Willem J. H. van Berkel

Sorghum grain as human food in Africa: relevance of content of starch and amylase activities

Sorghum is a staple food grain in many semi-arid and tropic areas of the world, notably in Sub-Saharan Africa because of its good adaptation to hard environments and its good yield of production. Among important biochemical components for sorghum processing are levels of starch (amylose and amylopectin) and starch depolymerizing enzymes. Current research focus on identifying varieties meeting specific agricultural and food requirements from the great biodiversity of sorghums to insure food security. Results show that some sorghums are rich sources of micronutrients (minerals and vitamins) and macronutrients (carbohydrates, proteins and fat). Sorghum has a resistant starch, which makes it interesting for obese and diabetic people. In addition, sorghum may be an alternative food for people who are allergic to gluten. Malts of some sorghum varieties display

Identification of a Baeyer-Villiger monooxygenase sequence motif

Baeyer–Villiger monooxygenases (BVMOs) form a distinct class of flavoproteins that catalyze the insertion of an oxygen atom in a C–C bond using dioxygen and NAD(P)H. Using newly characterized BVMO sequences, we have uncovered a BVMO‐identifying sequence motif: FXGXXXHXXXW(P/D). Studies with site‐directed mutants of 4‐hydroxyacetophenone monooxygenase from Pseudomonas fluorescens ACB suggest that this fingerprint sequence is critically involved in catalysis. Further sequence analysis showed that the BVMOs belong to a novel superfamily that comprises three known classes of FAD‐dependent monooxygenases: the so‐called flavin‐containing monooxygenases (FMOs), the N‐hydroxylating monooxygenases (NMOs), and the BVMOs. Interestingly, FMOs contain an almost identical sequence motif when compared to the BVMO sequences: FXGXXXHXXX(Y/F). Using these novel amino acid sequence fingerprints, BVMOs and FMOs can be readily identified in the protein sequence databank.

Flavin dependent monooxygenases.

Flavin-dependent monooxygenases catalyze a wide variety of chemo-, regio- and enantioselective oxygenation reactions. As such, they are involved in key biological processes ranging from catabolism, detoxification and biosynthesis, to light emission and axon guidance. Based on fold and function, flavin-dependent monooxygenases can be distributed into eight groups. Groups A and B comprise enzymes that rely on NAD(P)H as external electron donor. Groups C-F are two-protein systems, composed of a monooxygenase and a flavin reductase. Groups G and H comprise internal monooxygenases that reduce the flavin cofactor through substrate oxidation. Recently, many new flavin-dependent monooxygenases have been discovered. In addition to posing basic enzymological questions, these proteins attract attention of pharmaceutical and fine-chemical industries, given their importance as regio- and enantioselective biocatalysts. In this review we present an update of the classification of flavin-dependent monooxygenases and summarize the latest advances in our understanding of their catalytic and structural properties.

Mical links semaphorins to F-actin disassembly

How instructive cues present on the cell surface have their precise effects on the actin cytoskeleton is poorly understood. Semaphorins are one of the largest families of these instructive cues and are widely studied for their effects on cell movement, navigation, angiogenesis, immunology and cancer. Semaphorins/collapsins were characterized in part on the basis of their ability to drastically alter actin cytoskeletal dynamics in neuronal processes, but despite considerable progress in the identification of semaphorin receptors and their signalling pathways, the molecules linking them to the precise control of cytoskeletal elements remain unknown. Recently, highly unusual proteins of the Mical family of enzymes have been found to associate with the cytoplasmic portion of plexins, which are large cell-surface semaphorin receptors, and to mediate axon guidance, synaptogenesis, dendritic pruning and other cell morphological changes. Mical enzymes perform reduction–oxidation (redox) enzymatic reactions and also contain domains found in proteins that regulate cell morphology. However, nothing is known of the role of Mical or its redox activity in mediating morphological changes. Here we report that Mical directly links semaphorins and their plexin receptors to the precise control of actin filament (F-actin) dynamics. We found that Mical is both necessary and sufficient for semaphorin–plexin-mediated F-actin reorganization in vivo. Likewise, we purified Mical protein and found that it directly binds F-actin and disassembles both individual and bundled actin filaments. We also found that Mical utilizes its redox activity to alter F-actin dynamics in vivo and in vitro, indicating a previously unknown role for specific redox signalling events in actin cytoskeletal regulation. Mical therefore is a novel F-actin-disassembly factor that provides a molecular conduit through which actin reorganization—a hallmark of cell morphological changes including axon navigation—can be precisely achieved spatiotemporally in response to semaphorins.

Multiple pathways guide oxygen diffusion into flavoenzyme active sites

Dioxygen (O2) and other gas molecules have a fundamental role in a variety of enzymatic reactions. However, it is only poorly understood which O2 uptake mechanism enzymes employ to promote efficient catalysis and how general this is. We investigated O2 diffusion pathways into monooxygenase and oxidase flavoenzymes, using an integrated computational and experimental approach. Enhanced-statistics molecular dynamics simulations reveal spontaneous protein-guided O2 diffusion from the bulk solvent to preorganized protein cavities. The predicted protein-guided diffusion paths and the importance of key cavity residues for oxygen diffusion were verified by combining site-directed mutagenesis, rapid kinetics experiments, and high-resolution X-ray structures. This study indicates that monooxygenase and oxidase flavoenzymes employ multiple funnel-shaped diffusion pathways to absorb O2 from the solvent and direct it to the reacting C4a atom of the flavin cofactor. The difference in O2 reactivity among dehydrogenases, monooxygenases, and oxidases ultimately resides in the fine modulation of the local environment embedding the reactive locus of the flavin.

Phenol Hydroxylase from Bacillus thermoglucosidasius A7, a Two-protein Component Monooxygenase with a Dual Role for FAD

A novel phenol hydroxylase (PheA) that catalyzes the first step in the degradation of phenol in Bacillus thermoglucosidasius A7 is described. The two-protein system, encoded by the pheA1 and pheA2 genes, consists of an oxygenase (PheA1) and a flavin reductase (PheA2) and is optimally active at 55 °C. PheA1 and PheA2 were separately expressed in recombinant Escherichia coli BL21(DE3) pLysS cells and purified to apparent homogeneity. The pheA1 gene codes for a protein of 504 amino acids with a predicted mass of 57.2 kDa. PheA1 exists as a homodimer in solution and has no enzyme activity on its own. PheA1 catalyzes the efficient ortho-hydroxylation of phenol to catechol when supplemented with PheA2 and FAD/NADH. The hydroxylase activity is strictly FAD-dependent, and neither FMN nor riboflavin can replace FAD in this reaction. The pheA2 gene codes for a protein of 161 amino acids with a predicted mass of 17.7 kDa. PheA2 is also a homodimer, with each subunit containing a highly fluorescent FAD prosthetic group. PheA2 catalyzes the NADH-dependent reduction of free flavins according to a Ping Pong Bi Bi mechanism. PheA2 is structurally related to ferric reductase, an NAD(P)H-dependent reductase from the hyperthermophilic Archaea Archaeoglobus fulgidus that catalyzes the flavin-mediated reduction of iron complexes. However, PheA2 displays no ferric reductase activity and is the first member of a newly recognized family of short-chain flavin reductases that use FAD both as a substrate and as a prosthetic group.

Covalent flavinylation is essential for efficient redox catalysis in vanillyl-alcohol oxidase.

By mutating the target residue of covalent flavinylation in vanillyl-alcohol oxidase, the functional role of the histidyl-FAD bond was studied. Three His422mutants (H422A, H422T, and H422C) were purified, which all contained tightly but noncovalently bound FAD. Steady state kinetics revealed that the mutants have retained enzyme activity, although the turnover rates have decreased by 1 order of magnitude. Stopped-flow analysis showed that the H422A mutant is still able to form a stable binary complex of reduced enzyme and a quinone methide product intermediate, a crucial step during vanillyl-alcohol oxidase-mediated catalysis. The only significant change in the catalytic cycle of the H422A mutant is a marked decrease in reduction rate. Redox potentials of both wild type and H422A vanillyl-alcohol oxidase have been determined. During reduction of H422A, a large portion of the neutral flavin semiquinone is observed. Using suitable reference dyes, the redox potentials for the two one-electron couples have been determined: −17 and −113 mV. Reduction of wild type enzyme did not result in any formation of flavin semiquinone and revealed a remarkably high redox potential of +55 mV. The marked decrease in redox potential caused by the missing covalent histidyl-FAD bond is reflected in the reduced rate of substrate-mediated flavin reduction limiting the turnover rate. Elucidation of the crystal structure of the H422A mutant established that deletion of the histidyl-FAD bond did not result in any significant structural changes. These results clearly indicate that covalent interaction of the isoalloxazine ring with the protein moiety can markedly increase the redox potential of the flavin cofactor, thereby facilitating redox catalysis. Thus, formation of a histidyl-FAD bond in specific flavoenzymes might have evolved as a way to contribute to the enhancement of their oxidative power.

3DM: Systematic analysis of heterogeneous superfamily data to discover protein functionalities

Ten years of experience with molecular class–specific information systems (MCSIS) such as with the hand‐curated G protein–coupled receptor database (GPCRDB) or the semiautomatically generated nuclear receptor database has made clear that a wide variety of questions can be answered when protein‐related data from many different origins can be flexibly combined. MCSISes revolve around a multiple sequence alignment (MSA) that includes “all” available sequences from the entire superfamily, and it has been shown at many occasions that the quality of these alignments is the most crucial aspect of the MCSIS approach. We describe here a system called 3DM that can automatically build an entire MCSIS. 3DM bases the MSA on a multiple structure alignment, which implies that the availability of a large number of superfamily members with a known three‐dimensional structure is a requirement for 3DM to succeed well. Thirteen MCSISes were constructed and placed on the Internet for examination. These systems have been instrumental in a large series of research projects related to enzyme activity or the understanding and engineering of specificity, protein stability engineering, DNA‐diagnostics, drug design, and so forth. Proteins 2010.

Novel peptides with tyrosinase inhibitory activity.

Tyrosinase inhibition by peptides may find its application in food, cosmetics or medicine. In order to identify novel tyrosinase inhibitory peptides, protein-based peptide libraries made by SPOT synthesis were used to screen for peptides that show direct interaction with tyrosinase. One of the peptide libraries studied consists of overlapping, octameric peptides derived from industrial proteins as beta-casein, alpha-lactalbumin, beta-lactoglobulin, ovalbumin, gliadin, glycinin, and beta-conglycinin. On-membrane activity staining resulted in a set of peptides that are not only able to bind to tyrosinase, but are able to inhibit tyrosinase as well. Peptides containing aspartic or glutamic acid residues usually do not bind very well to tyrosinase. Strong tyrosinase-binding peptides always contain one or more arginine residues, often in combination with phenylalanine, while lysine residues can be found equally among nonbinding peptides as well as moderate tyrosinase-binding peptides. The presence of the hydrophobic, aliphatic residues valine, alanine or leucine appears to be important for tyrosinase inhibition. Therefore, good tyrosinase inhibitory peptides preferably contain arginine and/or phenylalanine in combination with valine, alanine and/or leucine.

The growing VAO flavoprotein family.

The VAO flavoprotein family is a rapidly growing family of oxidoreductases that favor the covalent binding of the FAD cofactor. In this review we report on the catalytic properties of some newly discovered VAO family members and their mode of flavin binding. Covalent binding of the flavin is a self-catalytic post-translational modification primarily taking place in oxidases. Covalent flavinylation increases the redox potential of the cofactor and thus its oxidation power. Recent findings have revealed that some members of the VAO family anchor the flavin via a dual covalent linkage (6-S-cysteinyl-8alpha-N1-histidyl FAD). Some VAO-type aldonolactone oxidoreductases favor the non-covalent binding of the flavin cofactor. These enzymes act as dehydrogenases, using cytochrome c as electron acceptor.