The 1.6 ? Crystal Structure of Pyranose Dehydrogenase from Agaricus meleagris Rationalizes Substrate Specificity and Reveals a Flavin Intermediate

Pyranose dehydrogenases (PDHs) are extracellular flavin-dependent oxidoreductases secreted by litter-decomposing fungi with a role in natural recycling of plant matter. All major monosaccharides in lignocellulose are oxidized by PDH at comparable yields and efficiencies. Oxidation takes place as single-oxidation or sequential double-oxidation reactions of the carbohydrates, resulting in sugar derivatives oxidized primarily at C2, C3 or C2/3 with the concomitant reduction of the flavin. A suitable electron acceptor then reoxidizes the reduced flavin. Whereas oxygen is a poor electron acceptor for PDH, several alternative acceptors, e.g., quinone compounds, naturally present during lignocellulose degradation, can be used. We have determined the 1.6-Å crystal structure of PDH from Agaricus meleagris. Interestingly, the flavin ring in PDH is modified by a covalent mono- or di-atomic species at the C(4a) position. Under normal conditions, PDH is not oxidized by oxygen; however, the related enzyme pyranose 2-oxidase (P2O) activates oxygen by a mechanism that proceeds via a covalent flavin C(4a)-hydroperoxide intermediate. Although the flavin C(4a) adduct is common in monooxygenases, it is unusual for flavoprotein oxidases, and it has been proposed that formation of the intermediate would be unfavorable in these oxidases. Thus, the flavin adduct in PDH not only shows that the adduct can be favorably accommodated in the active site, but also provides important details regarding the structural, spatial and physicochemical requirements for formation of this flavin intermediate in related oxidases. Extensive in silico modeling of carbohydrates in the PDH active site allowed us to rationalize the previously reported patterns of substrate specificity and regioselectivity. To evaluate the regioselectivity of D-glucose oxidation, reduction experiments were performed using fluorinated glucose. PDH was rapidly reduced by 3-fluorinated glucose, which has the C2 position accessible for oxidation, whereas 2-fluorinated glucose performed poorly (C3 accessible), indicating that the glucose C2 position is the primary site of attack.     

Monooxygenation of aromatic compounds by flavin‐dependent monooxygenases

Many flavoenzymes catalyze hydroxylation of aromatic compounds especially phenolic compounds have been isolated and characterized. These enzymes can be classified as either single‐component or two‐component flavin‐dependent hydroxylases (monooxygenases). The hydroxylation reactions catalyzed by the enzymes in this group are useful for modifying the biological properties of phenolic compounds. This review aims to provide an in‐depth discussion of the current mechanistic understanding of representative flavin‐dependent monooxygenases including 3‐hydroxy‐benzoate 4‐hydroxylase (PHBH, a single‐component hydroxylase), 3‐hydroxyphenylacetate 4‐hydroxylase (HPAH, a two‐component hydroxylase), and other monooxygenases which catalyze reactions in addition to hydroxylation, including 2‐methyl‐3‐hydroxypyridine‐5‐carboxylate oxygenase (MHPCO, a single‐component enzyme that catalyzes aromatic‐ring cleavage), and HadA monooxygenase (a two‐component enzyme that catalyzes additional group elimination reaction). These enzymes have different unique structural features which dictate their reactivity toward various substrates and influence their ability to stabilize flavin intermediates such as C4a‐hydroperoxyflavin. Understanding the key catalytic residues and the active site environments important for governing enzyme reactivity will undoubtedly facilitate future work in enzyme engineering or enzyme redesign for the development of biocatalytic methods for the synthesis of valuable compounds.     

Hydrogen peroxide elimination from C4a-hydroperoxyflavin in a flavoprotein oxidase occurs through a single proton transfer from flavin N5 to a peroxide leaving group

C4a-hydroperoxyflavin is found commonly in the reactions of flavin-dependent monooxygenases, in which it plays a key role as an intermediate that incorporates an oxygen atom into substrates. Only recently has evidence for its involvement in the reactions of flavoprotein oxidases been reported. Previous studies of pyranose 2-oxidase (P2O), an enzyme catalyzing the oxidation of pyranoses using oxygen as an electron acceptor to generate oxidized sugars and hydrogen peroxide (H(2)O(2)), have shown that C4a-hydroperoxyflavin forms in P2O reactions before it eliminates H(2)O(2) as a product (Sucharitakul, J., Prongjit, M., Haltrich, D., and Chaiyen, P. (2008) Biochemistry 47, 8485-8490). In this report, the solvent kinetic isotope effects (SKIE) on the reaction of reduced P2O with oxygen were investigated using transient kinetics. Our results showed that D(2)O has a negligible effect on the formation of C4a-hydroperoxyflavin. The ensuing step of H(2)O(2) elimination from C4a-hydroperoxyflavin was shown to be modulated by an SKIE of 2.8 ± 0.2, and a proton inventory analysis of this step indicates a linear plot. These data suggest that a single-proton transfer process causes SKIE at the H(2)O(2) elimination step. Double and single mixing stopped-flow experiments performed in H(2)O buffer revealed that reduced flavin specifically labeled with deuterium at the flavin N5 position generated kinetic isotope effects similar to those found with experiments performed with the enzyme pre-equilibrated in D(2)O buffer. This suggests that the proton at the flavin N5 position is responsible for the SKIE and is the proton-in-flight that is transferred during the transition state. The mechanism of H(2)O(2) elimination from C4a-hydroperoxyflavin is consistent with a single proton transfer from the flavin N5 to the peroxide leaving group, possibly via the formation of an intramolecular hydrogen bridge.     

Three-dimensional organization of the brain and distribution of serotonin in the brain and ovary,and its effects on ovarian steroidogenesis in the giant freshwater prawn, <Emphasis Type="Italic">Macrobrachium rosenbergii</Emphasis>

The giant freshwater prawn, Macrobrachium rosenbergii, is an economically important crustacean species which has also been extensively used as a model in neuroscience research. The crustacean central nervous system is a highly complex structure, especially the brain. However, little information is available on the brain structure, especially the three-dimensional organization. In this study, we demonstrated the three-dimensional structure and histology of the brain of M. rosenbergii together with the distribution of serotonin (5-HT) in the brain and ovary as well as its effects on ovarian steroidogenesis. The brain of M. rosenbergii consists of three parts: protocerebrum, deutocerebrum and tritocerebrum. Histologically, protocerebrum comprises of neuronal clusters 6–8 and prominent anterior and posterior medial protocerebral neuropils (AMPN/PMPN). The protocerebrum is connected posteriorly to the deutocerebrum which consists of neuronal clusters 9–13, medial antenna I neuropil, a paired lateral antenna I neuropils and olfactory neuropils (ON). Tritocerebrum comprises of neuronal clusters 14–17 with prominent pairs of antenna II (AnN), tegumentary and columnar neuropils (CN). All neuronal clusters are paired structures except numbers 7, 13 and 17 which are single clusters located at the median zone. These neuronal clusters and neuropils are clearly shown in three-dimensional structure of the brain. 5-HT immunoreactivity (-ir) was mostly detected in the medium-sized neurons and neuronal fibers of clusters 6/7, 8, 9, 10 and 14/15 and in many neuropils of the brain including anterior/posterior medial protocerebral neuropils (AMPN/PMPN), protocerebral tract, protocerebral bridge, central body, olfactory neuropil (ON), antennal II neuropil (Ann) and columnar neuropil (CN). In the ovary, the 5-HT-ir was light in the oocyte step 1(Oc₁) and very intense in Oc₂–Oc₄. Using an in vitro assay of an explant of mature ovary, it was shown that 5-HT was able to enhance ovarian estradiol-17β (E₂) and progesterone (P₄) secretions. We suggest that 5-HT is specifically localized in specific brain areas and ovary of this prawn and it plays a pivotal role in ovarian maturation via the induction of female sex steroid secretions, in turn these steroids may enhance vitellogenesis resulting in oocyte growth and maturation.     

Production and characterization of chitosan obtained from <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> grown on potato chip processing waste

Potato chip processing waste of trimmed potato, potato peel and substandard (low-quality) potato chips, obtained from a potato chip processing plant, were used as substrates for chitosan production from Rhizopus oryzae. It was cultured on each waste product at 30 ± 2°C and 70% moisture content for 21 days. Fermented potato peel had the highest yield after 5 days of fermentation. The cultivation condition of chitosan obtained from R. oryzae was optimum for a peel size of less than 6 mesh, 70% moisture content and a pH of 5. Furthermore, the best extraction condition was using 46% sodium hydroxide at 46°C for 13 h followed by 2% acetic acid at 95°C for 8 h. The maximum chitosan yield obtained by these conditions was 10.8 g/kg substrate. Fungal chitosan properties were found to be 86–90% degree of deacetylation, molecular weight of 80–128 kDa and viscosity of 3.1–6.1 mPa s. Therefore, potato peel could be applied as a low cost substrate for chitosan production from R. oryzae.     

N‐linked mannose glycoconjugates on shrimp thrombospondin, pmTSP‐II,and their involvement in the sperm acrosome reaction

Glycoconjugates in egg extracellular matrices are known to serve several functions in reproductive processes. Here, the presence of N‐linked mannose (Man) glycoconjugates on shrimp thrombospondin ( pmTSP‐II) and their physiological functions were investigated in the black tiger shrimp Penaeus monodon. A molecular analysis of pmTSP‐II demonstrated anchorage sites for N‐linked glycans in both the chitin‐binding and TSP3 domains. The presence of Man residues was verified by concanavalin A lectin histochemistry on the purified fraction of pmTSP‐II (250 kDa with protease inhibitor). The function of the Man glycoconjugates was evident by the Con A interference with the pmTSP‐II‐induced acrosome reaction (AR) as well as by the ability to recover the induction of the AR by the inclusion of Mans in the treatment mixture. In addition, the recombinant proteins of the three signature pmTSP‐II domains expressed in E. coli (lacking glycosylation) and mannosidase‐treated pmTSP‐II showed a minimal ability to initiate the AR response. Together, these results provide evidence of the pivotal role that Man‐linked pmTSP‐II plays in modulating the shrimp sperm AR, a novel role for a TSP family protein in shrimp reproductive biology.     

Microbial degradation of halogenated aromatics: molecular mechanisms and enzymatic reactions

Halogenated aromatics are used widely in various industrial, agricultural and household applications. However, due to their stability, most of these compounds persist for a long time, leading to accumulation in the environment. Biological degradation of halogenated aromatics provides sustainable, low-cost and environmentally friendly technologies for removing these toxicants from the environment. This minireview discusses the molecular mechanisms of the enzymatic reactions for degrading halogenated aromatics which naturally occur in various microorganisms. In general, the biodegradation process (especially for aerobic degradation) can be divided into three main steps: upper, middle and lower metabolic pathways which successively convert the toxic halogenated aromatics to common metabolites in cells. The most difficult step in the degradation of halogenated aromatics is the dehalogenation step in the middle pathway. Although a variety of enzymes are involved in the degradation of halogenated aromatics, these various pathways all share the common feature of eventually generating metabolites for utilizing in the energy-producing metabolic pathways in cells. An in-depth understanding of how microbes employ various enzymes in biodegradation can lead to the development of new biotechnologies via enzyme/cell/metabolic engineering or synthetic biology for sustainable biodegradation processes.