Interaction of Structurally Diverse Phenolic Compounds with Porcine Pancreatic α-Amylase.

A blood glucose level lowering effect is postulated for polyphenols (PPs), which is in part attributed to the inhibition of α-amylase. To estimate structure-effect relationships, chlorogenic acid (CA), phlorizin (PHL), epigallocatechin gallate (EGCG), epicatechin (EC), and malvidin-3-glucoside (Mlv-3-glc) were used as inhibitors in an enzyme assay, on the basis of the conversion of GalG2CNP by α-amylase. The detection of CNP was performed by UV/vis spectroscopy. The data reveal that the inhibitor strength decreases as follows: EGCG > Mlv-3-glc > EC > PHL ∼ CA. Detection of the substrate conversion by isothermal titration calorimetry supports these results. All PPs showed mixed inhibition, except for CA and EGCG wherein the competitive proportion was predominant. Investigations by saturation transfer difference NMR revealed interaction of PPs with α-amylase prevalently based on interactions with the aromatic or conjugated system. A correlation between the extent of the conjugated system and the IC50 of the PP could be found.

Identification and characterization of cytochrome P450 1232A24 and 1232F1 from Arthrobacter sp. and their role in the metabolic pathway of papaverine.

Cytochrome P450 monooxygenases (P450s) play crucial roles in the cell metabolism and provide an unsurpassed diversity of catalysed reactions. Here, we report the identification and biochemical characterization of two P450s from Arthrobacter sp., a Gram-positive organism known to degrade the opium alkaloid papaverine. Combining phylogenetic and genomic analysis suggested physiological roles for P450s in metabolism and revealed potential gene clusters with redox partners facilitating the reconstitution of the P450 activities in vitro. CYP1232F1 catalyses the para demethylation of 3,4-dimethoxyphenylacetic acid to homovanillic acid while CYP1232A24 continues demethylation to 3,4-dihydroxyphenylacetic acid. Interestingly, the latter enzyme is also able to perform both demethylation steps with preference for the meta position. The crystal structure of CYP1232A24, which shares only 29% identity to previous published structures of P450s helped to rationalize the preferred demethylation specificity for the meta position and also the broader substrate specificity profile. In addition to the detailed characterization of the two P450s using their physiological redox partners, we report the construction of a highly active whole-cell Escherichia coli biocatalyst expressing CYP1232A24, which formed up to 1.77 g l-1 3,4-dihydroxyphenylacetic acid. Our results revealed the P450s' role in the metabolic pathway of papaverine enabling further investigation and application of these biocatalysts.

(De)hydratases-recent developments and future perspectives.

Hydratases have gained attention as alternative to chemical catalysts for their ability to add and eliminate water with high regio-selectivity, stereo-selectivity and enantio-selectivity. Recently, especially cofactor-independent hydratases came into research focus as they are of particular interest for industrial application. The investigation of the substrate scope as well as mutagenesis studies combined with high-resolution crystal structures and bioinformatic methods shed light on this promising enzyme class. This review presents latest findings in the field of fatty acid hydratases, linalool dehydratase isomerase and carotenoid hydratases focusing on mechanistic und structural aspects as well as the expansion of the substrate scope and new applications in organic synthesis.

Structural and functional insights into asymmetric enzymatic dehydration of alkenols.

The asymmetric dehydration of alcohols is an important process for the direct synthesis of alkenes. We report the structure and substrate specificity of the bifunctional linalool dehydratase isomerase (LinD) from the bacterium Castellaniella defragrans that catalyzes in nature the hydration of ß-myrcene to linalool and the subsequent isomerization to geraniol. Enzymatic kinetic resolutions of truncated and elongated aromatic and aliphatic tertiary alcohols (C5-C15) that contain a specific signature motif demonstrate the broad substrate specificity of LinD. The three-dimensional structure of LinD from Castellaniella defragrans revealed a pentamer with active sites at the protomer interfaces. Furthermore, the structure of LinD in complex with the product geraniol provides initial mechanistic insights into this bifunctional enzyme. Site-directed mutagenesis confirmed active site amino acid residues essential for its dehydration and isomerization activity. These structural and mechanistic insights facilitate the development of hydrating catalysts, enriching the toolbox for novel bond-forming biocatalysis.