RESUMO
The multicopper oxidase CueO is involved in copper homeostasis and copper (Cu) tolerance in Escherichia coli. The laccase activity of CueO G304K mutant is higher than wild-type CueO. To explain this increase in activity, we solved the crystal structure of G304K mutant at 1.49 Å. Compared with wild-type CueO, the G304K mutant showed dramatic conformational changes in methionine-rich helix and the relative regulatory loop (R-loop). We further solved the structure of Cu-soaked enzyme, and found that the addition of Cu ions induced further conformational changes in the R-loop and methionine-rich helix as a result of the new Cu-binding sites on the enzyme's surface. We propose a mechanism for the enhanced laccase activity of the G304K mutant, where movements of the R-loop combined with the changes of the methionine-rich region uncover the T1 Cu site allowing greater access of the substrate. Two of the G304K double mutants showed the enhanced or decreased laccase activity, providing further evidence for the interaction between the R-loop and the methionine-rich region. The cuprous oxidase activity of these mutants was about 20% that of wild-type CueO. These structural features of the G304K mutant provide clues for designing specific substrate-binding mutants in the biotechnological applications.
Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Lacase/química , Oxirredutases/química , Conformação Proteica , Sequência de Aminoácidos/genética , Sítios de Ligação/genética , Cobre/química , Cristalografia por Raios X , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/ultraestrutura , Lacase/genética , Metionina/genética , Modelos Moleculares , Mutação , Oxirredutases/genética , Oxirredutases/ultraestrutura , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
Of the 20 common amino acids, 18 are encoded by multiple synonymous codons. These synonymous codons are not redundant; in fact, all of codons contribute substantially to protein expression, structure and function. In this study, the codon usage pattern of genes in the E. coli was learned from the sequenced genomes of E. coli. A machine learning based method, Presyncodon was proposed to predict synonymous codon selection in E. coli based on the learned codon usage patterns of the residue in the context of the specific fragment. The predicting results indicate that Presycoden could be used to predict synonymous codon selection of the gene in the E. coli with the high accuracy. Two reporter genes (egfp and mApple) were designed with a combination of low- and high-frequency-usage codons by the method. The fluorescence intensity of eGFP and mApple expressed by the (egfp and mApple) designed by this method was about 2.3- or 1.7- folds greater than that from the genes with only high-frequency-usage codons in E. coli. Therefore, both low- and high-frequency-usage codons make positive contributions to the functional expression of the heterologous proteins. This method could be used to design synthetic genes for heterologous gene expression in biotechnology.
