Extracellular Oxidase from the Neonothopanus nambi Fungus as a Promising Enzyme for Analytical Applications.

The extracellular enzyme with oxidase function was extracted from the Neonothopanus nambi luminescent fungus by using mild processing of mycelium with ß-glucosidase and then isolated by gel-filtration chromatography. The extracted enzyme is found to be a FAD-containing protein, catalyzing phenol co-oxidation with 4-aminoantipyrine without addition of H2O2, which distinguishes it from peroxidases. This fact allowed us to assume that this enzyme may be a mixed-function oxidase. According to gel-filtration chromatography and SDS-PAGE, the oxidase has molecular weight of 60 kDa. The enzyme exhibits maximum activity at 55-70 °C and pH 5.0. Kinetic parameters Km and Vmax of the oxidase for phenol were 0.21 mM and 0.40 µM min-1. We suggest that the extracted enzyme can be useful to develop a simplified biosensor for colorimetric detection of phenol in aqueous media, which does not require using hydrogen peroxide.

Purification of the key enzyme complexes of the anammox pathway from DEMON sludge.

Anaerobic Ammonium Oxidation ("anammox") is a bacterial process in which nitrite and ammonium are converted into nitrogen gas and water, yielding energy for the cell. Anammox is an important branch of the global biological nitrogen cycle, being responsible for up to 50% of the yearly nitrogen removal from the oceans. Strikingly, the anammox process uniquely relies on the extremely reactive and toxic compound hydrazine as a free intermediate. Given its global importance and biochemical novelty, there is considerable interest in the enzymes at the heart of the anammox pathway. Unfortunately, obtaining these enzymes in sufficiently large amounts for biochemical and structural studies is problematic, given the slow growth of pure cultures of anammox bacteria when high cell densities are required. However, the anammox process is being applied in wastewater treatment to remove nitrogenous waste in processes like DEamMONification (DEMON). In plants using such processes, which rely on a combination of aerobic ammonia-oxidizers and anammox organisms, kilogram amounts of anammox bacteria-containing sludge are readily available. Here, we report a protein isolation protocol starting from anammox cells present in DEMON sludge from a wastewater treatment plan that readily yields pure preparations of key anammox proteins in the tens of milligrams, including hydrazine synthase HZS and hydrazine dehydrogenase (HDH), as well as hydroxylamine oxidoreductase (HAO). HDH and HAO were active and of sufficient quality for biochemical studies and for HAO, the crystal structure could be determined. The method presented here provides a viable way to obtain materials for the study of proteins not only from the central anammox metabolism but also for the study of other exciting aspects of anammox bacteria, such as for example, their unusual ladderane lipids.

Discovery of fungal oligosaccharide-oxidising flavo-enzymes with previously unknown substrates, redox-activity profiles and interplay with LPMOs.

Oxidative plant cell-wall processing enzymes are of great importance in biology and biotechnology. Yet, our insight into the functional interplay amongst such oxidative enzymes remains limited. Here, a phylogenetic analysis of the auxiliary activity 7 family (AA7), currently harbouring oligosaccharide flavo-oxidases, reveals a striking abundance of AA7-genes in phytopathogenic fungi and Oomycetes. Expression of five fungal enzymes, including three from unexplored clades, expands the AA7-substrate range and unveils a cellooligosaccharide dehydrogenase activity, previously unknown within AA7. Sequence and structural analyses identify unique signatures distinguishing the strict dehydrogenase clade from canonical AA7 oxidases. The discovered dehydrogenase directly is able to transfer electrons to an AA9 lytic polysaccharide monooxygenase (LPMO) and fuel cellulose degradation by LPMOs without exogenous reductants. The expansion of redox-profiles and substrate range highlights the functional diversity within AA7 and sets the stage for harnessing AA7 dehydrogenases to fine-tune LPMO activity in biotechnological conversion of plant feedstocks.

Discovery of an ene-reductase for initiating flavone and flavonol catabolism in gut bacteria.

Gut microbial transformations of flavonoids, an enormous class of polyphenolic compounds abundant in plant-based diets, are closely associated with human health. However, the enzymes that initiate the gut microbial metabolism of flavones and flavonols, the two most abundant groups of flavonoids, as well as their underlying molecular mechanisms of action remain unclear. Here, we discovered a flavone reductase (FLR) from the gut bacterium, Flavonifractor plautii ATCC 49531 (originally assigned as Clostridium orbiscindens DSM 6740), which specifically catalyses the hydrogenation of the C2-C3 double bond of flavones/flavonols and initiates their metabolism as a key step. Crystal structure analysis revealed the molecular basis for the distinct catalytic property of FLR. Notably, FLR and its widespread homologues represent a class of ene-reductases that has not been previously identified. Genetic and biochemical analyses further indicated the importance of FLR in gut microbial consumption of dietary and medicinal flavonoids, providing broader insight into gut microbial xenobiotic transformations and possible guidance for personalized nutrition and medicine.

Cloning and characterization of steroid 5ß-reductase from the venom gland of Bufo bufo gargarizans.

Bufadienolides are the main active ingredients of Venenum Bufonis, which is a widely used traditional Chinese medicine secreted from parotoid gland and skin glands of Bufo bufo gargarizans. According to the transcriptome analysis, "cholesterol-bile acid-bufadienolidies pathway" was proposed as animal-derived bufadienolides biosynthesis pathway by us previously. In this pathway 3ß-hydroxysteroid dehydrogenase (3ßHSD) and steroid 5ß-reductase (SRD5ß) might be the key enzymes to convert the A/B ring to cis-configuration. Therefore, as the second report of our group, here we report the cloning of the full length of SRD5ß cDNA of B. bufo gargarizans (Bbg-SRD5ß) from the parotoid gland of B. bufo gargarizans for the first time, and site-directed mutagenesis was used to explored the character of Bbg-SRD5ß. Bbg-SRD5ß had an open reading frame of 981 bp and encoded 326 amino acids residues. The expression conditions of the recombinant Bbg-SRD5ß in E. coli BL21 (DE3) harbored with pCold-Bbg-SRD5ß was optimized as induction for 10 h at 15 °C with 0.1 mM IPTG. With NADPH as a cofactor, Bbg-SRD5ß can reduce the Δ4,5 double bonds of progesterone to generate dihydroprogesterone õwithout substrate inhibition effect. The catalytic rate of mutant type Bbg-SRD5ß-Y132G was 1.8 times higher than that of wild type Bbg-SRD5ß. Although Bbg-SRD5ß was almost unable to reduce the progesterone to dihydroprogesterone after mutation of V309, the affinity of enzyme with NADPH changed significantly. Bbg-SRD5ß is the key enzymes to convert the A/B ring of steroid to cis-configuration, and V309 is a key site affecting the binding affinity of enzyme with NADPH, and the mutation of Y132 can adjust the catalytic rate of Bbg-SRD5ß.

Co-purification of nitrate reductase 1 with components of the cytochrome bcc-aa3 oxidase supercomplex from spores of Streptomyces coelicolor A3(2).

In order to reduce nitrate in vivo, the spore-specific respiratory nitrate reductase, Nar1, of Streptomyces coelicolor relies on an active cytochrome bcc-aa3 oxidase supercomplex (bcc-aa3 supercomplex). This suggests that membrane-associated Nar1, comprising NarG1, NarH1, and NarI1 subunits, might not act as a classical menaquinol oxidase but could either receive electrons from the bcc-aa3 supercomplex, or require the supercomplex to stabilize the reductase in the membrane to allow it to function. To address the biochemical basis for this dependence on the bcc-aa3 supercomplex, we purified two different Strep-tagged variants of Nar1 and enriched the native enzyme complex from spore extracts using different chromatographic and electrophoretic procedures. Polypeptides associated with the isolated Nar1 complexes were identified using mass spectrometry and included components of the bcc-aa3 supercomplex, along with an alternative, spore-specific cytochrome b component, QcrB3. Surprisingly, we also co-enriched the Nar3 enzyme with Nar1 from the wild-type strain of S. coelicolor. Two differentially migrating active Nar1 complexes could be identified after clear native polyacrylamide gel electrophoresis; these had masses of approximately 450 and 250 kDa. The distribution of active Nar1 in these complexes was influenced by the presence of cytochrome bd oxidase and by QcrB3; the presence of the latter shifted Nar1 into the larger complex. Together, these data suggest that several respiratory complexes can associate in the spore membrane, including Nar1, Nar3, and the bcc-aa3 supercomplex. Moreover, these findings provide initial support for the hypothesis that Nar1 and the bcc-aa3 supercomplex physically associate.

Screening and characterization of a diverse panel of metagenomic imine reductases for biocatalytic reductive amination.

Finding faster and simpler ways to screen protein sequence space to enable the identification of new biocatalysts for asymmetric synthesis remains both a challenge and a rate-limiting step in enzyme discovery. Biocatalytic strategies for the synthesis of chiral amines are increasingly attractive and include enzymatic asymmetric reductive amination, which offers an efficient route to many of these high-value compounds. Here we report the discovery of over 300 new imine reductases and the production of a large (384 enzymes) and sequence-diverse panel of imine reductases available for screening. We also report the development of a facile high-throughput screen to interrogate their activity. Through this approach we identified imine reductase biocatalysts capable of accepting structurally demanding ketones and amines, which include the preparative synthesis of N-substituted ß-amino ester derivatives via a dynamic kinetic resolution process, with excellent yields and stereochemical purities.

Identification and Characterization of an Aspergillus niger Amine Oxidase that Detoxifies Intact Fumonisins.

Fumonisin contamination of maize damaged by Fusarium verticillioides and related species is a major problem when it is grown under warm and dry conditions. Consumption of fumonisin contaminated food and feed is harmful to both humans and livestock. Novel tools for reducing or eliminating fumonisin toxicity may be useful to the agri-feed sector to deal with this worldwide problem. Enzymes capable of catabolizing fumonisins have been identified from microorganisms that utilize fumonisins as an energy source. However, fumonisin detoxifying enzymes produced by the very species that biosynthesize the toxin have yet to be reported. Here we describe the identification and characterization of a novel amine oxidase synthesized by the fumonisin-producing fungus Aspergillus niger. We have recombinantly expressed this A. niger enzyme in E. coli and demonstrated its ability to oxidatively deaminate intact fumonisins without requiring exogenous cofactors. This enzyme, termed AnFAO (A. niger fumonisin amine oxidase), displays robust fumonisin deamination activity across a broad range of conditions, has a high native melting temperature, and can be purified to >95% homogeneity at high yield in a one-step enrichment. AnFAO is a promising tool to remediate fumonisin-contaminated feed including maize destined for ethanol production.

Hierarchical detection of diverse Clade II (atypical) nosZ genes using new primer sets for classical- and multiplex PCR array applications.

The reduction of nitrous oxide (N2O) to N2 represents the key terminal step in canonical denitrification. Nitrous oxide reductase (NosZ), the enzyme associated with this biological step, however, is not always affiliated with denitrifying microorganisms. Such organisms were shown recently to possess a Clade II (atypical) nosZ gene, in contrast to Clade I (typical) nosZ harbored in more commonly studied denitrifiers. Subsequent phylogenetic analyses have shown that Clade II NosZ are affiliated with a much broader diversity of microorganisms than those with Clade I NosZ, the former including both non-denitrifiers and denitrifiers. Most studies attempting to characterize the nosZ gene diversity using DNA-based PCR approaches have only focused on Clade I nosZ, despite recent metagenomic sequencing studies that have demonstrated the dominance of Clade II nosZ genes in many ecosystems, particularly soil. As a result, these studies have greatly underestimated the genetic potential for N2O reduction present in ecosystems. Because the high diversity of Clade II NosZ makes it impossible to design a universal primer set that would effectively amplify all nosZ genes in this clade, we developed a suite of primer sets to specifically target seven of ten designated subclades of Clade II nosZ genes. The new primer sets yield suitable product sizes for paired end amplicon sequencing and qPCR, demonstrated here in their use for both conventional single-reaction and multiplex array platforms. In addition, we show the utility of these primers for detecting nosZ gene transcripts from mRNA extracted from soil.

Multicopper oxidase of Acinetobacter baumannii: Assessing its role in metal homeostasis, stress management and virulence.

A putative multicopper oxidase, encoded as CopA in the proteome of Acinetobacter baumannii 19606, and designated as AbMCO, was expressed heterologously in E. coli (pET-28a) and purified by Ni-NTA affinity chromatography. The purified AbMCO exhibited in vitro oxidase activities upon exogenous addition of ≥1 µM copper ions. Kinetic studies revealed its phenol oxidase activity as it could catalyze the oxidation of substrates viz. 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), guaiacol, pyrogallol and catechol. Additionally, AbMCO displayed siderophore oxidase activity which depicted its role in metal homeostasis and protection from the toxic redox states of copper and iron. Importantly, expression of abMCO increased manifold upon challenge with high concentrations of copper sulphate (CuSO4, 1.5 mM) and sodium chloride (NaCl, 700 mM) which suggested its protective role in stress adaptation and management. Intra-macrophage assay of abMCO-expressing and abMCO-non expressing cells depicted no significant change in the survival rate of A. baumannii inside the macrophages. These findings indicate that A. baumannii encodes a multicopper oxidase, conferring copper tolerance and survival under stress conditions but had no role in virulence of this pathogen.

Human Tyrosinase: Temperature-Dependent Kinetics of Oxidase Activity.

Human tyrosinase (Tyr) is involved in pigment biosynthesis, where mutations in its corresponding gene TYR have been linked to oculocutaneous albinism 1, an autosomal recessive disorder. Although the enzymatic capabilities of Tyr have been well-characterized, the thermodynamic driving forces underlying melanogenesis remain unknown. Here, we analyze protein binding using the diphenol oxidase behavior of Tyr and van 't Hoff temperature-dependent analysis. Recombinant Tyr was expressed and purified using a combination of affinity and size-exclusion chromatography. Michaelis-Menten constants were measured spectrophotometrically from diphenol oxidase reactions of Tyr, using L-3,4-dihydroxyphenylalanine (L-DOPA) as a substrate, at temperatures: 25, 31, 37, and 43 °C. Under the same conditions, the Tyr structure and the L-DOPA binding activity were simulated using 3 ns molecular dynamics and docking. The thermal Michaelis-Menten kinetics data were subjected to the van 't Hoff analysis and fitted with the computational model. The temperature-dependent analysis suggests that the association of L-DOPA with Tyr is a spontaneous enthalpy-driven reaction, which becomes unfavorable at the final step of dopachrome formation.

The carboxy-terminal insert in the Q-loop is needed for functionality of Escherichia coli cytochrome bd-I.

Cytochrome bd, a component of the prokaryotic respiratory chain, is important under physiological stress and during pathogenicity. Electrons from quinol substrates are passed on via heme groups in the CydA subunit and used to reduce molecular oxygen. Close to the quinol binding site, CydA displays a periplasmic hydrophilic loop called Q-loop that is essential for quinol oxidation. In the carboxy-terminal part of this loop, CydA from Escherichia coli and other proteobacteria harbors an insert of ~60 residues with unknown function. In the current work, we demonstrate that growth of the multiple-deletion strain E. coli MB43∆cydA (∆cydA∆cydB∆appB∆cyoB∆nuoB) can be enhanced by transformation with E. coli cytochrome bd-I and we utilize this system for assessment of Q-loop mutants. Deletion of the cytochrome bd-I Q-loop insert abolished MB43∆cydA growth recovery. Swapping the cytochrome bd-I Q-loop for the Q-loop from Geobacillus thermodenitrificans or Mycobacterium tuberculosis CydA, which lack the insert, did not enhance the growth of MB43∆cydA, whereas swapping for the Q-loop from E. coli cytochrome bd-II recovered growth. Alanine scanning experiments identified the cytochrome bd-I Q-loop insert regions Ile318-Met322, Gln338-Asp342, Tyr353-Leu357, and Thr368-Ile372 as important for enzyme functionality. Those mutants that completely failed to recover growth of MB43∆cydA also lacked oxygen consumption activity and heme absorption peaks. Moreover, we were not able to isolate cytochrome bd-I from these inactive mutants. The results indicate that the cytochrome bd Q-loop exhibits low plasticity and that the Q-loop insert in E. coli is needed for complete, stable, assembly of cytochrome bd-I.

A Priming Cassette Generates Hydroxylated Acyl Starter Units in Mupirocin and Thiomarinol Biosynthesis.

Mupirocin, a commercially available antibiotic produced by Pseudomonas fluorescens NCIMB 10586, and thiomarinol, isolated from the marine bacterium Pseudoalteromonas sp. SANK 73390, both consist of a polyketide-derived monic acid homologue esterified with either 9-hydroxynonanoic acid (mupirocin, 9HN) or 8-hydroxyoctanoic acid (thiomarinol, 8HO). The mechanisms of formation of these deceptively simple 9HN and 8HO fatty acid moieties in mup and tml, respectively, remain unresolved. To define starter unit generation, the purified mupirocin proteins MupQ, MupS, and MacpD and their thiomarinol equivalents (TmlQ, TmlS and TacpD) have been expressed and shown to convert malonyl coenzyme A (CoA) and succinyl CoA to 3-hydroxypropionoyl (3-HP) or 4-hydroxybutyryl (4-HB) fatty acid starter units, respectively, via the MupQ/TmlQ catalyzed generation of an unusual bis-CoA/acyl carrier protein (ACP) thioester, followed by MupS/TmlS catalyzed reduction. Mix and match experiments show MupQ/TmlQ to be highly selective for the correct CoA. MacpD/TacpD were interchangeable but alternate trans-acting ACPs from the mupirocin pathway (MacpA/TacpA) or a heterologous ACP (BatA) were nonfunctional. MupS and TmlS selectivity was more varied, and these reductases differed in their substrate and ACP selectivity. The solution structure of MacpD determined by NMR revealed a C-terminal extension with partial helical character that has been shown to be important for maintaining high titers of mupirocin. We generated a truncated MacpD construct, MacpD_T, which lacks this C-terminal extension but retains an ability to generate 3-HP with MupS and MupQ, suggesting further downstream roles in protein-protein interactions for this region of the ACP.

Identification and characterisation of a fluorinase from Actinopolyspora mzabensis.

The incorporation of fluorine has been shown to improve the biophysical and bioactive properties of several organic compounds. However, sustainable strategies of fluorination are needed. Fluorinases have the unique ability to catalyse a C-F bond, hence, have vast potential to be applied as biocatalysts in the preparation of fine chemicals. But fluorinases are extremely rare in nature with only five representatives isolated thus far. Moreover, the heterologous expression of fluorinases is challenged by low yields of soluble protein. This study describes the identification of a fluorinase from Actinopolyspora mzabensis. Overexpression of the Am-fluorinase in E. coli BL21 (DE3) resulted in the formation of inclusion bodies (IBs). The enzyme was recovered from IBs, solubilised in 8 M urea, and successfully refolded into a biologically active form. Following hydrophobic interaction chromatography, >80 mg of the active fluorinase was obtained at a purity suitable for biocatalytic applications. An additional gel filtration step gave ≥95% pure Am-fluorinase. Using LC-MS/MS, the optimal pH for activity was found at 7.2 while the optimal temperature was 65 °C. At these conditions, the enzyme exhibited an activity of 0.44 ±â€¯0.03 µM min-1 mg-1. Furthermore, the Am-fluorinase showed exceptional stability at 25 °C. Preliminary results suggest that the newly identified Am-fluorinase is relatively thermostable.

Single amino acid mutation altered substrate specificity for L-glucose and inositol in scyllo-inositol dehydrogenase isolated from Paracoccus laeviglucosivorans.

scyllo-inositol dehydrogenase, isolated from Paracoccus laeviglucosivorans (Pl-sIDH), exhibits a broad substrate specificity: it oxidizes scyllo- and myo-inositols as well as L-glucose, converting L-glucose to L-glucono-1,5-lactone. Based on the crystal structures previously reported, Arg178 residue, located at the entry port of the catalytic site, seemed to be important for accepting substrates. Here, we report the role of Arg178 by using an alanine-substituted mutant for kinetic analysis as well as to determine the crystal structures. The wild-type Pl-sIDH exhibits the activity for scyllo-inositol most preferably followed by myo-inositol and L-glucose. On the contrary, the R178A mutant abolished the activities for both inositols, but remained active for L-glucose to the same extent as its wild-type. Based on the crystal structures of the mutant, the side chain of Asp191 flipped out of the substrate binding site. Therefore, Arg178 is important in positioning Asp191 correctly to exert its catalytic activities.Abbreviations: IDH: inositol dehydrogenase; LB: Luria-Bertani; kcat: catalyst rate constant; Km: Michaelis constant; NAD: nicotinamide dinucleotide; NADH: nicotinamide dinucleotide reduced form; PDB; Protein Data Bank; PDB entry: 6KTJ, 6KTK, 6KTL.

Oxalate oxidase from Abortiporus biennis - protein localisation and gene sequence analysis.

We have described for the first time the localisation of oxalate oxidase (OXO, EC 1.2.3.4) in Abortiporus biennis cells, using histochemical and immunochemical methods coupled with transmission electron microscopy. Rabbit anti-oxalate oxidase immunoglobulins with anti-rabbit secondary antibody conjugated with 10-nm gold particles were used. Moreover, the formation of electron dense precipitation of reaction of diaminobenzidine (DAB) with horseradish peroxidase (HRP) for histochemical localisation of the enzyme was found. OXO was localised close to the membranous structures of the cell membranes, in membranous vesicles located close to the outer cell membrane, and vacuolar membranes surrounding vacuoles. The positive immunoreaction to OXO was also intense in cell wall areas. Moreover, we proved that gene coding for OXO was expressed in the same cultures. Corresponding mRNA was isolated, full length cDNA was synthesized, cloned and sequenced. Two copies of cupin domains were found in the sequence of amino-acids conserved domain coding for the cupin enzyme. Comparison of the genomic DNA and cDNA sequences has revealed the presence of seventeen introns in the gene. The isoelectric point of the protein was estimated at pH 4.5 and several possible N-glycosylation sites were predicted.

Rapid detection of drug-resistant Mycobacterium tuberculosis directly from clinical specimens using allele-specific polymerase chain reaction assay.

Background & objectives: Rapid detection of drug resistance in Mycobacterium tuberculosis (MTB) is essential for the efficient control of tuberculosis. Hence, in this study a nested-allele-specific (NAS) PCR, nested multiple allele-specific PCR (NMAS-PCR) and multiple allele-specific (MAS) PCR assays were evaluated that enabled detection of the most common mutations responsible for isoniazid (INH) and rifampicin (RIF) resistance in MTB isolates directly from clinical specimens. Methods: Six pairs of primers, mutated and wild type, were used for the six targets such as codon 516, 526 and 531 of rpoB, codon 315 of katG and C15-T substitution in the promoter region of mabA-inhA using allele-specific (AS) PCR assays (NAS-PCR, NMAS-PCR and MAS-PCR). The performance of AS PCR method was compared with phenotypic drug susceptibility testing (DST). Results: The usefulness of AS PCR assays was evaluated with 391 clinical specimens (251 Acid fast bacilli smear positive and MTB culture positive; 93 smear negative and MTB culture positive; 47 smear positive and MTB culture negative) and 344 MTB culture positive isolates. With culture-based phenotypic DST as a reference standard, the sensitivity and specificity of the NAS-PCR, NMAS-PCR and MAS-PCR assay for drug resistance-related genetic mutation detection were 98.6 and 97.8 per cent for INH, 97.5 and 97.9 per cent for RIF and 98.9 and 100 per cent for multidrug resistance (MDR). Interpretation & conclusions: The performance of AS PCR assays showed that those could be less expensive and technically executable methods for rapid detection of MDR-TB directly from clinical specimens.

Soluble expression, purification and biochemical characterization of a C-7 cholesterol dehydrogenase from Drosophila melanogaster.

The C-7 cholesterol dehydrogenase (NVD), which converts cholesterol to 7-dehydrocholesterol (7-DHC) by 7,8-dehydrogenation, plays a pivotal role in the metabolism of cholesterol and steroid intermediates. The NVD protein was successfully expressed in insect Sf9 cells. To reduce the production cost for industrial application, the NVD gene was cloned into E. coli BL21(DE3). However, the His-tagged NVD protein showed poor binding to Ni-NTA resin, mainly due to the formation of inclusion bodies. Consequently, the expression and solubility of NVD were optimized by respectively fusing it with maltose-binding protein (MBP), glutathione S-transferase (GST), and the nonspecific DNA binding protein from Sulfolobus solfataricus (Sso7d) as solubility tags. The NVD fusion with MBP at the N-terminus and His-tag at the C-terminus achieved a high yield of the soluble enzyme. It was further purified by ion-exchange chromatography with 95.4% purity and with a 10.4% yield. The product 7-DHC, which is produced in a reaction catalyzed by NVD and ferredoxin reductase KshB, was initially identified by GC-MS. An analysis of the amino acid sequence alignment revealed a distinct Rieske-type iron-sulfur cluster and non-heme Fe2+-binding domain, which are evolutionarily conserved among NVD family enzymes.

Hyperthermophilic flavin reductase from Sulfolobus solfataricus P2: Production and biochemical characterization.

Nicotinamide adenine dinucleotide phosphate (NAD(P)H)-flavin oxidoreductases (flavin reductases) catalyze the reduction of flavin by NAD(P)H and provide the reduced form of flavin mononucleotide (FMN) to flavin-dependent monooxygenases. Based on bioinformatics analysis, we identified a putative flavin reductase gene, sso2055, in the genome of hyperthermophilic archaeon Sulfolobus solfataricus P2, and further cloned this target sequence into an expression vector. The cloned flavin reductase (EC. 1.5.1.30) was purified to homogeneity and characterized further. The purified enzyme exists as a monomer of 17.8 kDa, free of chromogenic cofactors. Homology modeling revealed this enzyme as a TIM barrel, which is also supported by circular dichroism measurements revealing a beta-sheet rich content. The optimal pH for SSO2055 activity was pH 6.5 in phosphate buffer and the highest activity observed was at 120 °C within the measurable temperature. We showed that this enzyme can use FMN and flavin adenine dinucleotide (FAD) as a substrate to generate their reduced forms. The purified enzyme is predicted to be a potential flavin reductase of flavin-dependent monooxygenases that could be involved in the biodesulfurization process of S. solfataricus P2.

Evidence for mouse sulfhydryl oxidase-assisted cross-linking of major seminal vesicle proteins.

Copulatory plug formation in animals is a general phenomenon by which competition is reduced among rival males. In mouse, the copulatory plug formation results from the coagulation of highly viscous seminal vesicle secretion (SVS) that is rich in proteins, such as dimers of SVS I, SVS I + II + III, and SVS II. These high-molecular-weight complexes (HMWCs) are also reported to be the bulk of proteins in the copulatory plug of the female mouse following copulation. In addition, mouse SVS contributes to the existence of sulfhydryl oxidase (Sox), which mediates the disulfide bond formation between cysteine residues. In this study, flavin adenine dinucleotide (FAD)-dependent Sox was purified from mouse SVS using ion exchange and high-performance liquid chromatography. The purified enzyme was identified to be Sox, based on western blot analysis with Sox antiserum and its capability of oxidizing dithiothreitol as substrate. The pH optima and thermal stability of the enzyme were determined. Among the metal ions tested, zinc showed an inhibitory effect on Sox activity. A prosthetic group of the enzyme was identified as FAD. The Km and Vmax of the enzyme was also determined. In addition to purification and biochemical characterization of seminal vesicle Sox, the major breakthrough of this study was proving its cross-linking activity among SVS I-III monomers to form HMWCs in SVS.