutin-induced cultures of the plant pathogen Monilinia fructicola were isolated using a protein-based strategy. The largest cDNA (Mfcut1) was found to contain an open reading frame of 603 bp that predicted a 20.2-kDa protein of 201 amino acids with a 20-amino-acid secretory signal peptide and a pl of 8.4. The predicted protein contained cutinase/lipase consensus sequences with active site serines and potential protein kinase phosphorylation sites. Comparison of the deduced amino sequence from Mfcut1 with other fungal cutinase sequences revealed new features, which include conserved cysteines, C-terminal aromatic residues, and a novel histidine substitution in the D-H active site motif. The presence in the growth medium of antioxidants, such as caffeic acid, suppressed mRNA accumulation and enzyme activity of a cutinase from M. fructicola. MFCUT1 was expressed at high levels as a His-tagged fusion protein in Pichia pastoris and purified to apparent homogeneity in a single step by Ni2+-nitrilotriacetic acid affinity chromatography. Analysis of variant MFCUT1 mutants in which the novel serine and histidine residues were replaced by site-directed mutagenesis indicated that these residues had an important effect on enzyme activity.

KW - Brown rot

KW - Cutinase

KW - Monilinia fructicola

KW - MOPAC-PCR

KW - Redox regulation

UR - http://www.scopus.com/inward/record.url?scp=0036348909&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0036348909&partnerID=8YFLogxK

U2 - 10.1006/fgbi.2001.1320

DO - 10.1006/fgbi.2001.1320

M3 - Article

C2 - 11929215

AN - SCOPUS:0036348909

VL - 35

SP - 261

EP - 276

JO - Fungal Genetics and Biology

JF - Fungal Genetics and Biology

SN - 1087-1845

IS - 3

ER -