M. Schulein.
"Protein engineering of cellulases"
Biochimica et Biophysica Acta-Protein Structure and Molecular Enzymology, 1543(2), 239-252 (2000)
Abstract
Cellulases are enzymes which hydrolyse the beta -1, 4- glucosidic linkages of cellulose. They fall into 13 of the 82 glycoside hydrolase families identified by sequence analysis, but they are traditionally divided into two classes termed 'endoglucanases' (EC 3.2.1.4) and 'cellobiohydrolases' (3.2.1.91). Both types of cellulases degrade soluble cellodextrins and amorphous cellulose but, with a few notable exceptions, it is only the cellobiohydrolases which degrade crystalline cellulose efficiently. Site-directed mutagenesis has been central to the characterisation of cellulases, ranging from the identification and characterisation of putative catalytic and binding residues, the trapping of enzyme-substrate complexes by crystallography through to the construction of new and improved biocatalysts including 'glycosynthases'. Whilst studies on soluble substrates and substrate analogues have provided a wealth of information, understanding the mechanism of degradation of the natural substrate, crystalline cellulose, remains a great challenge.
F. Xu; A.E. Palmer; D.S. Yaver; R.M. Berka; G.A. Gambetta; S.H. Brown; E.I. Solomon.
"Targeted mutations in a Trametes villosa laccase: Axial perturbations of the T1 copper."
J. Biol. Chem., 274, 12372-12375 (1999)
AbstractTrametes villosa laccase was mutated on a tetrapeptide segment near the type 1 site. The mutations F463M and F463L were at the position corresponding to the type 1 copper axial methionine (M517) ligand in Zucchini ascorbate oxidase. The mutations E460S and A461E were near the T1 copper site. The mutated Trametes laccases were expressed in an Aspergillus oryzae host and characterized. The E460S mutation failed to produce a transformant with meaningful expression. The F463L and A461E mutations did not significantly alter the molecular and enzymological properties of the laccase. In contrast, the F463M mutation resulted in a type 1 copper site with an EPR signal intermediate between that of the wild type laccase and plastocyanin, an altered UV-visible spectrum, and a decreased redox potential (by 0.1 V). In oxidizing phenolic substrate, the mutation led to a more basic optimal pH as well as an increase in kcat and Km. These effects are attributed to a significant perturbation of the T1 copper center caused by the coordination of the axial methionine (M463) ligand.
F. Xu; R.M. Berka; J.A. Wahleithner; B.A. Nelson; J.R. Shuster; S.H. Brown; A.E. Palmer; E.I. Solomon.
"Site-directed mutations in fungal laccase: Effect on redox potential, activity and pH profile."
Biochem. J., 334, 63-70 (1998)
Abstract
A Myceliophthora thermophila laccase and a Rhizoctonia solani laccase were mutated on a pentapeptide segment believed to be near the type-1 Cu site. The mutation L513F in Myceliophthora laccase and the mutation L470F in Rhizoctonia laccase took place at a position corresponding to the type-1 Cu axial methionine (M517) ligand in Zucchini ascorbate oxidase. The triple mutations V509L,S510E,G511A in Myceliophthora laccase and L466V,E467S,A468G in Rhizoctonia laccase involved a sequence segment whose homologue in ascorbate oxidase is flanked by the M517 and a type-1 Cu-ligating histidine (H512). The single mutation did not yield significant changes in the enzymic properties (including any significant increase in the redox potential of the type-1 Cu). In contrast, the triple mutation resulted in several significant changes. In comparison with the wild type, the Rhizoctonia and Myceliophthora laccase triple mutants had a phenol-oxidase activity whose pH optimum shifted 1 unit lower and higher, respectively. Although the redox potentials were not significantly altered, the Km, kcat and fluoride inhibition of the laccases were greatly changed by the mutations. The observed effects are interpreted as possible mutation-induced structural perturbations on the molecular recognition between the reducing substrate and laccase and on the electron transfer from the substrate to the type-1 Cu centre.