A comparative study of the binding and inhibition of four copper-containing amine oxidases by azide: implications for the role of copper during the oxidative half-reaction

Copper amine oxidases (CuAOs) catalyze the oxidative deamination of primary amines operating through a ping-pong bi-bi mechanism. In this work, azide (an exogenous monodentate ligand) was used to probe the role of copper during the oxidative half-reaction of CuAO catalysis. The effects of azide on both the reductive and oxidative half-reactions of pea seedling amine oxidase (PSAO), the recombinant human kidney diamine oxidase (rhDAO), Arthrobacter globiformis amine oxidase (AGAO), and Pichia pastoris amine oxidase (PPLO) have been examined. For the reductive half-reaction, defined as the oxidation of amine substrate to an aldehyde, azide was discovered to exhibit either noncompetitive or competitive inhibition with respect to the amine, depending on the enzyme source. With regard to the oxidative half-reaction, defined as the reoxidation of the enzyme via reduction of O(2) to H(2)O(2), azide has been determined to exhibit competitive inhibition with respect to O(2) in PSAO with a calculated K(i) value that is in excellent agreement with the experimentally determined K(d) value for the Cu(II)-N(3)(-) complex. Azide was found to exhibit mixed-type/partially competitive inhibition with respect to substrate O(2) in rhDAO, with an apparent K(i) that is similar to the K(d) value for the Cu(II)-N(3)(-) complex. The competitive inhibition for PSAO and the partially competitive inhibition for rhDAO are consistent with O(2) interacting directly with copper during enzymatic reoxidation. For the enzymes AGAO and PPLO, pure noncompetitive and mixed-type/partially competitive inhibition is observed. K(i) values for reductive and oxidative half-reactions are equivalent and are lower than measured K(d) values for the Cu(II)-N(3)(-) complexes in oxidized and substrate-reduced forms of these enzymes. Given these observations, it appears that substantial inhibition of the reductive half-reaction occurs at the concentrations of azide used for the oxidative half-reaction experiments, thereby complicating kinetic interpretation. At this time, the data do not permit us to distinguish between two possibilities: (1) inhibition by azide with respect to O(2) is intrinsically competitive in CuAOs, but this effect cannot always be deconvolved experimentally from the effects of azide on the reductive half-reaction; or (2) CuAOs differ in some steps of their reoxidation mechanisms.     

Cobalt substitution supports an inner-sphere electron transfer mechanism for oxygen reduction in pea seedling amine oxidase

Copper amine oxidases (CAOs) are a large family of proteins that use molecular oxygen to oxidize amines to aldehydes with the concomitant production of hydrogen peroxide and ammonia. CAOs utilize two cofactors for this reaction: topaquinone (TPQ) and a Cu(II) ion. Two mechanisms for oxygen reduction have been proposed for these enzymes. In one mechanism (involving inner-sphere electron transfer to O₂), Cu(II) is reduced by TPQ, forming Cu(I), to which O₂ binds, forming a copper–superoxide complex. In an alternative mechanism (involving outer-sphere electron transfer to O₂), O₂ is directly reduced by TPQ, without reduction of Cu(II). Substitution of Cu(II) with Co(II) has been used to distinguish between the two mechanisms in several CAOs. Because it is unlikely that Co(II) could be reduced to Co(I) in this environment, an inner-sphere mechanism, as described above, is prevented. We adapted metal replacement methods used for other CAOs to the amine oxidase from pea seedlings (PSAO). Cobalt-substituted PSAO (CoPSAO) displayed nominal catalytic activity: k _cat is 4.7% of the native k _cat, and K _M (O₂) for CoPSAO is substantially (22-fold) higher. The greatly reduced turnover number for CoPSAO suggests that PSAO uses the inner-sphere mechanism, as has been predicted from ¹⁸O isotope effect studies (Mukherjee et al. in J Am Chem Soc 130:9459–9473, 2008), and is catalytically compromised when constrained to operate via outer-sphere electron transfer to O₂. This study, together with previous work, provides strong evidence that CAOs use both proposed mechanisms, but each homolog may prefer one mechanism over the other.     

Fission Yeast Sec3 Bridges the Exocyst Complex to the Actin Cytoskeleton

The exocyst complex tethers post‐Golgi secretory vesicles to the plasma membrane prior to docking and fusion. In this study, we identify Sec3, the missing component of the Schizosaccharomyces pombe exocyst complex (SpSec3). SpSec3 shares many properties with its orthologs, and its mutants are rescued by human Sec3/EXOC1. Although involved in exocytosis, SpSec3 does not appear to mark the site of exocyst complex assembly at the plasma membrane. It does, however, mark the sites of actin cytoskeleton recruitment and controls the organization of all three yeast actin structures: the actin cables, endocytic actin patches and actomyosin ring. Specifically, SpSec3 physically interacts with For3 and sec3 mutants have no actin cables as a result of a failure to polarize this nucleating formin. SpSec3 also interacts with actin patch components and sec3 mutants have depolarized actin patches of reduced endocytic capacity. Finally, the constriction and disassembly of the cytokinetic actomyosin ring is compromised in these sec3 mutant cells. We propose that a role of SpSec3 is to spatially couple actin machineries and their independently polarized regulators. As a consequence of its dual role in secretion and actin organization, Sec3 appears as a major co‐ordinator of cell morphology in fission yeast .     

Direct hemin transfer from IsdA to IsdC in the iron-regulated surface determinant (Isd) heme acquisition system of Staphylococcus aureus

The iron-regulated surface determinants (Isd) of Staphylococcus aureus, including surface proteins IsdA, IsdB, IsdC, and IsdH and ATP-binding cassette transporter IsdDEF, constitute the machinery for acquiring heme as a preferred iron source. Here we report hemin transfer from hemin-containing IsdA (holo-IsdA) to hemin-free IsdC (apo-IsdC). The reaction has an equilibrium constant of 10 +/- 5 at 22 degrees C in favor of holo-IsdC formation. During the reaction, holo-IsdA binds to apo-IsdC and then transfers the cofactor to apo-IsdC with a rate constant of 54.3 +/- 1.8 s(-1) at 25 degrees C. The transfer rate is >70,000 times greater than the rate of simple hemin dissociation from holo-IsdA into solvent (k transfer = 54.3 s(-1) versus k -hemin = 0.00076 s(-1)). The standard free energy change, Delta G 0, is -27 kJ/mol for the formation of the holo-IsdA-apo-IsdC complex. IsdC has a higher affinity for hemin than IsdA. These results indicate that the IsdA-to-IsdC hemin transfer is through the activated holo-IsdA-apo-IsdC complex and is driven by the higher affinity of apo-IsdC for the cofactor. These findings demonstrate for the first time in the Isd system that heme transfer is rapid, direct, and affinity-driven from IsdA to IsdC. These results also provide the first example of heme transfer from one surface protein to another surface protein in Gram-positive bacteria and, perhaps most importantly, indicate that the mechanism of activated heme transfer, which we previously demonstrated between the streptococcal proteins Shp and HtsA, may apply in general to all bacterial heme transport systems.     

Mutagenicity of cyanate, a decomposition product of MNU

Knox reported that the short-term effects of the carcinogen methylnitrosourea (MNU) were due to the formation of its decomposition product, the cyanate ion. He showed that cell survival and DNA synthesis decreased as the concentration of MNU and the cyanate ion (NCO-) increased in the medium. Further, the product of MNU decomposition comigrated with NCO- when added to his chromatographic test system. However, Knox did not study the mutagenicity of MNU or its breakdown products. We compared the mutagenicity of MNU and potassium cyanate (KNCO) in mammalian cells. Our results demonstrate that, although it is toxic to cells, KNCO does not induce ouabain-resistant mutants in cultured Chinese hamster cells (V79).     

Chromosome 16 microdeletion in a patient with juvenile neuronal ceroid lipofuscinosis (Batten disease).

The gene that is involved in juvenile neuronal ceroid lipofuscinosis (JNCL), or Batten disease--CLN3--has been localized to 16p12, and the mutation shows a strong association with alleles of microsatellite markers D16S298, D16S299, and D16S288. Recently, haplotype analysis of a Batten patient from a consanguineous relationship indicated homozygosity for a D16S298 null allele. PCR analysis with different primers on DNA from the patient and his family suggests the presence of a cytogenetically undetectable deletion, which was confirmed by Southern blot analysis. The microdeletion is embedded in a region containing chromosome 16-specific repeated sequences. However, putative candidates for CLN3, members of the highly homologous sulfotransferase gene family, which are also present in this region in several copies, were not deleted in the patient. If the microdeletion in this patient is responsible for Batten disease, then we conclude that the sulfotransferase genes are probably not involved in JNCL. By use of markers and probes flanking D16S298, the maximum size of the microdeletion was determined to be approximately 29 kb. The microdeletion may affect the CLN3 gene, which is expected to be in close proximity to D16S298.     

Occurrence and molecular genotyping of Cryptosporidium spp. in surface waters in Northern Ireland

AIMS: To investigate the incidence and genotype of Cryptosporidium parvum oocysts in drinking water sources in Northern Ireland for the period 1996-1999, and to compare conventional and molecular methods of detection. METHODS AND RESULTS: Four hundred and seventy-four waters were investigated by conventional methods, namely immuno-fluorescent antibody detection (IFA; 380) and immuno-magnetic separation-IFA (IMS-IFA; 94), of which 14/474 (3%) were positive. Two hundred and fourteen samples (214/474) were also investigated by PCR techniques, targeting both the 18S rRNA and TRAP-C2 genes, of which 11/214 (5.1%) were positive. These 11 samples were classified as genotype II following sequence analysis of the TRAP-C2 amplicon. CONCLUSIONS: This study demonstrated the low incidence of oocysts of C. parvum in water sources in Northern Ireland. SIGNIFICANCE AND IMPACT OF THE STUDY: Such molecular-based techniques offer a number of advantages over conventional detection methodologies, namely greater sensitivity and specificity as well as the ability to provide accurate genotyping data rapidly, which may be valuable in directing operational management in potential outbreak situations.