The α-helix (L351-G378) Of Bacterial Laccase Affects Catalytic Efficiency

Laccase (EC1.10.3.2) is a member of the muticopper oxidase family which widely exists in nature, and catalyzes the oxidizing reaction of a category of phenol and non-phenolic compounds. In the past, laccases were mainly found in plants, insects and fungi. However, laccases are also found recently in prokaryotic bacteria. Bacterial laccases may perhaps display different enzyme properties and kinetics as compared with those of fungi which are widely studied. Moreover, bacterial laccases don’t require glycosylation modification and most of them are thermodynamic stable, and catalyze oxidizing reaction in a wide range of pH. Therefore, the finding and utilization bacterial laccase is becoming popular nationwide and aboard.Klebsiella sp.601displaying laccase activity has been isolated from the forest soil in our laboratory. The gene encoding laccase is highly homologous to Escherichia coli CueO with a similarity of90%and an identity of78%. Structural comparison between bacterial CueO and fungal laccases has suggested that a charged residue Glu (E106) in CueO replaces the corresponding residue Phe in fungal laccases at the gate of the tunnel connecting type II copper to the protein surface and an extra a-helix(L351-G378) near the type I copper site covers the substrate biding pocket and might compromise the electron transfer from substrate to type I copper. To test this hypothesis, several mutants were made in Klebsiella sp.601multicopper oxidase, and we gauged these enzymes’kinetics.The E106F mutant gave smaller Km (2.4-7fold) and kcat (1-4.4fold) values for all three substrates DMP, ABTS, and SGZ as compared with those for the wild-type enzyme. Its slightly larger kcat/Km values for three substrates mainly come from the decreased Km. Deleting α-helix(L351-G378) resulted in the formation of inactive inclusion body when the mutant Δα351-378was expressed in Escherichia coli. Another mutant α351-378M was then made via substitution of seven amino acid residues in the α-helix(L351-G378) region. The a351-378M mutant was active, and displayed a far-UV CD spectrum markedly different from that for wild-type enzyme.Kinetic studies showed the α351-378M mutant gave very low Km values for DMP, ABTS and SGZ,4.5-and1.9-and7-fold less than those for the wild type. In addition, kcat/Km values were increased,9.4-fold for DMP, similar for ABTS and3-fold for SGZ. The Glu residue at position106appears not to be the only factor affecting the copper binding, and it may also play a role in maintaining enzyme conformation. The a-helix(L351-G378) may not only block access to the type I copper site but also play a role in substrate specificities of bacterial MCOs. The α351-378M mutant catalyzing oxidation of the phenolic substrate DMP effectively would be very useful in green chemistry.