Purification and characterization of an H 2 O-forming NADH oxidase from Clostridium aminovalericum : existence of an oxygen-detoxifying enzyme in an obligate anaerobic bacteria

Abstract

Clostridium aminovalericum, an obligate anaerobe, is unable to form colonies on PYD agar plates in the presence of 1% O(2). When grown anaerobically in PYD liquid medium, the strain can continue normal growth after the shift from anoxic (sparged with O(2)-free N(2) carrier-gas) to microoxic (sparged with 3% O(2)/97% N(2) mixed carrier-gas) growth conditions in the mid exponential phase (OD(660)=1.0). When the strain grew under 3% O(2)/97% N(2), the medium remains anoxic. Thirty minutes after beginning aeration with 3% O(2), the activity of NADH oxidase in cell-free extracts increased more than five-fold from the level before aeration. We purified NADH oxidase to determine the characteristics of this enzyme in an obligate anaerobe. The purified NADH oxidase dominated the NADH oxidase activity detected in cell-free extracts. The enzyme is a homotetramer composed of a subunit with a molecular mass of 45 kDa. The enzyme shows a spectrum typical of a flavoprotein, and flavin adenine dinucleotide (FAD) was identified as a cofactor. The final product of NADH oxidation was H(2)O, and the estimated K(m) for oxygen was 61.9 microM. These data demonstrate that an O(2)-response enzyme that is capable of detoxifying oxygen to water exists in C. aminovalericum.

A synthetic biochemistry molecular purge valve module that maintains redox balance

“…1). To this end, we employ a mixture of both an NAD þ -utilizing wild-type (WT) PDH (PDH NADH ), a mutant PDH that utilizes NADP þ (PDH NADPH ), and a water-generating NADH oxidase (NoxE) that specifically oxidizes NADH, but not NADPH 31,32 . By employing this metabolic node, we generate NADPH needed for PHB production from pyruvate, but also dissipate excess reducing equivalents in an autoregulatory manner.…”

“…We chose NoxE from Lactococcus lactis as it is a water-forming NADH oxidase so it does not generate any toxic products such as hydrogen peroxide 31,32,35 . As shown in Table 1, the K cat of NoxE is 248.8 times greater with NADH than NADPH and k cat /K m is 9,900 times greater.…”

Characterization of an Exceedingly Active NADH Oxidase from the Anaerobic Hyperthermophilic Bacterium Thermotoga maritima

“…There was a fairly constant NADH oxidase activity of 1.0 Ϯ 0.1 U/mg from T. maritima cells grown in the absence of oxygen, even with the addition of cysteine HCl (0.4 g/liter) and sodium thiosulfate (3.2 g/liter) to the growth medium. This activity is still the highest compared to 0.13 U/mg in T. hypogea (51), 0.55 U/mg in Thermotoga neapolitana (our unpublished data), 0.29 U/mg in Thermococcus guaymasensis (our unpublished data), 0.042 U/mg in Clostridium aminovalericum (19), and 0.073 U/mg in Amphibacillus xylanus (31).…”

“…However, the nature of the cellular system facilitating this oxygen tolerance has not been determined. In addition to enzymes such as superoxide dismutase (12), superoxide reductase (16), and peroxidase and catalase (10,12,49), NADH oxidase is considered to be an important enzyme involved in oxygen scavenging systems because of its potential to reduce the oxygen that is transiently encountered by anaerobes (14,19,20).…”

Cofactor Recycling Using a Thermostable NADH Oxidase

“…One catalyzes a two-electron reduction of oxygen to give hydrogen peroxide, and the other catalyzes a four electron reduction of oxygen to give water with NADH oxidation. So far, NOXs have been isolated from anaerobic bacteria such as Streptococcus (Matsumoto et al, 1996;Higuchi et al, 1993Higuchi et al, , 1994Ross and Claiborne, 1992;Schmidt et al, 1986), Thermotoga (Yang & Ma, 2005, Clostridium (Kawasaki et al, 2004;Maeda et al, 1992), Eubacterium (Herles et al, 2002), and Lactobacillus (Hummel et al, 2003b;Riebel et al, 2002Riebel et al, , 2003, and from archaea such as…”