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Substrate Acceptance (substrate + acceptance)

Distribution by Scientific Domains

Chemistry	80%

Selected Abstracts

Mapping the Limits of Substrate Specificity of the Adenylation Domain of TycA

CHEMBIOCHEM, Issue 4 2009
Benoit R. M. Villiers
Abstract The limits and potential of substrate promiscuity of the adenylation domain of tyrocidine synthetase 1 were systematically explored. Substrate acceptance is governed by hydrophobic effects (as shown by the correlation of kcat/KM and side-chain log,P), shape complementarity and steric exclusion. The quantification of these factors provides ground rules for understanding and possibly evolving substrate specificity in this class of enzymes. The catalytic potential of tyrocidine synthetase 1 (TycA) was probed by the kinetic characterization of its adenylation activity. We observed reactions with 30 substrates, thus suggesting some substrate promiscuity. However, although the TycA adenylation (A) domain was able to accommodate alternative substrates, their kcat/KM values ranged over six orders of magnitude. A comparison of the activities allowed the systematic mapping of the substrate specificity determinants of the TycA A-domain. Hydrophobicity plays a major role in the recognition of substrate analogues but can be combined with shape complementarity, conferring higher activity, and/or steric exclusion, leading to substantial discrimination against larger substrates. A comparison of the kcat/KM values of the TycA A-domain and phenylalanyl-tRNA synthetase showed that the level of discrimination was comparable in the two enzymes for the adenylation reaction and suggested that TycA was also subjected to high selective pressure. The specificity patterns observed and the quantification of alternative activities provide a basis for exploring possible paths for the future directed evolution of A-domain specificity. [source]

Resistance of apple trees to Cydia pomonella egg-laying due to leaf surface metabolites

ENTOMOLOGIA EXPERIMENTALIS ET APPLICATA, Issue 1 2008
Nadia Lombarkia
Abstract During host plant selection and particularly after alighting on a plant, chemical cues from the plant surface influence an insect's acceptance of the plant and, subsequently, its egg-laying behaviour. Primary metabolites in the phylloplane may be more important than hitherto known. We have shown that soluble carbohydrates, such as glucose, fructose, and sucrose, and sugar alcohols, such as sorbitol, quebrachitol, and myo -inositol, can be detected by insects after contacting the plant and that they positively influence egg-laying of the codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), on apple trees. We addressed the question whether a lack of these substances could also explain apple tree resistance to C. pomonella in terms of reduced egg-laying. Leaf surface washings were collected in an apple orchard by spraying water on the resistant cultivar X65-11 and on the susceptible cultivar P5R50A4. The washings were tested on a nylon cloth on isolated females under no-choice conditions. The washings were analysed and synthetic blends, each consisting of the six metabolites in the proportions established in the leaf surface washings of both cultivars, were then tested for their effect on egg-laying of C. pomonella. Dose,response egg-laying tests were carried out on substrates impregnated with the X65-11 leaf surface blend at 1, 100, 1 000, and 10 000 times the natural dose. Egg-laying behaviour in the bioassays with leaf surface washings of both cultivars closely resembled egg-laying in the orchard. Washings of P5R50A4 stimulated egg-laying to a greater extent than those of X65-11 and the water control. Synthetic blends reduced substrate acceptance and egg-laying, compared to the washings of X65-11. Ratios between components within the blend are responsible for this resistance. In conclusion, quantities and ratios of the six primary metabolites found on the leaf surface may influence host preference of C. pomonella as well as their egg-laying behaviour, thus they may play a role in the trees' resistance to the codling moth. [source]

Addressing the Numbers Problem in Directed Evolution

CHEMBIOCHEM, Issue 11 2008
Manfred T. Reetz Prof. Dr.
Abstract Our previous contribution to increasing the efficiency of directed evolution is iterative saturation mutagenesis (ISM) as a systematic means of generating focused libraries for the control of substrate acceptance, enantioselectivity, or thermostability of enzymes. We have now introduced a crucial element to knowledge-guided targeted mutagenesis in general that helps to solve the numbers problem in directed evolution. We show that the choice of the amino acid (aa) alphabet, as specified by the utilized codon degeneracy, provides the experimenter with a powerful tool in designing "smarter" randomized libraries that require considerably less screening effort. A systematic comparison of two different codon degeneracies was made by examining the relative quality of the identically sized enzyme libraries in relation to the degree of oversampling required in the screening process. The specific example in our case study concerns the conventional NNK codon degeneracy (32 codons/20 aa) versus NDT (12 codons/12 aa). The model reaction is the hydrolytic kinetic resolution of a chiral trans -disubstituted epoxide, catalyzed by the epoxide hydrolase from Aspergillus niger. The NDT library proves to be of much higher quality, as measured by the dramatically higher frequency of positive variants and by the magnitude of catalyst improvement (enhanced rate and enantioselectivity). We provide a statistical analysis that constitutes a useful guide for the optimal design and generation of "smarter" focused libraries. This type of approach accelerates the process of laboratory evolution considerably and can be expected to be broadly applicable when engineering functional proteins in general. [source]

Prediction of the Candida antarctica Lipase A Protein Structure by Comparative Modeling and Site-Directed Mutagenesis

CHEMBIOCHEM, Issue 12 2007
Alex Kasrayan Dr.
Abstract A number of model structures of the CalA suggested by comparative modeling were tested by site-directed mutagenesis. Enzyme variants were created where amino acids predicted to play key roles for the lipase activity in the different models were replaced by an inert amino acid (alanine). The results from activity measurements of the overproduced and purified mutant enzymes indicate a structure where the active site consists of amino acid residues Ser184, His366, and Asp334 and in which there is no lid. This model can be used for future targeted modifications of the enzyme to obtain new substrate acceptance, better thermostability, and higher enantioselectivity. [source]

Recombinant Whole-Cell Mediated Baeyer,Villiger Oxidation of Perhydropyran-Type Ketones

CHEMISTRY & BIODIVERSITY, Issue 3 2008
Marko
Abstract Recombinant Escherichia coli cells expressing eight Baeyer,Villiger monooxygenases of bacterial origin have been utilized to oxidize prochiral heterocyclic ketones containing a pyran ring system. Within the biotransformation, two stereogenic centers were introduced with high control of enantioselectivity. The chemoselectivity of the enzymatic reaction was found to be high in favor of the Baeyer,Villiger process when using substituted ketone precursors incorporating functional groups labile to oxidation. A significantly different behavior was observed for two groups of monooxygenases with respect to substrate acceptance, which is consistent with our previous classification into two enzyme clusters. [source]