Heterologous expression of athermostable manganese peroxidase from Dichomitus squalens in Phanerochaete chrysosporium.
Dichomitus squalens belongs to a group of white-rot fungi which express manganese peroxidase (MnP) and laccase but do not express lignin peroxidase (LiP). To facilitate structure/function studies of MnP from D. squalens, we heterologously expressed the enzyme in the well-studied basidiomycete, Phanerochaete chrysosporium. The glyceraldehyde-3-phosphate-dehydrogenase (gpd) promoter of P. chrysosporium was fused to the coding region of the mnp2 gene of D. squalens, 5 bp upstream of the translation start site, and placed in a vector containing the ural gene as a selectable marker. Purified recombinant protein (rDsMnP) was similar in kinetic and spectral characteristics to both the wild-type MnPs from D. squalens and P. chrysosporium (PcMnP). The N-terminal amino acid sequence of the rDsMnP was determined and was identical to the predicted sequence. Cleavage of the propeptide followed a conserved amino acid motif (A-A-P-S/T) in both rDsMnP and PcMnP. However, the protein from D. squalens was considerably more thermostable than its P. chrysosporium homolog with half-lives 15- to 40-fold longer at 55 degrees C. As previously demonstrated for PcMnP, addition of exogenous MnII and CdII conferred additional thermal stability to rDsMnP. However, unlike PcMnP, ZnII also confers some additional thermal stability to rDsMnP at 55 degrees C. Some differences in the metal-specific effects on thermal stability of rDsMnP at 65 degrees C were noted.
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Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, Beaverton 97006-8921, USA.
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D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001487:Basidiomycota / Q000201:enzymology* / Q000235:genetics*; D004795:Enzyme Stability; D015870:Gene Expression; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006207:Half-Life; D007700:Kinetics; D008314:Malonates / Q000378:metabolism; D008670:Metals / Q000378:metabolism; D008969:Molecular Sequence Data; D010544:Peroxidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D020075:Phanerochaete / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D020539:Sequence Analysis, Protein; D013054:Spectrophotometry, Atomic; D013696:Temperature
D008314:Malonates; D008670:Metals; D011993:Recombinant Fusion Proteins; C030290:malonic acid; D010544:Peroxidases; C051129:manganese peroxidase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases