Probing the mechanism of flavin action in the oxidative decarboxylation catalyzed by salicylate hydroxylase.

Salicylate hydroxylase (NahG) is a FAD-dependent monooxygenase in which the reduced flavin activates O2 coupled to the oxidative decarboxylation of salicylate to catechol or uncoupled from substrate oxidation to afford H2O2. This chapter presents different methodologies in equilibrium studies, steady-state kinetics, and identification of reaction products, which were important to understand the SEAr mechanism of catalysis in NahG, the role of the different FAD parts for ligand binding, the extent of uncoupled reaction, and the catalysis of salicylate's oxidative decarboxylation. These features are likely familiar to many other FAD-dependent monooxygenases and offer a potential asset for developing new tools and strategies in catalysis.

The role of remote flavin adenine dinucleotide pieces in the oxidative decarboxylation catalyzed by salicylate hydroxylase.

Salicylate hydroxylase (NahG) has a single redox site in which FAD is reduced by NADH, the O2 is activated by the reduced flavin, and salicylate undergoes an oxidative decarboxylation by a C(4a)-hydroperoxyflavin intermediate to give catechol. We report experimental results that show the contribution of individual pieces of the FAD cofactor to the observed enzymatic activity for turnover of the whole cofactor. A comparison of the kinetic parameters and products for the NahG-catalyzed reactions of FMN and riboflavin cofactor fragments reveal that the adenosine monophosphate (AMP) and ribitol phosphate pieces of FAD act to anchor the flavin to the enzyme and to direct the partitioning of the C(4a)-hydroperoxyflavin reaction intermediate towards hydroxylation of salicylate. The addition of AMP or ribitol phosphate pieces to solutions of the truncated flavins results in a partial restoration of the enzymatic activity lost upon truncation of FAD, and the pieces direct the reaction of the C(4a)-hydroperoxyflavin intermediate towards hydroxylation of salicylate.

Transformation of xylan into value-added biocommodities using Thermobacillus composti GH10 xylanase.

Enzymatic transformation of xylans into renewable fuels and value-added products is mediated by xylanases. Here we describe the biochemical and X-ray structural characterization of Thermobacillus composti GH10 xylanase (TcXyn10A) at 2.1 Å resolution aiming to unravel details of its recognition of glucurono- and arabinoxylan at a molecular level. TcXyn10A improves the efficiency of pretreated lignocellulosic biomass hydrolysis by a commercial enzyme cocktail causing a 15.35 % increase in xylose release and 4.38 % glucose release after 24 h of reaction. The enzyme releases predominantly xylobiose and xylotriose, as well as MeGlcA3 × 3 (from beechwood glucuronoxylan) and a range of decorated xylooligosaccharides (XOS) from rye arabinoxylan, with Ara2 × 2 being the major product. The enzyme liberates XOS with the yields of 29.09 % for beechwood glucuronoxylan and 16.98 % for rye arabinoxylan. Finally, TcXyn10A has a high thermal stability, halotolerance, and resistance to ethanol, biochemical properties that can be desirable for a number of industrial applications.

Crystallographic structure and molecular dynamics simulations of the major endoglucanase from Xanthomonas campestris pv. campestris shed light on its oligosaccharide products release pattern.

Cellulases are essential enzymatic components for the transformation of plant biomass into fuels, renewable materials and green chemicals. Here, we determined the crystal structure, pattern of hydrolysis products release, and conducted molecular dynamics simulations of the major endoglucanase from the Xanthomonas campestris pv. campestris (XccCel5A). XccCel5A has a TIM barrel fold with the catalytic site centrally placed in a binding groove surrounded by aromatic side chains. Molecular dynamics simulations show that productive position of the substrate is secured by a network of hydrogen bonds in the four main subsites, which differ in details from homologous structures. Capillary zone electrophoresis and computational studies reveal XccCel5A can act both as endoglucanase and licheninase, but there are preferable arrangements of substrate regarding ß-1,3 and ß-1,4 bonds within the binding cleft which are related to the enzymatic efficiency.

Catalytic mechanism for the conversion of salicylate into catechol by the flavin-dependent monooxygenase salicylate hydroxylase.

Salicylate hydroxylase (NahG) is a flavin-dependent monooxygenase that catalyzes the decarboxylative hydroxylation of salicylate into catechol in the naphthalene degradation pathway in Pseudomonas putida G7. We explored the mechanism of action of this enzyme in detail using a combination of structural and biophysical methods. NahG shares many structural and mechanistic features with other versatile flavin-dependent monooxygenases, with potential biocatalytic applications. The crystal structure at 2.0 Šresolution for the apo form of NahG adds a new snapshot preceding the FAD binding in flavin-dependent monooxygenases. The kcat/Km for the salicylate reaction catalyzed by the holo form is >105 M-1 s-1 at pH 8.5 and 25 °C. Hammett plots for Km and kcat using substituted salicylates indicate change in rate-limiting step. Electron-donating groups favor the hydroxylation of salicylate by a peroxyflavin to yield a Wheland-like intermediate, whereas the decarboxylation of this intermediate is faster for electron-withdrawing groups. The mechanism is supported by structural data and kinetic studies at different pHs. The salicylate carboxyl group lies near a hydrophobic region that aids decarboxylation. A conserved histidine residue is proposed to assist the reaction by general base/general acid catalysis.

Structural and Kinetic Properties of the Aldehyde Dehydrogenase NahF, a Broad Substrate Specificity Enzyme for Aldehyde Oxidation.

The salicylaldehyde dehydrogenase (NahF) catalyzes the oxidation of salicylaldehyde to salicylate using NAD(+) as a cofactor, the last reaction of the upper degradation pathway of naphthalene in Pseudomonas putida G7. The naphthalene is an abundant and toxic compound in oil and has been used as a model for bioremediation studies. The steady-state kinetic parameters for oxidation of aliphatic or aromatic aldehydes catalyzed by 6xHis-NahF are presented. The 6xHis-NahF catalyzes the oxidation of aromatic aldehydes with large kcat/Km values close to 10(6) M(-1) s(-1). The active site of NahF is highly hydrophobic, and the enzyme shows higher specificity for less polar substrates than for polar substrates, e.g., acetaldehyde. The enzyme shows α/ß folding with three well-defined domains: the oligomerization domain, which is responsible for the interlacement between the two monomers; the Rossmann-like fold domain, essential for nucleotide binding; and the catalytic domain. A salicylaldehyde molecule was observed in a deep pocket in the crystal structure of NahF where the catalytic C284 and E250 are present. Moreover, the residues G150, R157, W96, F99, F274, F279, and Y446 were thought to be important for catalysis and specificity for aromatic aldehydes. Understanding the molecular features responsible for NahF activity allows for comparisons with other aldehyde dehydrogenases and, together with structural information, provides the information needed for future mutational studies aimed to enhance its stability and specificity and further its use in biotechnological processes.

Crystal Structures of Apo and Liganded 4-Oxalocrotonate Decarboxylase Uncover a Structural Basis for the Metal-Assisted Decarboxylation of a Vinylogous ß-Keto Acid.

The enzymes in the catechol meta-fission pathway have been studied for more than 50 years in several species of bacteria capable of degrading a number of aromatic compounds. In a related pathway, naphthalene, a toxic polycyclic aromatic hydrocarbon, is fully degraded to intermediates of the tricarboxylic acid cycle by the soil bacteria Pseudomonas putida G7. In this organism, the 83 kb NAH7 plasmid carries several genes involved in this biotransformation process. One enzyme in this route, NahK, a 4-oxalocrotonate decarboxylase (4-OD), converts 2-oxo-3-hexenedioate to 2-hydroxy-2,4-pentadienoate using Mg(2+) as a cofactor. Efforts to study how 4-OD catalyzes this decarboxylation have been hampered because 4-OD is present in a complex with vinylpyruvate hydratase (VPH), which is the next enzyme in the same pathway. For the first time, a monomeric, stable, and active 4-OD has been expressed and purified in the absence of VPH. Crystal structures for NahK in the apo form and bonded with five substrate analogues were obtained using two distinct crystallization conditions. Analysis of the crystal structures implicates a lid domain in substrate binding and suggests roles for specific residues in a proposed reaction mechanism. In addition, we assign a possible function for the NahK N-terminal domain, which differs from most of the other members of the fumarylacetoacetate hydrolase superfamily. Although the structural basis for metal-dependent ß-keto acid decarboxylases has been reported, this is the first structural report for that of a vinylogous ß-keto acid decarboxylase and the first crystal structure of a 4-OD.