RESUMEN
Nox-1 from Streptococcus mutans, the bacteria which cause dental caries, was previously identified as an H2O2-forming reduced nicotinamide adenine dinucleotide (NADH) oxidase. Nox-1 is homologous with the flavoprotein component, AhpF, of Salmonella typhimurium alkyl hydroperoxide reductase. A partial open reading frame upstream of nox1, homologous with the other (peroxidase) component, ahpC, from the S. typhimurium system, was also identified. We report here the complete sequence of S. mutans ahpC. Analyses of purified AhpC together with Nox-1 have verified that these proteins act as a cysteine-based peroxidase system in S. mutans, catalyzing the NADH-dependent reduction of organic hydroperoxides or H2O2 to their respective alcohols and/or H2O. These proteins also catalyze the four-electron reduction of O2 to H2O2, clarifying the role of Nox-1 as a protective protein against oxygen toxicity. Major differences between Nox-1 and AhpF include: (i) the absolute specificity of Nox-1 for NADH; (ii) lower amounts of flavin semiquinone and a more prominent FADH2 to NAD+ charge transfer absorbance band stabilized by Nox-1; and (iii) even higher redox potentials of disulfide centers relative to flavin for Nox-1. Although Nox-1 and AhpC from S. mutans were shown to play a protective role against oxidative stress in vitro and in vivo in Escherichia coli, the lack of a significant effect on deletion of these genes from S. mutans suggests the presence of additional antioxidant proteins in these bacteria.
Asunto(s)
Proteínas Bacterianas/metabolismo , Genes Bacterianos , Peróxido de Hidrógeno/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , Streptococcus mutans/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Secuencia de Bases , Proteínas de Escherichia coli , Bacterias Gramnegativas/enzimología , Bacterias Grampositivas/enzimología , Datos de Secuencia Molecular , NADH NADPH Oxidorreductasas/clasificación , NADH NADPH Oxidorreductasas/genética , NADH NADPH Oxidorreductasas/aislamiento & purificación , NADPH Oxidasa 1 , Peroxidasas/genética , Peroxidasas/metabolismo , Peroxirredoxinas , Proteínas Recombinantes de Fusión/metabolismo , Salmonella typhimurium/enzimología , Salmonella typhimurium/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Streptococcus mutans/genéticaRESUMEN
Aldehyde oxidase was purified to homogeneity from bovine liver and primary structural information obtained by sequencing a series of cleavage peptides permitted the cloning of the corresponding cDNA. The cDNA is 4,630 base pairs long, and it consists of a 102-base pair 5'-untranslated region followed by a 4017-base pair coding region and a 511-base pair 3'-untranslated region. The open reading frame predicts a 1339-amino acid polypeptide with a calculated molecular weight of 147,441, which is consistent with the size of the aldehyde oxidase monomeric subunit. The aldehyde oxidase polypeptide contains consensus sequences for iron-sulfur centers and a molybdopterin binding site. The amino acid sequence deduced from the cDNA shows significant similarity with that of xanthine dehydrogenases from various sources. The primary structure of bovine aldehyde oxidase is remarkably similar (approximately 86%) to that of the translation product of a cDNA recently isolated by Wright et al. (Wright, R. M., Vaitaitis, G. M., Wilson, C. M., Repine, T. B., Terada, L. S., and Repine, J. E. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10690-10694) and reported to represent human xanthine dehydrogenase. With the help of a monospecific antibody raised against the purified protein and the isolated cDNA, the tissue distribution of the bovine aldehyde oxidase protein and corresponding mRNA was determined. Aldehyde oxidase is expressed at high levels in liver, lung, and spleen, and, at a much lower level, in many other organs.