Gene organization and molecular modeling of copper amine oxidase from Aspergillus niger: re-evaluation of the cofactor structure

Amine oxidase AO-I from Aspergillus niger AKU 3302 has been reported to contain topa quinone (TPQ) as a cofactor; however, analysis of the p-nitrophenylhydrazine-derivatized enzyme and purified active site peptides showed the presence of a carboxylate ester linkage of TPQ to a glutamate. The catalytic functionality of such a cross-linked cofactor has recently been shown unlikely by spectroscopic and voltammetric studies on synthesized model compounds. We have obtained resonance Raman spectra of native and substrate-reduced AO-I demonstrating that the catalytically active cofactor is unmodified TPQ. The primary structure of the enzyme (GenBank acc. no. U31869) has been reviewed and updated by repeated isolation and sequencing of AO-I cDNA. This allowed rectification of several errors that account for previously reported low homology to other amine oxidases in the regions around copper binding histididyl residues. The results were confirmed by cloning the ao-1 structural gene (GenBank acc. no. AF362473). Analysis of the gene 5'-upstream region of the gene revealed potential binding sites for an analog of NIT2, the nitrogen metabolism regulatory protein found in Neurospora crassa and other fungi. The molecular structure of AO-I was modeled by a comparative method using published crystal structures of amine oxidases as templates.     

Mapping the primary structure of copper/topaquinone-containing methylamine oxidase fromAspergillus niger

The amino acid sequence of methylamine oxidase (MeAO) from the fungus Aspergillus niger was analyzed using mass spectrometry (MS). First, MeAO was characterized by an accurate molar mass of 72.4 kDa of the monomer measured using MALDI-TOF-MS and by a pI value of 5.8 determined by isoelectric focusing. MALDI-TOF-MS revealed a clear peptide mass fingerprint after tryptic digestion, which did not provide any relevant hit when searched against a nonredundant protein database and was different from that of A. niger amine oxidase AO-I. Tandem mass spectrometry with electrospray ionization coupled to liquid chromatography allowed unambiguous reading of six peptide sequences (11-19 amino acids) and seven sequence tags (4-15 amino acids), which were used for MS BLAST homology searching. MeAO was found to be largely homologous to a hypothetical protein AN7641.2 (EMBL/GenBank protein-accession code EAA61827) from Aspergillus nidulans FGSC A4 with a theoretical molar mass of 76.46 kDa and pI 6.14, which belongs to the superfamily of copper amine oxidases. The protein AN7641.2 is only little homologous to the amine oxidase AO-I (32% identity, 49 % similarity).     

Functional expression of amine oxidase from <Emphasis Type="Italic">Aspergillus niger</Emphasis> (AO-I) in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The aim of this work was to prepare recombinant amine oxidase from Aspergillus niger after overexpressing in yeast. The yeast expression vector pDR197 that includes a constitutive PMA1 promoter was used for the expression in Saccharomyces cerevisiae. Recombinant amine oxidase was extracted from the growth medium of the yeast, purified to homogeneity and identified by activity assay and MALDI-TOF peptide mass fingerprinting. Similarity search in the newly published A. niger genome identified six genes coding for copper amine oxidase, two of them corresponding to the previously described enzymes AO-I a methylamine oxidase and three other genes coding for FAD amine oxidases. Thus, A. niger possesses an enormous metabolic gear to grow on amine compounds and thus support its saprophytic lifestyle.     

Purification and characterization of methylamine oxidase induced in Aspergillus niger AKU 3302

Crude extract of Aspergillus niger AKU 3302 mycelia incubated with methylamine showed a single amine oxidase activity band in a developed polyacrylamide gel that weakly cross-reacted with the antibody against a copper/topa quinone-containing amine oxidase (AO-II) from the same strain induced by n-butylamine. Since the organism cannot grow on methylamine and the already known quinoprotein amine oxidases of the organism cannot catalyze oxidation of methylamine, the organism was forced to produce another enzyme that could oxidize methylamine when the mycelia were incubated with methylamine. The enzyme was separated and purified from the already known two quinoprotein amine oxidases formed in the same mycelia. The purified enzyme showed a sharp symmetric sedimentation peak in analytical ultracentrifugation showing S20,w0 of 6.5s. The molecular mass of 133 kDa estimated by gel chromatography and 66.6 kDa found by SDS-PAGE confirmed the dimeric structure of the enzyme. The purified enzyme was pink in color with an absorption maximum at 494 nm. The enzyme readily oxidized methylamine, n-hexylamine, and n-butylamine, but not benzylamine, histamine, or tyramine, favorite substrates for the already known two quinoprotein amine oxidases. Inactivation by carbonyl reagents and copper chelators suggested the presence of a copper/topa quinone cofactor. Spectrophotometric titration by p-nitrophenylhydrazine showed one reactive carbonyl group per subunit and redox-cyclic quinone staining confirmed the presence of a quinone cofactor. pH-dependent shift of the absorption spectrum of the enzyme-p-nitrophenylhydrazone (469 nm at neutral to 577 nm at alkaline pH) supported the identity of the cofactor with topaquinone. Nothern blot analysis indicated that the methylamine oxidase encoding gene is largely different from the already known amine oxidase in the organism.     

Reductive trapping of substrate to methylamine oxidase from Arthrobacter P1

Methylamine oxidase (EC 1.4.3.6) from Arthrobacter P1 was inactivated by NaCNBH3 in the presence of [14C]benzylamine, leading to the incorporation of 1 mol of radiolabeled substrate/mol of enzyme subunit at complete inactivation. By contrast, no labeling of enzyme was observed using [3H]NaCNBH3 as reductant. These results are analogous to those previously reported for the eukaryotic enzyme, bovine serum plasma amine oxidase [(1987) J. Biol. Chem. 262, 962-965]. The observed pattern of labeling is consistent with the presence of dicarbonyl cofactor at the active site of methylamine oxidase. Further, these studies suggest that our reductive trapping technique, in which the pattern of radiolabeling of an enzyme is compared using C-14 substrate vs tritiated reductant, may serve as a general assay for covalently bound dicarbonyl structures.     

Increase in putrescine,amine oxidase,and acrolein in plasma of renal failure patients

Since polyamines have been suggested to be one of the uremic "toxins," the levels of each polyamine, its oxidized product, acrolein, and amine oxidase in plasma of patients with renal failure were investigated. The level of putrescine was increased, whereas the level of spermine was decreased in the plasma of patients with renal failure. The patients also had increased serum amine oxidase activity leading to increased degradation of spermine. Both levels of free and protein-conjugated acrolein were also increased in plasma of patients with renal failure. The accumulated acrolein found as protein conjugates was equivalent to 180 microM, which was 6-fold higher than in plasma of normal subjects. It was found that acrolein is mainly produced by polyamine oxidase in plasma. A cell lysate containing polyamine oxidase was cytotoxic in the presence of spermine. Our results indicate that the level of acrolein is well correlated with the degree of seriousness of chronic renal failure.     

Reassessment of the active site quino-cofactor proposed to occur in the Aspergillus niger amine oxidase AO-I from the properties of model compounds

Quino-cofactors have been found in a wide variety of prokaryotic and eukaryotic organisms. Two variants have, thus far, been demonstrated to derive from tyrosine precursors: these are the 2,4, 5-trihydroxyphenylalanine quinone (topa quinone or TPQ) [Janes, S. M. , et al. (1990) Science 248, 98] and an o-quinone analogue containing the side chain of a lysine residue (lysyltyrosine quinone or LTQ) [Wang, S. Z., et al. (1996) Science 273, 1078]. Additionally, a third variant of the family of tyrosine-derived cofactors has been reported to exist in an Aspergillus niger amine oxidase AO-I. This was described as an o-quinone cross-linked to the side chain of a glutamate residue [Frebort, I. (1996) Biochim. Biophys. Acta 1295, 59]. We have synthesized model compounds related to the proposed structure. Characterization of the redox properties for the model compound and spectral properties of its 4-nitrophenylhydrazine derivative lead us to conclude that the cofactor in A. niger amine oxidase AO-I has been misidentified. A TPQ carboxylate ester is considered an unlikely candidate for a biologically functional quino-cofactor.     

Lipopolysaccharide (LPS) labeled with Alexa 488 hydrazide as a novel probe for LPS binding studies

BACKGROUND: Lipopolysaccharide (LPS) comprises the outer cell wall of all gram-negative bacteria. It consists of an oligosaccharide core and lipid A. All LPS-induced biological responses are lipid A-dependent. Once released, LPS triggers a host systemic inflammatory response that leads to septic shock. Binding studies have helped to reveal some of the molecular interactions behind septic shock. Such studies have employed methods of labeling bacterial LPS with either radiochemicals or fluorescent dyes. Poor labeling of the LPS has resulted in the use of high concentrations of LPS in order to detect its binding. METHODS: In this study, we have devised a new methodology for labeling LPS, using hydrazide and galactose oxidase in order to oxidize galactose residues to aldehyde groups in the oligosaccharide core of the LPS. RESULTS: We have managed to generate a conjugate that is highly fluorescent (LPS-to-Alexa 488 labeling ratio of 1:5) and biologically active. CONCLUSIONS: For the first time, this probe has enabled us to detect LPS binding even at pg/ml concentrations. Using this methodology, any Alexa-hydrazide dye can be conjugated to LPS, providing us with novel probes for imaging studies.     

Localization of NADH oxidase on the surface of human polymorphonuclear leukocytes by a new cytochemical method

The ultrastructural localization of NADH oxidase, a possible enzyme in the increased oxidative activity of polymorphonuclear leukocytes (PMN) during phagocytosis, was studied. A new cytochemical technique for the localization of H2O2, a product of NADH oxidase activity, was developed. Cerous ions, in the presence of peroxide, form an electron-dense precipitate. Resting and phagocytically stimulated PMN were exposed to cerous ions at pH 7.5 to demonstrate sites of NADH-dependent, cyanide-insensitive H2O2 production. Resting PMN exhibites slight activity on the plasma membrane; phagocytizing PMN had extensive deposits of reaction product localized within the phagosome and on the plasma membrane. Peroxide involvement was demonstrated by the inhibitory effect of catalase on cerium precipitation; the surface localization of the enzyme responsible was confirmed by using nonpenetrating inhibitors of enzymatic activity. A correlative study was performed with an NADH-dependent, tetrazolium-reduction system. As with cerium, formazan deposition on the surface of the cell was NADH dependent, cyanide insensitive, and stimulated by phagocytosis. Superoxide dismutase did not inhibit tetrazolium reduction, as observed cytochemically, indicating direct enzymatic dye reduction without superoxide interposition. These findings, combined with oxygen consumption studies on resting and stimulated PMN in the presence or absence of NADH, indicate that NADH oxidase is a surface enzyme in human PMN. It is internalized during phagocytosis and retains its peroxide-generating capacity within the phagocytic vacuole.     

Simulation of motor-driven cochlear outer hair cell electromotility.

We propose a three-dimensional (3D) model to simulate outer hair cell electromotility. In our model, the major components of the composite cell wall are explicitly represented. We simulate the activity of the particles/motor complexes in the plasma membrane by generating active strains inside them and compute the overall response of the cell. We also consider the constrained wall and compute the generated active force. We estimate the parameters of our model by matching the predicted longitudinal and circumferential electromotile strains with those observed in the microchamber experiment. In addition, we match the earlier estimated values of the active force and cell wall stiffness. The computed electromotile strains in the plasma membrane and other components of the wall are in agreement with experimental observations in trypsinized cells and in nonmotile cells transfected with Prestin. We discover several features of the 3D mechanism of outer hair cell electromotilty. Because of the constraints under which the motors operate, the motor-related strains have to be 2-3 times larger than the observable strains. The motor density has a strong effect on the electromotile strain. Such effect on the active force is significantly lower because of the interplay between the active and passive properties of the cell wall.     

Expression and purification of enzymatically active forms of the human lysyl oxidase-like protein 4

The lysyl oxidase-like protein 4 (LOXL4) is the latest member of the emerging family of lysyl oxidases, several of which were shown to function as copper-dependent amine oxidases catalyzing lysine-derived cross-links in extracellular matrix proteins. LOXL4 contains four scavenger receptor cysteine-rich domains in addition to the characteristic domains of the LOX family, including the copper-binding domain, the cytokine receptor-like domain, and the residues of the lysyl-tyrosyl quinone cofactor. In an effort to assess its amine oxidase activity, we expressed LOXL4 as recombinant forms attached with hexa-histidine residues at the carboxyl terminus by using an Escherichia coli expression system. The recombinant proteins were purified with nickel-chelating affinity chromatography and converted into enzymatically active forms by stepwise dialysis. The purified LOXL4 proteins showed beta-aminopropionitrile-inhibitable activity of 0.022-0.032 units/mg toward a nonpeptidyl substrate, benzylamine. These results indicate that LOXL4, with the four scavenger receptor cysteine rich domains, may also function as an active amine oxidase. Availability of the pure and active forms of LOXL4 will be significantly helpful in functional studies related to substrate specificity and crystal structure of this amine oxidase, which should provide significant insights into functional differences within the LOX family members.     

Autolytic formation of protoplasts (autoplasts) of Streptococcus faecalis; location of active and latent autolysin.

Over 80% of the active and porteinase-activatable, latent forms of the autolytic N-acetylmuramide glycanhydrolase of Streptococcus faecalis ATCC 9790 were released to the supernatant buffer during the autolytic formation of protoplasts (autoplasts) in the presence of absence of trypsin. Autolysin activity was not found in association with released mesosomal vesicles and had little affinity for binding to membranes or to the outer surface of the wall. Isolated walls were able to bind over four times as much autolysin activity as that present on wall exponential-phase cells. Using a rapid technique for wall isolation, evidence was obtained that the latent form (as well as the active form) was wall bound in intact cells. In addition, isotope labeling and ultrastructural studies were able to show that latent autolysin was concentrated in the newer, septally associated portion of the wall.     

Effect of the active immunization to plasma amine oxidase on the passive avoidance conditioned reflex

The influence of immune suppression of blood serum amine oxidase on mnemonic processes was studied during passive avoidance conditioning in white rats. The monoaminergic system is known to participate in conditioning. Our previous studies showed that the active immunization against serum amine oxidase results in suppression of endogenous serum amine oxidase and brain mitochondrial monoamine oxidase activities. We also revealed the specific changes in catecholamine concentrations. In this study, we observed a positive effect of immune suppression of serum amine oxidase on the passive avoidance conditioning during its reproduction on the next day and 45 days later. Thus, the obtained results suggest the possibility of using the active immunization against endogenous amine oxidase for regulation of mnestic processes.     

The history of renalase from amine oxidase to α-NAD(P)H-oxidase/anomerase

Renalase is a recently discovered secretory protein, which plays a certain (still poorly understood) role in regulation of blood pressure. The review summarizes own and literature data on structure and catalytic properties of renalase accumulated since the first publication on this protein (2005). Initial reports on FADdependent amine oxidase activity were not confirmed in independent experiments performed in different laboratories. In addition, proposed amine oxidase activity of circulating extracellular renalase requires the presence of FAD, which has not been detected either in blood or urinary renalase. Moreover, renalase excreted into urine lacks its N-terminal peptide, which is ultimately needed for accommodation of the FAD cofactor. Results of the Aliverti’s group on NAD(P)H binding by renalase and weak diaphorase activity of this enzyme stimulated further studies of renalase as NAD(P)H oxidase catalyzing reaction of catecholamine co-oxidation. However, physiological importance of such extracellular catecholamine-metabolizing activity remains unclear due to existence of much more active enzymatic systems (e.g., neutrophil NAD(P)H oxidase, xanthine oxidase/xanthine) in circulation, which can perform such co-oxidation reactions. Recently α-NAD(P)H oxidase/anomerase activity of renalase, which also promotes oxidative conversion of β-NADH isomers inhibiting activity of NAD-dependent dehydrogenases, has been described. However, its possible contribution to the antihypertensive effect of renalase remains unclear. Thus, the antihypertensive effect of renalase still remains a phenomenon with unclear biochemical mechanim(s) and functions of intracellular and extracellular (circulating) renalases obviously differ.     

Origin of the 2-amino-2-deoxy-gluconate unit in Rhizobium leguminosarum lipid A. Expression cloning of the outer membrane oxidase LpxQ

An unusual feature of the lipid A from the plant endosymbionts Rhizobium etli and Rhizobium leguminosarum is the presence of a proximal sugar unit consisting of a 2-amino-2-deoxy-gluconate moiety in place of glucosamine. An outer membrane oxidase that generates the 2-amino-2-deoxy-gluconate unit from a glucosamine-containing precursor is present in membranes of R. leguminosarum and R. etli but not in S. meliloti or Escherichia coli. We now report the identification of a hybrid cosmid that directs the overexpression of this activity by screening 1800 lysates of individual colonies of a R. leguminosarum 3841 genomic DNA library in the host strain R. etli CE3. Two cosmids (p1S11D and p1U12G) were identified in this manner and transferred into S. meliloti, in which they also directed the expression of oxidase activity in the absence of any chromosomal background. Subcloning and sequencing of the oxidase gene on a 6.5-kb fragment derived from the approximately 20-kb insert in p1S11D revealed that the enzyme is encoded by a gene (lpxQ) that specifies a protein of 224 amino acid residues with a putative signal sequence cleavage site at position 28. Heterologous expression of lpxQ using the T7lac promoter system in E. coli resulted in the production of catalytically active oxidase that was localized in the outer membrane. A new outer membrane protein of the size expected for LpxQ was present in this construct and was subjected to microsequencing to confirm its identity and the site of signal peptide cleavage. LpxQ expressed in E. coli generates the same products as seen in R. leguminosarum membranes. LpxQ is dependent on O(2) for activity, as demonstrated by inhibition of the reaction under strictly anaerobic conditions. An ortholog of LpxQ is present in the genome of Agrobacterium tumefaciens, as shown by heterologous expression of oxidase activity in E. coli.     

DISSECTION OF TWO DISTINCT DEFENSE‐RELATED RESPONSES TO AGAR OLIGOSACCHARIDES IN GRACILARIA CHILENSIS (RHODOPHYTA) AND GRACILARIA CONFERTA (RHODOPHYTA)1

The two agar‐producing red algae, Gracilaria chilensis C. J. Bird, McLachlan & E. C. Oliveira and Gracilaria conferta (Schousboe ex Montagne) Montagne, responded with hydrogen peroxide (H₂O₂) release when agar oligosaccharides were added to the medium. In G. conferta, a transient release was observed, followed by a refractory state of 6 h. This response was sensitive to chemical inhibitors of NADPH oxidase, protein kinases, protein phosphatases, and calcium translocation in the cell, whereas it was insensitive to inhibitors of metalloenzymes. Transmission electron microscopic observations of the H₂O₂‐dependent formation of cerium peroxide from cerium chloride indicated oxygen activation at the plasma membrane of G. conferta. A putative system, consisting of a receptor specific to agar oligosaccharides and a plasma membrane‐located NADPH oxidase, appears to be responsible for the release of H₂O₂ in G. conferta. Subcellular examination of G. chilensis showed that the H₂O₂ release was located in the cell wall. It was sensitive to inhibitors of metalloenzymes and flavoenzymes, and no refractory state was observed. The release was correlated with accumulation of an aldehyde in the algal medium, suggesting that an agar oligosaccharide oxidase is present in the apoplast of G. chilensis. The presence of this enzyme could also be demonstrated by polyacrylamide electrophoresis under nondenaturating conditions and proven to be variable. Cultivation of G. chilensis at 16 to 17°C resulted in significantly stronger expression of agar oligosaccharide oxidase than cultivation at 12°C, which indicates that the enzyme is used under conditions that generally favor microbial agar macerating activity.     

Molecular characteristics of tissue-bound semicarbazide-sensitive amine oxidase (SSAO) in guinea pig tissues

Various mammalian tissues contain a tissue-bound amine oxidizing enzyme distinct from mitochondrial outer membrane enzyme, monoamine oxidase (MAO, EC 1.4.3.4), termed semicarbazide-sensitive amine oxidase (SSAO, EC 1.4.3.6). An increase in SSAO activity was found in patients suffering from vascular disorders such as diabetes and diabetic complications. It has previously been shown that 2-bromoethylamine (2-BEA) is a potent, and selective suicidal inhibitor of tissue-bound SSAO. The aim of this study was to investigate the interaction of this suicidal SSAO inhibitor with the tissue-bound enzyme in guinea pig lung, kidney, stomach, and heart homogenates. The conditions necessary for this inhibitor to titrate the concentrations of this enzyme were also determined. 2-BEA appears to interact with SSAO, as reported previously for this enzyme from different sources, in a manner consistent with an irreversible, "suicide" reaction. Because of this property, 2-BEA could be used to titrate the concentrations of SSAO active centers in these tissues under the appropriate conditions employed. Although some possible non-specific binding of the inhibitor to sites other than the active center of the enzyme, metabolism of this inhibitor and/or presence of enzyme subtypes was hypothesized, the molecular characteristics of SSAO in these tissues (Km, Vmax values, enzyme efficiencies, approximate enzyme concentrations, and molecular turnover numbers) towards the substrate kynuramine (0.1 mM) at pH 7.4 and 37 degrees C have been estimated.     

Inhibition of six copper-containing amine oxidases by the antidepressant drug tranylcypromine

Potential inhibitory effects of the clinically utilized monoamine oxidase inhibitor tranylcypromine (TCP) on mammalian, plant, bacterial, and fungal copper-containing amine oxidases have been examined. The following enzymes have been investigated: human kidney diamine oxidase (HKAO), bovine plasma amine oxidase (BPAO), equine plasma amine oxidase (EPAO), pea seedling amine oxidase (PSAO), Arthrobacter globiformis amine oxidase (AGAO), and Pichia pastoris lysyl oxidase (PPLO). Only BPAO, EPAO, and AGAO were found to lose significant levels of activity when incubated with varying amounts of TCP. Inhibition of BPAO was completely reversible, with dialysis restoring full activity. TCP inhibition of AGAO was also found to be ultimately reversible; however, dialysis did not remove all bound compounds. Chemical displacement with either substrate or a substrate analogue successfully removed all bound TCP, indicating that this compound has a high affinity for the active site of AGAO. The notable lack of TCP inhibition on HKAO argues against the inhibition of diamine oxidase as a potential source for some of the deleterious side effects occurring in patients treated with this antidepressant. The marked differences observed in behavior among these enzymes speaks to the importance of intrinsic structural differences between the active sites of copper amine oxidases (CAO) which affect reactivity with a given inhibitor.     

The human lysyl oxidase-like 2 protein functions as an amine oxidase toward collagen and elastin

The lysyl oxidase-like 2 (LOXL2) protein is a human paralogue of lysyl oxidase (LOX) that functions as an amine oxidase for formation of lysine-derived cross-links found in collagen and elastin. In addition to the C-terminal domains characteristic to the LOX family members, LOXL2 contains four scavenger receptor cysteine-rich (SRCR) domains in the N-terminus. In order to assess the amine oxidase activity of LOXL2, we expressed a series of recombinant LOXL2 proteins with deletions in the SRCR domains, using an Escherichia coli expression system. All of the purified recombinant LOXL2 proteins, with or without the SRCR domains in the N-terminus, showed significant amine oxidase activity toward several different types of collagen and elastin in in vitro amine oxidase assays, indicating deletion of the SRCR domains does not interfere with amine oxidase activity of LOXL2. Further, amine oxidase activity of LOXL2 was not susceptible to inhibition by β-aminopropionitrile, an irreversible inhibitor of LOX, suggesting a different enzymatic mechanism between these two paralogues.