TY - JOUR T1 - Examining the role of glutamic acid 183 in chloroperoxidase catalysis JF - J Biol Chem Y1 - 2003 A1 - Yi, X. A1 - A. Conesa A1 - Punt, P. J. A1 - Hager, L. P. KW - Aspergillus niger/metabolism Catalase/metabolism Catalysis Chloride Peroxidase/*chemistry/*metabolism Chlorine/metabolism Chromatography KW - Ion Exchange Circular Dichroism Crystallography KW - Polyacrylamide Gel Fungi/enzymology Glutamic Acid/*chemistry Histidine/chemistry/metabolism Hydrogen-Ion Concentration Immunoblotting Isoelectric Focusing Mutation Oxidoreductases/metabolism Plasmids/metabolism KW - X-Ray Electrophoresis AB - Site-directed mutagenesis has been used to investigate the role of glutamic acid 183 in chloroperoxidase catalysis. Based on the x-ray crystallographic structure of chloroperoxidase, Glu-183 is postulated to function on distal side of the heme prosthetic group as an acid-base catalyst in facilitating the reaction between the peroxidase and hydrogen peroxide with the formation of Compound I. In contrast, the other members of the heme peroxidase family use a histidine residue in this role. Plasmids have now been constructed in which the codon for Glu-183 is replaced with a histidine codon. The mutant recombinant gene has been expressed in Aspergillus niger. An analysis of the produced mutant gene shows that the substitution of Glu-183 with a His residue is detrimental to the chlorination and dismutation activity of chloroperoxidase. The activity is reduced by 85 and 50% of wild type activity, respectively. However, quite unexpectedly, the epoxidation activity of the mutant enzyme is significantly enhanced approximately 2.5-fold. These results show that Glu-183 is important but not essential for the chlorination activity of chloroperoxidase. It is possible that the increased epoxidation of the mutant enzyme is based on an increase in the hydrophobicity of the active site. VL - 278 UR - http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12576477 N1 - Yi, Xianwen Conesa, Ana Punt, Peter J Hager, Lowell P GM 07768/GM/NIGMS NIH HHS/United States Research Support, U.S. Gov’t, P.H.S. United States The Journal of biological chemistry J Biol Chem. 2003 Apr 18;278(16):13855-9. Epub 2003 Feb 7. ER - TY - JOUR T1 - Calnexin overexpression increases manganese peroxidase production in Aspergillus niger JF - Appl Environ Microbiol Y1 - 2002 A1 - A. Conesa A1 - Jeenes, D. A1 - Archer, D. B. A1 - van den Hondel, C. A. A1 - Punt, P. J. KW - Aspergillus niger/*enzymology/genetics Calcium-Binding Proteins/*metabolism Calnexin Culture Media *Fungal Proteins HSP70 Heat-Shock Proteins/metabolism Heme/metabolism Peroxidases/*biosynthesis/genetics Phanerochaete/enzymology/genetics Transformation KW - Genetic AB - Heme-containing peroxidases from white rot basidiomycetes, in contrast to most proteins of fungal origin, are poorly produced in industrial filamentous fungal strains. Factors limiting peroxidase production are believed to operate at the posttranslational level. In particular, insufficient availability of the prosthetic group which is required for peroxidase biosynthesis has been proposed to be an important bottleneck. In this work, we analyzed the role of two components of the secretion pathway, the chaperones calnexin and binding protein (BiP), in the production of a fungal peroxidase. Expression of the Phanerochaete chrysosporium manganese peroxidase (MnP) in Aspergillus niger resulted in an increase in the expression level of the clxA and bipA genes. In a heme-supplemented medium, where MnP was shown to be overproduced to higher levels, induction of clxA and bipA was also higher. Overexpression of these two chaperones in an MnP-producing strain was analyzed for its effect on MnP production. Whereas bipA overexpression seriously reduced MnP production, overexpression of calnexin resulted in a four- to fivefold increase in the extracellular MnP levels. However, when additional heme was provided in the culture medium, calnexin overexpression had no synergistic effect on MnP production. The possible function of these two chaperones in MnP maturation and production is discussed. VL - 68 UR - http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11823227 N1 - Conesa, Ana Jeenes, David Archer, David B van den Hondel, Cees A M J J Punt, Peter J United States Applied and environmental microbiology Appl Environ Microbiol. 2002 Feb;68(2):846-51. ER - TY - JOUR T1 - Filamentous fungi as cell factories for heterologous protein production JF - Trends Biotechnol Y1 - 2002 A1 - Punt, P. J. A1 - van Biezen, N. A1 - A. Conesa A1 - Albers, A. A1 - Mangnus, J. A1 - van den Hondel, C. KW - Fermentation/genetics/physiology Fungi/*genetics/*metabolism Humans Interleukin-6/analysis/*biosynthesis/genetics Peroxidases/analysis/*biosynthesis/genetics Protein Conformation Recombinant Proteins/analysis/*biosynthesis/genetics AB - Filamentous fungi have been used as sources of metabolites and enzymes for centuries. For about two decades, molecular genetic tools have enabled us to use these organisms to express extra copies of both endogenous and exogenous genes. This review of current practice reveals that molecular tools have enabled several new developments. But it has been process development that has driven the final breakthrough to achieving commercially relevant quantities of protein. Recent research into gene expression in filamentous fungi has explored their wealth of genetic diversity with a view to exploiting them as expression hosts and as a source of new genes. Inevitably, the progress in the ’genomics’ technology will further develop high-throughput technologies for these organisms. VL - 20 UR - http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11943375 N1 - Punt, Peter J van Biezen, Nick Conesa, Ana Albers, Alwin Mangnus, Jeroen van den Hondel, Cees Review England Trends in biotechnology Trends Biotechnol. 2002 May;20(5):200-6. ER - TY - JOUR T1 - Fungal peroxidases: molecular aspects and applications JF - J Biotechnol Y1 - 2002 A1 - A. Conesa A1 - Punt, P. J. A1 - van den Hondel, C. A. KW - Amino Acid Sequence Binding Sites Biotechnology Catalysis Fungi/*enzymology Molecular Sequence Data Peroxidases/chemistry/*genetics/metabolism Recombinant Proteins Sequence Homology Substrate Specificity AB - Peroxidases are oxidoreductases that utilize hydrogen peroxide to catalyze oxidative reactions. A large number of peroxidases have been identified in fungal species and are being characterized at the molecular level. In this manuscript we review the current knowledge on the molecular aspects of this type of enzymes. We present an overview of the research efforts undertaken in deciphering the structural basis of the catalytic properties of fungal peroxidases and discuss molecular genetics and protein homology aspects of this enzyme class. Finally, we summarize the potential biotechnological applications of these enzymes and evaluate recent advances on their expression in heterologous systems for production purposes. VL - 93 UR - http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11738721 N1 - Conesa, Ana Punt, Peter J van den Hondel, Cees A M J J Review Netherlands Journal of biotechnology J Biotechnol. 2002 Feb 14;93(2):143-58. ER - TY - JOUR T1 - C-terminal propeptide of the Caldariomyces fumago chloroperoxidase: an intramolecular chaperone? JF - FEBS Lett Y1 - 2001 A1 - A. Conesa A1 - Weelink, G. A1 - van den Hondel, C. A. A1 - Punt, P. J. KW - Amino Acid Sequence Ascomycota/*enzymology/genetics Aspergillus niger/genetics Base Sequence Chloride Peroxidase/biosynthesis/*chemistry/genetics DNA Primers/genetics Enzyme Precursors/biosynthesis/chemistry/genetics Gene Expression Molecular Chaperones/b AB - The Caldariomyces fumago chloroperoxidase (CPO) is synthesised as a 372-aa precursor which undergoes two proteolytic processing events: removal of a 21-aa N-terminal signal peptide and of a 52-aa C-terminal propeptide. The Aspergillus niger expression system developed for CPO was used to get insight into the function of this C-terminal propeptide. A. niger transformants expressing a CPO protein from which the C-terminal propeptide was deleted failed in producing any extracellular CPO activity, although the CPO polypeptide was synthesised. Expression of the full-length gene in an A. niger strain lacking the KEX2-like protease PclA also resulted in the production of CPO cross-reactive material into the culture medium, but no CPO activity. Based on these results, a function of the C-terminal propeptide in CPO maturation is indicated. VL - 503 UR - http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11513866 N1 - Conesa, A Weelink, G van den Hondel, C A Punt, P J Netherlands FEBS letters FEBS Lett. 2001 Aug 17;503(2-3):117-20. ER - TY - JOUR T1 - Expression of the Caldariomyces fumago chloroperoxidase in Aspergillus niger and characterization of the recombinant enzyme JF - J Biol Chem Y1 - 2001 A1 - A. Conesa A1 - van De Velde, F. A1 - van Rantwijk, F. A1 - Sheldon, R. A. A1 - van den Hondel, C. A. A1 - Punt, P. J. KW - Aspergillus niger/enzymology/genetics Catalysis Chloride Peroxidase/biosynthesis/*genetics Fungal Proteins/biosynthesis/*genetics Recombinant Proteins/biosynthesis/genetics Substrate Specificity AB - The Caldariomyces fumago chloroperoxidase was successfully expressed in Aspergillus niger. The recombinant enzyme was produced in the culture medium as an active protein and could be purified by a three-step purification procedure. The catalytic behavior of recombinant chloroperoxidase (rCPO) was studied and compared with that of native CPO. The specific chlorination activity (47 units/nmol) of rCPO and its pH optimum (pH 2.75) were very similar to those of native CPO. rCPO catalyzes the oxidation of various substrates in comparable yields and selectivities to native CPO. Indole was oxidized to 2-oxindole with 99% selectivity and thioanisole to the corresponding R-sulfoxide (enantiomeric excess >98%). Incorporation of (18)O from labeled H(2)18O(2) into the oxidized products was 100% in both cases. VL - 276 UR - http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11278701 N1 - Conesa, A van De Velde, F van Rantwijk, F Sheldon, R A van Den Hondel, C A Punt, P J Research Support, Non-U.S. Gov’t United States The Journal of biological chemistry J Biol Chem. 2001 May 25;276(21):17635-40. Epub 2001 Feb 22. ER - TY - JOUR T1 - The secretion pathway in filamentous fungi: a biotechnological view JF - Fungal Genet Biol Y1 - 2001 A1 - A. Conesa A1 - Punt, P. J. A1 - van Luijk, N. A1 - van den Hondel, C. A. KW - Animals Biotechnology/*methods Fungal Proteins/*genetics/*metabolism Fungi/*genetics/*metabolism Humans Recombinant Proteins/metabolism AB - The high capacity of the secretion machinery of filamentous fungi has been widely exploited for the production of homologous and heterologous proteins; however, our knowledge of the fungal secretion pathway is still at an early stage. Most of the knowledge comes from models developed in yeast and higher eukaryotes, which have served as reference for the studies on fungal species. In this review we compile the data accumulated in recent years on the molecular basis of fungal secretion, emphasizing the relevance of these data for the biotechnological use of the fungal cell and indicating how this information has been applied in attempts to create improved production strains. We also present recent emerging approaches that promise to provide answers to fundamental questions on the molecular genetics of the fungal secretory pathway. VL - 33 UR - http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11495573 N1 -

Conesa, A Punt, P J van Luijk, N van den Hondel, C A Review United States Fungal genetics and biology : FG & B Fungal Genet Biol. 2001 Aug;33(3):155-71.

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