Willem Van Berkel

Many agricultural and industrial food by-products are rich in cellulose and xylan. Their enzymatic degradation into monosaccharides is seen as a basis for the production of biofuels and bio-based chemicals. Lytic polysaccharide monooxygenases (LPMOs) constitute a group of recently discovered enzymes, classified as the auxiliary activity subgroups AA9, AA10, AA11 and AA13 in the CAZy database. LPMOs cleave cellulose, chitin, starch and β-(1 → 4)-linked substituted and non-substituted glucosyl units of hemicellulose under formation of oxidized gluco-oligosaccharides. Here, we demonstrate a new LPMO, obtained from Myceliophthora thermophila C1 (MtLPMO9A). This enzyme cleaves β-(1 → 4)-xylosyl bonds in xylan under formation of oxidized xylo-oligosaccharides, while it simultaneously cleaves β-(1 → 4)-glucosyl bonds in cellulose under formation of oxidized gluco-oligosaccharides. In particular, MtLPMO9A benefits from the strong interaction between low substituted linear xylan and cellulose. MtLPMO9A shows a strong synergistic effect with endoglucanase I (EGI) with a 16-fold higher release of detected oligosaccharides, compared to the oligosaccharides release of MtLPMO9A and EGI alone. Now, for the first time, we demonstrate the activity of a lytic polysaccharide monooxygenase (MtLPMO9A) that shows oxidative cleavage of xylan in addition to cellulose. The ability of MtLPMO9A to cleave these rigid regions provides a new paradigm in the understanding of the degradation of xylan-coated cellulose. In addition, MtLPMO9A acts in strong synergism with endoglucanase I. The mode of action of MtLPMO9A is considered to be important for loosening the rigid xylan-cellulose polysaccharide matrix in plant biomass, enabling increased accessibility to the matrix for hydrolytic enzymes. This discovery provides new insights into how to boost plant biomass degradation by enzyme cocktails for biorefinery applications.

NADPH binding to p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens is found to be strongly dependent on pH and ionic strength. In the ionic strength range of 0.02-0.15 M, optimal NADPH binding is observed at a pH value of 6.4. Extrapolation of the dissociation constants to infinite ionic strength shows that under these conditions optimal binding occurs at pH values greater than 8. Similar results were obtained for complexes between the enzyme and two NADPH analogues in the presence or absence of the substrate. The experimental data can be explained by a theoretical model in which monopole-monopole or monopole-dipole interactions between the enzyme and the ligand are dominant. Changes in the former interaction prevail at low ionic strength and low pH values while the changes in the latter prevail at high ionic strength and high pH values. The dipole moment of the enzyme in the direction of the NADPH binding site was calculated from the ionic strength and pH dependence of the complex formation. The calculated dipole moment of the enzyme is about 2000 Debye at pH 6 and decreases to about 1100 Debye at pH 8.5. The results are discussed with respect to published results, including data obtained from the enzyme from a different source.

l-Galactono-1,4-lactone dehydrogenase (GALDH; ferricytochrome c oxidoreductase; EC 1.3.2.3) is a mitochondrial flavoenzyme that catalyzes the final step in the biosynthesis of vitamin C (l-ascorbic acid) in plants. In the present study, we report on the biochemical properties of recombinant Arabidopsis thaliana GALDH (AtGALDH). AtGALDH oxidizes, in addition to l-galactono-1,4-lactone (K(m) = 0.17 mm, k(cat) = 134 s(-1)), l-gulono-1,4-lactone (K(m) = 13.1 mm, k(cat) = 4.0 s(-1)) using cytochrome c as an electron acceptor. Aerobic reduction of AtGALDH with the lactone substrate generates the flavin hydroquinone. The two-electron reduced enzyme reacts poorly with molecular oxygen (k(ox) = 6 x 10(2) m(-1).s(-1)). Unlike most flavoprotein dehydrogenases, AtGALDH forms a flavin N5 sulfite adduct. Anaerobic photoreduction involves the transient stabilization of the anionic flavin semiquinone. Most aldonolactone oxidoreductases contain a histidyl-FAD as a covalently bound prosthetic group. AtGALDH lacks the histidine involved in covalent FAD binding, but contains a leucine instead (Leu56). Leu56 replacements did not result in covalent flavinylation but revealed the importance of Leu56 for both FAD-binding and catalysis. The Leu56 variants showed remarkable differences in Michaelis constants for both l-galactono-1,4-lactone and l-gulono-1,4-lactone and released their FAD cofactor more easily than wild-type AtGALDH. The present study provides the first biochemical characterization of AtGALDH and some active site variants. The role of GALDH and the possible involvement of other aldonolactone oxidoreductases in the biosynthesis of vitamin C in A. thaliana are also discussed.

Aryl-alcohol oxidase provides H(2)O(2) for lignin biodegradation, a key process for carbon recycling in land ecosystems that is also of great biotechnological interest. However, little is known of the structural determinants of the catalytic activity of this fungal flavoenzyme, which oxidizes a variety of polyunsaturated alcohols. Different alcohol substrates were docked on the aryl-alcohol oxidase molecular structure, and six amino acid residues surrounding the putative substrate-binding site were chosen for site-directed mutagenesis modification. Several Pleurotus eryngii aryl-alcohol oxidase variants were purified to homogeneity after heterologous expression in Emericella nidulans, and characterized in terms of their steady-state kinetic properties. Two histidine residues (His502 and His546) are strictly required for aryl-alcohol oxidase catalysis, as shown by the lack of activity of different variants. This fact, together with their location near the isoalloxazine ring of FAD, suggested a contribution to catalysis by alcohol activation, enabling its oxidation by flavin-adenine dinucleotide (FAD). The presence of two aromatic residues (at positions 92 and 501) is also required, as shown by the conserved activity of the Y92F and F501Y enzyme variants and the strongly impaired activity of Y92A and F501A. By contrast, a third aromatic residue (Tyr78) does not seem to be involved in catalysis. The kinetic and spectral properties of the Phe501 variants suggested that this residue could affect the FAD environment, modulating the catalytic rate of the enzyme. Finally, L315 affects the enzyme k(cat), although it is not located in the near vicinity of the cofactor. The present study provides the first evidence for the role of aryl-alcohol oxidase active site residues.

Sulfite salts are widely used as antibrowning agents in food processing. Nevertheless, the exact mechanism by which sulfite prevents enzymatic browning has remained unknown. Here, we show that sodium hydrogen sulfite (NaHSO3) irreversibly blocks the active site of tyrosinase from the edible mushroom Agaricus bisporus, and that the competitive inhibitors tropolone and kojic acid protect the enzyme from NaHSO3 inactivation. LC-MS analysis of pepsin digests of NaHSO3 -treated tyrosinase revealed two peptides showing a neutral loss corresponding to the mass of SO3 upon MS(2) fragmentation. These peptides were found to be homologous peptides containing two of the three histidine residues that form the copper-B-binding site of mushroom tyrosinase isoform PPO3 and mushroom tyrosinase isoform PPO4, which were both present in the tyrosinase preparation used. Peptides showing this neutral loss behavior were not found in the untreated control. Comparison of the effects of NaHSO3 on apo-tyrosinase and holo-tyrosinase indicated that inactivation is facilitated by the active site copper ions. These data provide compelling evidence that inactivation of mushroom tyrosinase by NaHSO3 occurs through covalent modification of a single amino-acid residue, probably via addition of HSO3(-) to one of the copper-coordinating histidines in the copper-B site of the enzyme.

The substrate specificity of the flavoprotein vanillyl-alcohol oxidase from Penicillium simplicissimum was investigated. Vanillyl-alcohol oxidase catalyzes besides the oxidation of 4-hydroxybenzyl alcohols, the oxidative deamination of 4-hydroxybenzylamines and the oxidative demethylation of 4-(methoxymethyl)phenols. During the conversion of vanillylamine to vanillin, a transient intermediate, most probably vanillylimine, is observed. Vanillyl-alcohol oxidase weakly interacts with 4-hydroxyphenylglycols and a series of catecholamines. These compounds are converted to the corresponding ketones. Both enantiomers of (nor)epinephrine are substrates for vanillyl-alcohol oxidase, but the R isomer is preferred. Vanillyl-alcohol oxidase is most active with chavicol and eugenol. These 4-allylphenols are converted to coumaryl alcohol and coniferyl alcohol, respectively. Isotopic labeling experiments show that the oxygen atom inserted at the C gamma atom of the side chain is derived from water. The 4-hydroxycinnamyl alcohol products and the substrate analog isoeugenol are competitive inhibitors of vanillyl alcohol oxidation. The binding of isoeugenol to the oxidized enzyme perturbs the optical spectrum of protein-bound FAD. pH-dependent binding studies suggest that vanillyl-alcohol oxidase preferentially binds the phenolate form of isoeugenol (pKa < 6, 25 degrees C). From this and the high pH optimum for turnover, a hydride transfer mechanism involving a p-quinone methide intermediate is proposed for the vanillyl-alcohol-oxidase-catalyzed conversion of 4-allylphenols.

The crystal structure of the enzyme-substrate complex of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens shows that the hydroxyl group of 4-hydroxybenzoate interacts with the side chain of Tyr201, which is in close contact with the side chain of Tyr385. The role of this hydrogen bonding network in substrate activation was studied by kinetic and spectral analysis of Tyr-->Phe mutant enzymes. The catalytic properties of the enzymes with Tyr201 or Tyr385 replaced by Phe (Tyr201-->Phe and Tyr385-->Phe) with the physiological substrate are comparable with those of the corresponding mutant proteins of p-hydroxybenzoate hydroxylase from P. aeruginosa [Entsch, B., Palfey, B. A., Ballou, D. P. & Massey, V. (1991) J. Biol. Chem. 266, 17341-17349]. Enzyme Tyr201-->Phe has a high Km for NADPH and produces only 5% of 3,4-dihydroxybenzoate/catalytic cycle. Unlike the wild-type enzyme, the Tyr201-->Phe mutant does not stabilize the phenolate form of 4-hydroxybenzoate. With enzyme Tyr385-->Phe, flavin reduction is rate-limiting and the turnover rate is only 2% of wild type. Despite rather efficient hydroxylation, and deviating from the description of the corresponding P. aeruginosa enzyme, mutant Tyr385-->Phe prefers the binding of the phenolic form of 4-hydroxybenzoate. Studies with substrate analogs show that both tyrosines are important for the fine tuning of the effector specificity. Binding of 4-fluorobenzoate differentially stimulates the stabilization of the 4 alpha-hydroperoxyflavin intermediate. Unlike wild type, both Tyr mutants produce 3,4,5-trihydroxybenzoate from 3,4-dihydroxybenzoate. The affinity of enzyme Tyr201-->Phe for the dianionic substrate 2,3,5,6-tetrafluoro-4-hydroxybenzoate is very low, probably because of repulsion of the substrate phenolate in a more nonpolar microenvironment. In contrast to data reported for p-hydroxybenzoate hydroxylase from P. aeruginosa, binding of the inhibitor 4-hydroxycinnamate to wild-type and mutant proteins is not simply described by binary complex formation. A binding model is presented, including secondary binding of the inhibitor. Enzyme Tyr201-->Phe does not stabilize the phenolate form of the inhibitor. In enzyme Tyr385-->Phe, the phenolic pKa of bound 4-hydroxycinnamate is increased with respect to wild type. It is proposed that Tyr385-->Phe is involved in substrate activation by facilitating the deprotonation of Tyr201.