Timofeevski S L, Nie G, Reading N S, Aust S D
Biotechnology Center, Utah State University, Logan, Utah, 84322-4705, USA.
Biochem Biophys Res Commun. 1999 Mar 24;256(3):500-4. doi: 10.1006/bbrc.1999.0360.
Manganese peroxidase and lignin peroxidase are ligninolytic heme-containing enzymes secreted by the white-rot fungus Phanerochaete chrysosporium. Despite structural similarity, these peroxidases oxidize different substrates. Veratryl alcohol is a typical substrate for lignin peroxidase, while manganese peroxidase oxidizes chelated Mn2+. By a single mutation, S168W, we have added veratryl alcohol oxidase activity to recombinant manganese peroxidase expressed in Escherichia coli. The kcat for veratryl alcohol oxidation was 11 s-1, Km for veratryl alcohol approximately 0.49 mM, and Km for hydrogen peroxide approximately 25 microM at pH 2.3. The Km for veratryl alcohol was higher and Km for hydrogen peroxide was lower for this manganese peroxidase mutant compared to two recombinant lignin peroxidase isoenzymes. The mutant retained full manganese peroxidase activity and the kcat was approximately 2.6 x 10(2) s-1 at pH 4.3. Consistent with relative activities with respect to these substrates, Mn2+ strongly inhibited veratryl alcohol oxidation. The single productive mutation in manganese peroxidase suggested that this surface tryptophan residue (W171) in lignin peroxidase is involved in catalysis.
锰过氧化物酶和木质素过氧化物酶是白腐真菌黄孢原毛平革菌分泌的含血红素的木质素分解酶。尽管结构相似，但这些过氧化物酶氧化不同的底物。藜芦醇是木质素过氧化物酶的典型底物，而锰过氧化物酶氧化螯合的Mn2+。通过单一突变S168W，我们已将藜芦醇氧化酶活性添加到在大肠杆菌中表达的重组锰过氧化物酶中。在pH 2.3时，藜芦醇氧化的kcat为11 s-1，藜芦醇的Km约为0.49 mM，过氧化氢的Km约为25 μM。与两种重组木质素过氧化物酶同工酶相比，该锰过氧化物酶突变体的藜芦醇Km较高，过氧化氢Km较低。该突变体保留了完整的锰过氧化物酶活性，在pH 4.3时kcat约为2.6×10(2) s-1。与这些底物的相对活性一致，Mn2+强烈抑制藜芦醇氧化。锰过氧化物酶中的单一有效突变表明，木质素过氧化物酶中的这个表面色氨酸残基（W171）参与催化作用。
