Glucooligosaccharide oxidase from Acremonium strictum has been screened for potential applications in oligosaccharide acid production and alternative carbohydrate detection, because it catalyzes the oxidation of glucose, maltose, lactose, cellobiose and cello-and maltooligosaccharides. We report the crystal structures of the enzyme and of its complex with an inhibitor, 5-amino-5-deoxycellobiono-1,5-lactam at 1.55-and 1.98-Å resolution, respectively. Unexpectedly, the protein structure demonstrates the first known double attachment flavinylation, 6-S-cysteinyl, 8␣-N1-histidyl FAD. The FAD cofactor is cross-linked to the enzyme via the C Sugar oxidases and dehydrogenases that catalyze carbohydrate oxidation to the corresponding lactones are of considerable commercial importance as potential diagnostic reagents and industrial biocatalysts. For example, glucose oxidase (GOX) 4 is widely used in analytical biochemistry and in the food industry (1). A search of the BRENDA enzyme data base (2) reveals that most of these enzymes are specific for a variety of mono-and disaccharides, and only a few enzymes are highly selective for oligosaccharides. These include galactose oxidase, cellobiose dehydrogenase (CDH), and glucooligosaccharide oxidase (GOOX). GOOX from Acremonium strictum was screened with the aim of identifying enzymes with potential applications in oligosaccharide acid production and alternative carbohydrate assays (3). It is a monomeric glycoprotein with a covalently linked FAD and catalyzes the oxidization of a variety of carbohydrates with the concomitant reduction of molecular oxygen to hydrogen peroxide. A screening of more than 50 carbohydrates and derivatives showed that D-glucose, maltose, lactose, and cellobiose are good substrates. In addition, this enzyme can react with malto-and cellooligosaccharides, and hence the name of this novel oxidase. The broad substrate specificity of GOOX, particularly toward oligosaccharides, suggests that it may have great potential applicability.To facilitate further characterization and potential industrial use of A. strictum GOOX, we have cloned the encoding gene, which is composed of a 25-residue signal peptide and a 474-residue mature protein (4). Although GOOX shows a similar substrate specificity as GOX, CDH, and pyranose oxidase (POX), it shares no sequence similarity with them. However, GOOX displays significant sequence homology to the FADbinding domain (F domain) of berberine bridge enzyme-like proteins including three other sugar oxidases, a red alga hexose oxidase (CcHEOX), a tobacco nectar protein (nectarin V, NlNEC5), and a sunflower defense protein (HaCHOX) (5-7). Interestingly, although NlNEC5 catalyzes oxidation of glucose, and GOOX, CcHEOX and HaCHOX catalyze the oxidation of glucose, maltose, lactose, and cellobiose, the protein sequences of their substrate-binding domains (S domains) are quite diverse and apparently lack conserved carbohydrateinteracting residues. Moreover, structural and mutational studies demonstrated a consensus histidine for f...

Candida spp. are part of the natural human microbiota, but they also represent important opportunistic human pathogens. Biofilm-associated Candida albicans infections are clinically relevant due to their high levels of resistance to traditional antifungal agents. In this study, we investigated the ability of linalool to inhibit the formation of C. albicans biofilms and reduce existing C. albicans biofilms. Linalool exhibited antifungal activity against C. albicans ATCC 14053, with a minimum inhibitory concentration (MIC) of 8 mM. Sub-MIC concentrations of linalool also inhibited the formation of germ tubes and biofilms in that strain. The defective architecture composition of C. albicans biofilms exposed to linalool was characterized by scanning electron microscopy. The expression levels of the adhesin genes HWP1 and ALS3 were downregulated by linalool, as assessed by real-time RT-PCR. The expression levels of CYR1 and CPH1, which encode components of the cAMP-PKA and MAPK hyphal formation regulatory pathways, respectively, were also suppressed by linalool, as was the gene encoding their upstream regulator, Ras1. The expression levels of long-term hyphae maintenance associated genes, including UME6, HGC1, and EED1, were all suppressed by linalool. These results indicate that linalool may have therapeutic potential in the treatment of candidiasis associated with medical devices because it interferes with the morphological switch and biofilm formation of C. albicans.

Glucooligosaccharide oxidase from Acremonium strictum was screened for potential applications in oligosaccharide acid production and carbohydrate detection. This protein is a unique covalent flavoenzyme which catalyzes the oxidation of a variety of carbohydrates with high selectivity for cello-and maltooligosaccharides. Kinetic measurements suggested that this enzyme possesses an open carbohydrate-binding groove, which is mainly composed of two glucosyl-binding subsites. The encoding gene was subsequently cloned, and one intron was detected in the genomic DNA. Large amounts of active enzymes were expressed in Pichia pastoris, with a yield of 300 mg per liter medium. The protein was predicted to share structural homology with plant cytokinin dehydrogenase and related flavoproteins that share a conserved flavin adenine dinucleotide (FAD)-binding domain. The closest sequence matches are those of plant berberine bridge enzyme-like proteins, particularly the characteristic flavinylation site. Unexpectedly, mutation of the putative FAD-attaching residue, H70, to alanine, serine, cysteine, and tyrosine did not abolish the covalent FAD linkage and had little effect on the K m . Instead, the variants displayed k cat values that were 50-to 600-fold lower, indicating that H70 is crucial for efficient redox catalysis, perhaps through modulation of the oxidative power of the flavin.Sugar oxidases and dehydrogenases that catalyze the oxidation of glucose and other carbohydrates into the corresponding lactones are widely distributed in nature. These enzymes have received great attention as potential diagnostic reagents and industrial biocatalysts. For example, glucose oxidase (GOX) is widely used in analytical biochemistry and in the food industry (21,22). A search of the BRENDA enzyme database (23) revealed that most of these enzymes are specific for a variety of mono-and disaccharides, and only a few enzymes exhibit high specificity with oligosaccharides. The latter enzymes include galactose oxidase, cellobiose dehydrogenase (CDH), and glucooligosaccharide oxidase (GOOX). GOOX from Acremonium strictum was identified from sugar oxidase-producing microorganisms with the aim of identifying enzymes with potential applications in oligosaccharide acid production and alternative carbohydrate assays (16).GOOX is a monomeric glycoprotein with a covalently linked flavin adenine dinucleotide (FAD) (16). It catalyzes the oxidization of a variety of carbohydrates with concomitant reduction of molecular oxygen to hydrogen peroxide. Screening of 30 monosaccharides and derivatives of these compounds showed that D-glucose is the only good substrate. In terms of the disaccharides, maltose, cellobiose, and lactose with reducing-end glucosyl residues linked by an ␣ bond or ␤-1,4 bonds are good substrates, whereas disaccharides containing other linkage types are not. Moreover, GOOX can react with maltooligosaccharides composed of ␣-1,4-linked D-glucopyranosyl residues (up to at least seven units); hence, the name of this novel oxidase. The broad...

Recent studies have demonstrated beneficial effects of specific probiotics on alleviating obesity-related disorders. Here we aimed to identify probiotics with potential antiobesity activity among 88 lactic acid bacterial strains via in vitro screening assays, and a Lactobacillus plantarum strain K21 was found to harbor abilities required for hydrolyzing bile salt, reducing cholesterol, and inhibiting the accumulation of lipid in 3T3-L1 preadipocytes. Furthermore, effects of K21 on diet-induced obese (DIO) mice were examined. Male C57Bl/6J mice received a normal diet, high-fat diet (HFD), or HFD with K21 administration (109 CFU in 0.2 mL PBS/day) for eight weeks. Supplementation of K21, but not placebo, appeared to alleviate body weight gain and epididymal fat mass accumulation, reduce plasma leptin levels, decrease cholesterol and triglyceride levels, and mitigate liver damage in DIO mice. Moreover, the hepatic expression of peroxisome proliferator-activated receptor-γ (PPAR-γ) related to adipogenesis was significantly downregulated in DIO mice by K21 intervention. We also found that K21 supplementation strengthens intestinal permeability and modulates the amount of Lactobacillus spp., Bifidobacterium spp., and Clostridium perfringens in the cecal contents of DIO mice. In conclusion, our results suggest that dietary intake of K21 protects against the onset of HFD-induced obesity through multiple mechanisms of action.