Design and evolution of new catalytic activity with an existing protein scaffold.

The design of enzymes with new functions and properties has long been a goal in protein engineering. Here, we report a strategy to change the catalytic activity of an existing protein scaffold. This was achieved by simultaneous incorporation and adjustment of functional elements through insertion, deletion, and substitution of several active site loops, followed by point mutations to fine-tune the activity. Using this approach, we were able to introduce beta-lactamase activity into the alphabeta/betaalpha metallohydrolase scaffold of glyoxalase II. The resulting enzyme, evMBL8 (evolved metallo beta-lactamase 8), completely lost its original activity and, instead, catalyzed the hydrolysis of cefotaxime with a (kcat/Km)app of 1.8 x 10(2) (mole/liter)(-1) second(-1), thus increasing resistance to Escherichia coli growth on cefotaxime by a factor of about 100.

Evolutionary relationship and application of a superfamily of cyclic amidohydrolase enzymes.

Cyclic amidohydrolases belong to a superfamily of enzymes that catalyze the hydrolysis of cyclic C-N bonds. They are commonly found in nucleotide metabolism of purine and pyrimidine. These enzymes share similar catalytic mechanisms and show considerable structural homologies, suggesting that they might have evolved from a common ancestral protein. Homology searches based on common mechanistic properties and three-dimensional protein structures provide clues to the evolutionary relationships of these enzymes. Among the superfamily of enzymes, hydantoinase has been highlighted by its potential for biotechnological applications in the production of unnatural amino acids. The enzymatic process for the production of optically pure amino acids consists of three enzyme steps: hydantoin racemase, hydantoinase, and N-carbamoylase. For efficient industrial application, some critical catalytic properties such as thermostability, catalytic activity, enantioselectivity, and substrate specificity require further improvement. To this end, isolation of new enzymes with desirable properties from natural sources and the optimization of enzymatic processes were attempted. A combination of directed evolution techniques and rational design approaches has made brilliant progress in the redesign of industrially important catalytic enzymes; this approach is likely to be widely applied to the creation of designer enzymes with desirable catalytic properties.

Inhibition assay of biomolecules based on fluorescence resonance energy transfer (FRET) between quantum dots and gold nanoparticles.

An inhibition assay method was developed based on the modulation in the FRET efficiency between quantum dots (QDs) and gold nanoparticles (AuNPs) in the presence of the molecules which inhibit the interactions between QD- and AuNP-conjugated biomolecules. For the functionalization, AuNPs were first stabilized by chemisorption of n-alkanethiols and then capped with the first generation polyamidoamine (G1 PAMAM) dendrimers. By employing a streptavidin-biotin couple as a model system, avidin was quantitatively analyzed as an inhibitor by sensing the change in photoluminescence (PL) quenching of SA-QDs by biotin-AuNPs. The detection limit for avidin was about 10 nM. It is anticipated that the PL quenching-based sensing system can be used for the quantitative analysis and high throughput screening of molecules which inhibit the specific biomolecular interactions.

Functional tuning of a salvaged green fluorescent protein variant with a new sequence space by directed evolution.

We previously reported a method, designated functional salvage screen (FSS), to generate protein lineages with new sequence spaces through the functional or structural salvage of a defective protein by employing green fluorescent protein (GFP) as a model protein. Here, in an attempt to mimic a step in the natural evolution process of proteins, the functionally salvaged mutant GFP-I5 with new sequence space, but showing low fluorescence intensity and stability, was selected and fine-tuned by directed evolution. During a course of functional tuning, GFP-I5 was found to evolve rapidly, recovering the spectral traits to those of the parent GFPuv. The mutant 3E4 from the third round of directed evolution possessed four substitutions; three (F64L, E111V and K166Q) were at the original GFP gene and the other (K8N) at the inserted segment. The fluorescence intensity of 3E4 was approximately 28-fold stronger than GFP-I5, and other spectral properties were retained. Biochemical and biophysical investigations suggested that the fine-tuning by directed evolution led the salvaged variant GFP-I5 to a functionally favorable structure, resulting in recovery of stability and fluorescence. Site-directed mutagenesis of the mutated amino acid residues in both GFPuv and GFP-I5 revealed that each amino acid residue has a different effect on the fluorescence intensity, which implies that 3E4 adopted a new evolutionary path with respect to fluorescence characteristics compared with the parent GFPuv. Directed evolution in conjunction with FSS is expected to be used for generating protein lineages with new fitness landscapes.

Improvement of oxidative and thermostability of N-carbamyl-d-amino Acid amidohydrolase by directed evolution.

N-Carbamyl-D-amino acid amidohydrolase (N-carbamoylase), which is currently employed in the industrial production of unnatural D-amino acid in conjunction with D-hydantoinase, has low oxidative and thermostability. We attempted the simultaneous improvement of the oxidative and thermostability of N-carbamoylase from Agrobacterium tumefaciens NRRL B11291 by directed evolution using DNA shuffling. In a second generation of evolution, the best mutant 2S3 with improved oxidative and thermostability was selected, purified and characterized. The temperature at which 50% of the initial activity remains after incubation for 30 min was 73 degrees C for 2S3, whereas it was 61 degrees C for wild-type enzyme. Treatment of wild-type enzyme with 0.2 mM hydrogen peroxide for 30 min at 25 degrees C resulted in a complete loss of activity, but 2S3 retained about 79% of the initial activity under the same conditions. The K(m) value of 2S3 was estimated to be similar to that of wild-type enzyme; however k(cat) was decreased, leading to a slightly reduced value of k(cat)/K(m), compared with wild-type enzyme. DNA sequence analysis revealed that six amino acid residues were changed in 2S3 and substitutions included Q23L, V40A, H58Y, G75S, M184L and T262A. The stabilizing effects of each amino acid residue were investigated by incorporating mutations individually into wild-type enzyme. Q23L, H58Y, M184L and T262A were found to enhance both oxidative and thermostability of the enzyme and of them, T262A showed the most significant effect. V40A and G75S gave rise to an increase only in oxidative stability. The positions of the mutated amino acid residues were identified in the structure of N-carbamoylase from Agrobacterium sp. KNK 712 and structural analysis of the stabilizing effects of each amino acid substitution was also carried out.

Directed evolution of N-carbamyl-D-amino acid amidohydrolase for simultaneous improvement of oxidative and thermal stability.

Directed evolution of N-carbamyl-D-amino acid amidohydrolase from Agrobacterium tumefaciens NRRL B11291 was attempted in order to simultaneously improve oxidative and thermal stability. A mutant library was generated by DNA shuffling, and positive clones with improved oxidative and thermal stability were screened on the basis of the activity staining method on a solid agar plate containing pH indicator (phenol red) and substrate (N-carbamyl-D-p-hydroxyphenylglycine). Two rounds of directed evolution resulted in the best mutant 2S3 with a significantly improved stability. Oxidative stability of the evolved enzyme 2S3 was about 18-fold higher than that of the wild type, and it also showed an 8-fold increased thermostability. The K(m) value of 2S3 was comparable to that of wild-type enzyme, but k(cat) was slightly decreased. DNA sequence analysis revealed that six amino acid residues (Q23L, V40A, H58Y, G75S, M184L, and T262A) were substituted in 2S3. From the mutational analysis, four mutations (Q23L, H58Y, M184L, and T262A) were found to lead to an improvement of both oxidative and thermal stability. Of them, T262A had the most significant effect, and V40A and G75S only increased the oxidative stability.