scholarly journals Molecular Cloning and Heterologous Expression of Manganese(II)-Oxidizing Enzyme from Acremonium strictum Strain KR21-2

Catalysts ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 686
Author(s):  
Fuyumi Tojo ◽  
Ayumi Kitayama ◽  
Naoyuki Miyata ◽  
Kunihiro Okano ◽  
Jun Fukushima ◽  
...  

Diverse ascomycete fungi oxidize manganese(II) [Mn(II)] and produce Mn(III, IV) oxides in terrestrial and freshwater environments. Although multicopper oxidase (MCO) is considered to be a key catalyst in mediating Mn(II) oxidation in ascomycetes, the responsible gene and its product have not been identified. In this study, a gene, named mco1, encoding Mn(II)-oxidizing MCO from Acremonium strictum strain KR21-2 was cloned and heterologously expressed in the methylotrophic yeast Pichia pastoris. Based on the phylogenetic relationship, similarity of putative copper-binding motifs, and homology modeling, the gene product Mco1 was assigned to a bilirubin oxidase. Mature Mco1 was predicted to be composed of 565 amino acids with a molecular mass of 64.0 kDa. The recombinant enzyme oxidized Mn(II) to yield spherical Mn oxides, several micrometers in diameter. Zinc(II) ions added to the reaction mixture were incorporated by the Mn oxides at a Zn/Mn molar ratio of 0.36. The results suggested that Mco1 facilitates the growth of the micrometer-sized Mn oxides and affects metal sequestration through Mn(II) oxidation. This is the first report on heterologous expression and identification of the Mn(II) oxidase enzyme in Mn(II)-oxidizing ascomycetes. The cell-free, homogenous catalytic system with recombinant Mco1 could be useful for understanding Mn biomineralization by ascomycetes and the sequestration of metal ions in the environment

Author(s):  
Lisa Klug ◽  
Pablo Tarazona ◽  
Clemens Gruber ◽  
Karlheinz Grillitsch ◽  
Brigitte Gasser ◽  
...  

Genetics ◽  
1999 ◽  
Vol 151 (4) ◽  
pp. 1379-1391
Author(s):  
Monique A Johnson ◽  
Hans R Waterham ◽  
Galyna P Ksheminska ◽  
Liubov R Fayura ◽  
Joan Lin Cereghino ◽  
...  

Abstract We have developed two novel schemes for the direct selection of peroxisome-biogenesis-defective (pex) mutants of the methylotrophic yeast Pichia pastoris. Both schemes take advantage of our observation that methanol-induced pex mutants contain little or no alcohol oxidase (AOX) activity. AOX is a peroxisomal matrix enzyme that catalyzes the first step in the methanol-utilization pathway. One scheme utilizes allyl alcohol, a compound that is not toxic to cells but is oxidized by AOX to acrolein, a compound that is toxic. Exposure of mutagenized populations of AOX-induced cells to allyl alcohol selectively kills AOX-containing cells. However, pex mutants without AOX are able to grow. The second scheme utilizes a P. pastoris strain that is defective in formaldehyde dehydrogenase (FLD), a methanol pathway enzyme required to metabolize formaldehyde, the product of AOX. AOX-induced cells of fld1 strains are sensitive to methanol because of the accumulation of formaldehyde. However, fld1 pex mutants, with little active AOX, do not efficiently oxidize methanol to formaldehyde and therefore are not sensitive to methanol. Using these selections, new pex mutant alleles in previously identified PEX genes have been isolated along with mutants in three previously unidentified PEX groups.


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