Amphibacillus xylanus NADH Oxidase and Salmonella typhimurium Alkyl-hydroperoxide Reductase Flavoprotein Components Show Extremely High Scavenging Activity for Both Alkyl Hydroperoxide and Hydrogen Peroxide in the Presence of S. typhimurium Alkyl-hydroperoxide Reductase 22-kDa Protein Component

Abstract

The flavoprotein NADH oxidase from Amphibacillus xylanus consumes oxygen to produce hydrogen peroxide. The amino acid sequence of this flavoprotein shows 51.2% identity to the F-52a component, denoted AhpF, of the alkyl-hydroperoxide reductase from Salmonella typhimurium. AhpF also catalyzes NADH-dependent hydrogen peroxide formation under aerobic conditions, albeit at a somewhat slower rate than the Amphibacillus protein. In the presence of the 22-kDa colorless component (AhpC) of the Salmonella alkyl-hydroperoxide reductase, both proteins catalyze the 4-electron reduction of oxygen to water. Both flavoproteins are active as AhpC reductases and mediate electron transfer, resulting in the NADH-dependent reduction of hydrogen peroxide and cumene hydroperoxide. Both enzymes' K m values for hydrogen peroxide, cumene hydroperoxide, and NADH are so low that they could not be determined accurately. V max values for hydrogen peroxide or cumene hydroperoxide reduction are >10,000 min ؊1 at 25°C. These values are almost the same as the reduction rate of the flavoprotein component by NADH. The involvement in catalysis of a redox-active disulfide of the A. xylanus flavoprotein was shown by construction of three mutant enzymes, C337S, C340S, and C337S/C340S. Very little activity for hydrogen peroxide or cumene hydroperoxide was found with the single mutants (C337S and C340S), and none with the double mutant (C337S/C340S).Analysis of the DNA sequence upstream of the Amphibacillus flavoprotein structural gene indicated the presence of a partial open reading frame homologous to the Salmonella ahpC structural gene (64.3% identical at the amino acid sequence level), suggesting that the NADH oxidase protein of A. xylanus is also part of a functional alkyl-hydroperoxide reductase system within these catalase-lacking bacteria.We recently have isolated a new group of facultatively anaerobic bacteria from alkaline compost (1). The bacteria have unique phenotypic and chemotaxonomic characteristics (2) as well as bioenergetic properties (3) and were named Amphibacillus xylanus (2). A. xylanus, lacking a respiratory system and hemeproteins, catalase, and peroxidase, grows well and has the same growth rate and cell yield under strictly anaerobic and aerobic conditions (2). This growth characteristic of A. xylanus is due to the presence of anaerobic and aerobic pathways producing similar amounts of ATP (4). Under aerobic conditions, NADH is thought to be responsible for maintenance of the intracellular redox balance (4).A flavoprotein functional as NADH oxidase was purified from aerobically grown A. xylanus (5). The flavoprotein is a homotetramer composed of subunits (M r ϭ 56,000) containing 1 mol of FAD and also catalyzes a thiol-disulfide interchange reaction, NADH:DTNB 1 oxidoreductase (6). The complete reduction of enzyme by dithionite requires 6 electrons/subunit (6). Such behavior indicates the presence of redox centers in addition to the FAD, and these were postulated to be disulfides (6). To assess the catalytic role of disulfide...

The Peroxiredoxin Repair Proteins

“…Substrate inactivation was also shown to occur in the peroxiredoxins (Prxs) from Kinetoplastida, including Leishmania major, L. donovani and Trypanosoma brucei, but not in Crithidia fasciculate (Nogoceke et al, 1997;Castro et al, 2002;Flohe et al, 2002;Budde et al, 2003). In contrast, the homologous bacterial peroxiredoxin AhpC efficiently detoxifies mM levels of hydrogen peroxide and cumene peroxide without any indication of peroxide-dependent inactivation (Niimura et al, 1995;Poole, 1996). Furthermore, in vivo peroxide stress studies in Salmonella typhimurium showed no indication of post-translational modification in AhpC (Christman et al, 1985;Morgan et al, 1986).…”

Adaptive response of Amphibacillus xylanus to normal aerobic and forced oxidative stress conditions

“…Interestingly, the two enzymes have different reactivities to oxygen (Niimura et al, 1995). Nox from A. xylanus is more reactive to oxygen than AhpF from S. typhimurium is (Niimura et al, 1995). Therefore, it is thought that the Nox-Prx system is involved not only in the removal of peroxides but also in oxygen metabolism in the aerobic metabolic pathway of A. xylanus (Niimura et al, 2000).…”

“…Although the physiological function of the NADH oxidase was first thought to be the in-vivo regeneration of NAD in the aerobic metabolism of the bacterium, the enzyme was shown to catalyse the reduction of oxygen by NADH to form hydrogen peroxide (Niimura et al, 1993). Also, the NADH oxidase that acts together with an alkyl hydroperoxide reductase C (AhpC) was found to catalyse the NADH-dependent reduction of both hydrogen peroxide and alkyl hydroperoxides (Niimura et al, 1995). A. xylanus NADH oxidase has 51.2 % amino acid sequence identity with alkyl hydroperoxide reductase F (AhpF) obtained from Salmonella typhimurium (Jacobson et al, 1989;Niimura et al, 1993).…”

“…AhpC is now commonly referred to as peroxiredoxin (Prx), and the NADH oxidases (Nox) and AhpF thus belong to a growing new family of peroxiredoxin oxidoreductases (PrxR) (Poole et al, 2000b). Interestingly, the two enzymes have different reactivities to oxygen (Niimura et al, 1995). Nox from A. xylanus is more reactive to oxygen than AhpF from S. typhimurium is (Niimura et al, 1995).…”

Differential Expression of Mycothiol Pathway Genes: Are they Affected by Antituberculosis Drugs?

“…Further evidence for this hypothesis is the genome sequence conservation between M. tuberculosis and M. bovis. In addition, a recent study suggested that molecules such as mycothiol may serve as partner molecules in the enzymatic cycle of the mycobacterial alkyl hydroperoxide reductase (AhpC) (27), an enzyme that has been shown to detoxify organic peroxides (24) and possibly H 2 O 2 (28). This enzyme has been implicated in the survival of INH resistant M. tuberculosis, as point mutations in the AhpC gene was associated with drug resistance (29) and its expression was shown to be up-regulated in M. tuberculosis drug resistant phenotypes (30).…”