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  • Following mutagenesis of the glucarpidase


    Following mutagenesis of the glucarpidase gene of Pseudomonas sp. strain RS-16 [30], approximately 73% of the clones retained enzyme activity, as indicated by the clear zones and yellow precipitate surrounding their colonies. However, there were very few that had ‘halos’ around colonies that were darker than that of the wild-type, which would be indicative either of more active glucarpidase variants or variants that over-produced the enzyme relative to the wild-type construct. DNA sequence analysis of the three mutants taken for further study indicated that each had a single point mutation leading to the alteration of a single amino Levofloxacin at the protein level (Supplemental Fig. S3). The fact that only single point mutations were present suggests that the error-prone PCR may have contributed more than the DNA shuffling procedure to the production of these particular mutants. Alternatively, it is possible that combining two or more individual mutations into a single gene may have resulted in mutant enzymes with little or no enzyme activity. The three mutant enzymes, named CPG2 I100 T, CPG2 G123S, and CPG2 T329 A, were then characterized in greater detail. In keeping with their colony phenotypes, each had a higher specific activity in enzyme assays, and this was supported by the results of studies to determine their kinetic parameters. Specifically, the three mutants had higher substrate affinity as shown by their lower Km values relative to the wild-type enzyme (Table 2). It remains to be seen if combining two or more of the mutations into the same CPG2 gene results in a further increase in enzyme activity. The analysis of the three web servers to predict the effect of a single mutant [[22], [23], [24]], the prediction shows a higher destabilizing effect in position 100 and 123 compared to 329 of the three mutants. In contrast, CD analysis of the third mutant, CPG2 T329 A, suggests that it has a marked increase in alpha-helical content, which, interestingly, might even lead to a modest increase in its structural stability, as indicated by analysis with the SDM software package (Table 4). It has previously been shown [31] that amino acids in a protein that is involved in enzyme catalysis are not optimized for stability. Thus, replacement of specific residues may reduce the activity of an enzyme but concomitantly increase its stability. Alternatively, the replacement of residues involved in protein stability could lead to higher enzyme activity. The results presented in this work are consistent with these findings. The three randomly produced glucarpidase mutation substitution, I100 T, G123S and T329 A, increased the enzyme activity in each case but are predicted to decrease the stability of each mutant (Fig. 3, Table 4). Our study may also suggest the involvement of the isoleucine, glycine and alanine at positions 100, 123 and 329, respectively, in glucarpidase catalysis. It has previously been shown that [32] proteins with specific sites known as flexibility hotspots are important for both binding and stability. The predicted structure (Fig. 5) shows that in the two mutations, I100 T and G123S, hydrophobic and neutral side chains respectively, are replaced replace with a polar functional group. As previously observed [33], only certain amino acids show a specific propensity to become part of an alpha helix [33]: according to the proposed scale, the helical penalty of threonine and serine are 0.66 Kcal/mol and 0.50 Kcal/mol. Such thermodynamic penalties can be related to steric clashes and the formation of new hydrogen bonds, as we propose in our models. The substitution with such amino acids can therefore lead to a destabilization to the wild type, and this reinforces the validity of the model calculated for the I100 T and G123S which were found to be more active to cleave the methotrexate compared to T329 A.
    Conflict of interest