Promiscuous aldolase activity has also been found for macrophomat

Promiscuous aldolase activity has also been found for macrophomate synthase which catalyses the addition of the enolate of pyruvate (generated on the enzyme by decarboxylation of oxaloacetate) with a wide range of structurally complex aldehydes to yield 3-deoxysugars [41]. This system has

advantages over known natural pyruvate-dependent aldolases as it has a broad substrate spectrum. The biological outcomes of the interactions of stereoisomers of small drug molecules with their targets can be dramatically different and the global market for enantiomerically pure, active pharmaceutical ingredients (APIs) Tanespimycin supplier is increasing rapidly. However, the chemical synthesis of enantiomerically pure compounds can be challenging, and most often relies on the classical resolution of a racemate. Harnessing enzymes as chiral

catalysts is EGFR inhibitor viable in both the small scale and industrial synthesis of enantiomerically enriched compounds. In this respect, protein engineering of enzymes to enhance or alter the stereochemical outcome of an enzyme reaction is of great importance and much attention has been focused on aldolases, as up to two stereo-centres may be generated during the carbon-carbon bond forming step [11]. In recent years there has been much progress in using many engineering methods ranging from directed evolution [42] to rational Decitabine in vivo redesign [43••, 44 and 45] to produce products with high stereochemical control. Improved biocatalysts have also been found by screening available environmental DNA libraries. In this way, a natural variant of DERA was discovered that produced (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranoside (see above), with a diastereomeric excess of 99.8% and an enantiomeric excess of 99.9% [ 46]. This variant also had a higher tolerance to the inhibiting substrate chloroacetaldehyde and was more efficient

than the E. coli variant, allowing lower quantities of the enzyme to be used in the process. Both these factors increase the commercial and industrial viability of the biocatalytic process. The ability to engineer or evolve the stereochemical outcome of an aldolase reaction was first demonstrated for tagatose-1,6-bisphosphate aldolase [47] and N-acetylneuraminic acid lyase [ 42]. More recently, rational redesign has been carried out on the Class II aldolase BphI to switch the stereochemical outcome of the reaction of pyruvate with acetaldehyde. First, the substrate specificity of BphI was changed to favour propionaldehyde over acetaldehyde [ 48] using site-directed mutagenesis based on modelling of the structure using the orthologous enzyme DmpG [ 49].

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