Formation and Conversion of Oxygen Metabolites by Lactococcus lactis subsp. lactis ATCC 19435 under Different Growth Conditions

A semidefined medium based on Casamino Acids allowed Lactococcus lactis ATCC 19435 to grow in the presence of oxygen at a slow rate (0.015 h⁻¹). Accumulation of H₂O₂ in the culture prevented a higher growth rate. Addition of asparagine to the medium increased the growth rate, whereby H₂O₂ accumulated only temporarily during the lag phase. H₂O₂ is an inhibitor for several glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase being the most sensitive. Strain ATCC 19435 contained NADH oxidase (maximum specific rate under aerobic conditions, 426 nmol of NADH min⁻¹ mg of protein⁻¹), which reduced oxygen to water, whereby superoxide was formed as a by-product. H₂O₂ originated from the dismutation of superoxide by superoxide dismutase. Although H₂O₂ was rapidly destroyed under high metabolic fluxes, neither NADH peroxidase nor any other enzymatic H₂O₂-reducing activity was detected. However, pyruvate, the end product of glycolysis, reacted nonenzymatically and rapidly with H₂O₂ and hence was a potential alternative for scavenging of this oxygen metabolite intracellularly. Indeed, intracellular concentrations of up to 93 mM pyruvate were detected in aerobic cultures growing at high rates. It is hypothesized that self-generated pyruvate may serve to protect L. lactis strain ATCC 19435 from H₂O₂.     

Fine tuning of the lactate and diacetyl production through promoter engineering in Lactococcus lactis

Lactococcus lactis is a well-studied bacterium widely used in dairy fermentation and capable of producing metabolites with organoleptic and nutritional characteristics. For fine tuning of the distribution of glycolytic flux at the pyruvate branch from lactate to diacetyl and balancing the production of the two metabolites under aerobic conditions, a constitutive promoter library was constructed by randomizing the promoter sequence of the H(2)O-forming NADH oxidase gene in L. lactis. The library consisted of 30 promoters covering a wide range of activities from 7,000 to 380,000 relative fluorescence units using a green fluorescent protein as reporter. Eleven typical promoters of the library were selected for the constitutive expression of the H(2)O-forming NADH oxidase gene in L. lactis, and the NADH oxidase activity increased from 9.43 to 58.17-fold of the wild-type strain in small steps of activity change under aerobic conditions. Meanwhile, the lactate yield decreased from 21.15 ± 0.08 mM to 9.94 ± 0.07 mM, and the corresponding diacetyl production increased from 1.07 ± 0.03 mM to 4.16 ± 0.06 mM with the intracellular NADH/NAD(+) ratios varying from 0.711 ± 0.005 to 0.383 ± 0.003. The results indicated that the reduced pyruvate to lactate flux was rerouted to the diacetyl with an almost linear flux variation via altered NADH/NAD(+) ratios. Therefore, we provided a novel strategy to precisely control the pyruvate distribution for fine tuning of the lactate and diacetyl production through promoter engineering in L. lactis. Interestingly, the increased H(2)O-forming NADH oxidase activity led to 76.95% lower H(2)O(2) concentration in the recombinant strain than that of the wild-type strain after 24 h of aerated cultivation. The viable cells were significantly elevated by four orders of magnitude within 28 days of storage at 4°C, suggesting that the increased enzyme activity could eliminate H(2)O(2) accumulation and prolong cell survival.     

Redirection of pyruvate catabolism in Lactococcus lactis by selection of mutants with additional growth requirements

Based on requirements for acetate or lipoic acid for aerobic (but not anaerobic) growth, Lactococcus lactis subsp. lactis mutants with impaired pyruvate catabolism were isolated following classical mutagenesis. Strains with defects in one or two of the enzymes, pyruvate formate-lyase (PFL), lactate dehydrogenase (LDH) and the pyruvate dehydrogenase complex (PDHC) were obtained. Growth and product formation of these strains were characterized. A PFL-defective strain (requiring acetate for anaerobic growth) displayed a two-fold increase in specific lactate production compared with the corresponding wild-type strain when grown anaerobically. LDH defective strains directed 91-96% of the pyruvate towards alpha-acetolactate, acetoin and diacetyl production when grown aerobically in the presence of acetate and absence of lipoic acid (a similar characteristic was observed in an LDH and PDHC defective strain in the presence of both acetate and lipoic acid) and more than 65% towards formate, acetate and ethanol production under anaerobic conditions. Another strain with defective PFL and LDH was strictly aerobic. However, a variant with strongly enhanced diacetyl reductase activities (NADH/NAD+ dependent diacetyl reductase, acetoin reductase and butanediol dehydrogenase activities) was selected from this strain under anaerobic conditions by supplementing the medium with acetoin. This strain is strictly aerobic, unless supplied with acetoin.     

Synthesis and posttranslational regulation of pyruvate formate-lyase in Lactococcus lactis

The enzyme pyruvate formate-lyase (PFL) from Lactococcus lactis was produced in Escherichia coli and purified to obtain anti-PFL antibodies that were shown to be specific for L. lactis PFL. It was demonstrated that activated L. lactis PFL was sensitive to oxygen, as in E. coli, resulting in the cleavage of the PFL polypeptide. The PFL protein level and its in vivo activity and regulation were shown by Western blotting, enzyme-linked immunosorbent assay, and metabolite measurement to be dependent on the growth conditions. The PFL level during anaerobic growth on the slowly fermentable sugar galactose was higher than that on glucose. This shows that variation in the PFL protein level may play an important role in the regulation of metabolic shift from homolactic to mixed-acid product formation, observed during growth on glucose and galactose, respectively. During anaerobic growth in defined medium, complete activation of PFL was observed. Strikingly, although no formate was produced during aerobic growth of L. lactis, PFL protein was indeed detected under these conditions, in which the enzyme is dispensable due to the irreversible inactivation of PFL by oxygen. In contrast, no oxygenolytic cleavage was detected during aerobic growth in complex medium. This observation may be the result of either an effective PFL deactivase activity or the lack of PFL activation. In E. coli, the PFL deactivase activity resides in the multifunctional alcohol dehydrogenase ADHE. It was shown that in L. lactis, ADHE does not participate in the protection of PFL against oxygen under the conditions analyzed. Our results provide evidence for major differences in the mechanisms of posttranslational regulation of PFL activity in E. coli and L. lactis.     

Regulation of product formation during glucose or lactose limitation in nongrowing cells of Streptococcus lactis

Nongrowing cells of Streptococcus lactis in a pH-stat were dosed with sugar to allow fermentation at the maximum rate or were fed a continuous supply of sugar at rates less than the maximum. Under anaerobic conditions, rapid fermentation of either glucose or lactose was essentially homolactic. However, with strain ML3, limiting the fermentation rate diverted approximately half of the pyruvate to formate, acetate, and ethanol. At limiting glucose fermentation rates, cells contained lower concentrations of lactate dehydrogenase activator (fructose 1,6-diphosphate) and pyruvate formate-lyase inhibitors (triose phosphates). As a result, pyruvate formate-lyase and pyruvate dehydrogenase play a greater role in pyruvate metabolism. In contrast to strain ML3, strain ML8 did not give the same diversion of products under anaerobic conditions, and cells retained higher concentrations of the above effector compounds. Lactose metabolism under aerobic conditions resulted in pyruvate excretion by both S. lactis ML3 and ML8. At 7% of the maximum utilization rate, pyruvate accounted for 69 and 35% of the lactose metabolized by ML3 and ML8, respectively. Acetate was also a major product, especially with ML8. The data suggest that NADH oxidase is involved in coenzyme recycling in the presence of oxygen and that pyruvate formate-lyase is inactivated, but the pyruvate dehydrogenase complex still functions.     

Regulation of product formation during glucose or lactose limitation in nongrowing cells of Streptococcus lactis.

Nongrowing cells of Streptococcus lactis in a pH-stat were dosed with sugar to allow fermentation at the maximum rate or were fed a continuous supply of sugar at rates less than the maximum. Under anaerobic conditions, rapid fermentation of either glucose or lactose was essentially homolactic. However, with strain ML3, limiting the fermentation rate diverted approximately half of the pyruvate to formate, acetate, and ethanol. At limiting glucose fermentation rates, cells contained lower concentrations of lactate dehydrogenase activator (fructose 1,6-diphosphate) and pyruvate formate-lyase inhibitors (triose phosphates). As a result, pyruvate formate-lyase and pyruvate dehydrogenase play a greater role in pyruvate metabolism. In contrast to strain ML3, strain ML8 did not give the same diversion of products under anaerobic conditions, and cells retained higher concentrations of the above effector compounds. Lactose metabolism under aerobic conditions resulted in pyruvate excretion by both S. lactis ML3 and ML8. At 7% of the maximum utilization rate, pyruvate accounted for 69 and 35% of the lactose metabolized by ML3 and ML8, respectively. Acetate was also a major product, especially with ML8. The data suggest that NADH oxidase is involved in coenzyme recycling in the presence of oxygen and that pyruvate formate-lyase is inactivated, but the pyruvate dehydrogenase complex still functions.     

Glucose/citrate cometabolism in Lactococcus lactis subsp. lactis biovar diacetylactis with impaired alpha-acetolactate decarboxylase

The pyruvate metabolism of a Lactococcus lactis subsp. lactis biovar diacetylactis mutant deficient in alpha-acetolactate decarboxylase and its wild-type strain was studied during batch cultivations. A chemically defined medium was used containing glucose as carbon- and energy-source. The alpha-acetolactate decarboxylase deficiency had no effect on the specific growth rate. Addition of citrate was found to increase the specific growth rate of both strains under aerobic and anaerobic conditions. The product formation was monitored throughout the cultivations. The carbon- and redox-balances were within the accuracy of the experimental data. When citrate was added, alpha-acetolactate, diacetyl, and acetoin were formed, and aeration was shown to have a positive effect on the formation of these metabolites. By omitting lipoic acid (required for a functional pyruvate dehydrogenase complex) from the growth medium, a similar stimulatory effect on alpha-acetolactate, diacetyl, and acetoin formation was observed under aerobic conditions. The strain with impaired alpha-acetolactate decarboxylase activity accumulated alpha-acetolactate which resulted in an increased diacetyl formation compared to the wild-type strain, under aerobic and anaerobic conditions.     

Physiological role of beta-phosphoglucomutase in Lactococcus lactis

A beta-phosphoglucomutase (beta-PGM) mutant of Lactococcus lactis subsp. lactis ATCC 19435 was constructed using a minimal integration vector and double-crossover recombination. The mutant and the wild-type strain were grown under controlled conditions with different sugars to elucidate the role of beta-PGM in carbohydrate catabolism and anabolism. The mutation did not significantly affect growth, product formation, or cell composition when glucose or lactose was used as the carbon source. With maltose or trehalose as the carbon source the wild-type strain had a maximum specific growth rate of 0.5 h(-1), while the deletion of beta-PGM resulted in a maximum specific growth rate of 0.05 h(-1) on maltose and no growth at all on trehalose. Growth of the mutant strain on maltose resulted in smaller amounts of lactate but more formate, acetate, and ethanol, and approximately 1/10 of the maltose was found as beta-glucose 1-phosphate in the medium. Furthermore, the beta-PGM mutant cells grown on maltose were considerably larger and accumulated polysaccharides which consisted of alpha-1,4-bound glucose units. When the cells were grown at a low dilution rate in a glucose and maltose mixture, the wild-type strain exhibited a higher carbohydrate content than when grown at higher growth rates, but still this content was lower than that in the beta-PGM mutant. In addition, significant differences in the initial metabolism of maltose and trehalose were found, and cell extracts did not digest free trehalose but only trehalose 6-phosphate, which yielded beta-glucose 1-phosphate and glucose 6-phosphate. This demonstrates the presence of a novel enzymatic pathway for trehalose different from that of maltose metabolism in L. lactis.     

Conversion of Lactococcus lactis from homolactic to homoalanine fermentation through metabolic engineering.

We report the engineering of Lactococcus lactis to produce the amino acid L-alanine. The primary end product of sugar metabolism in wild-type L. lactis is lactate (homolactic fermentation). The terminal enzymatic reaction (pyruvate + NADH-->L-lactate + NAD+) is performed by L-lactate dehydrogenase (L-LDH). We rerouted the carbon flux toward alanine by expressing the Bacillus sphaericus alanine dehydrogenase (L-AlaDH; pyruvate + NADH + NH4+ -->L-alanine + NAD+ + H2O). Expression of L-AlaDH in an L-LDH-deficient strain permitted production of alanine as the sole end product (homoalanine fermentation). Finally, stereospecific production (>99%) of L-alanine was achieved by disrupting the gene encoding alanine racemase, opening the door to the industrial production of this stereoisomer in food products or bioreactors.     

Metabolic behavior of Lactococcus lactis MG1363 in microaerobic continuous cultivation at a low dilution rate

Minute amounts of oxygen were supplied to a continuous cultivation of Lactococcus lactis subsp. cremoris MG1363 grown on a defined glucose-limited medium at a dilution rate of 0.1 h(-1). More than 80% of the carbon supplied with glucose ended up in fermentation products other than lactate. Addition of even minute amounts of oxygen increased the yield of biomass on glucose by more than 10% compared to that obtained under anaerobic conditions and had a dramatic impact on catabolic enzyme activities and hence on the distribution of carbon at the pyruvate branch point. Increasing aeration caused carbon dioxide and acetate to replace formate and ethanol as catabolic end products while hardly affecting the production of either acetoin or lactate. The negative impact of oxygen on the synthesis of pyruvate formate lyase was confirmed. Moreover, oxygen was shown to down regulate the protein level of alcohol dehydrogenase while increasing the enzyme activity levels of the pyruvate dehydrogenase complex, alpha-acetolactate synthase, and the NADH oxidases. Lactate dehydrogenase and glyceraldehyde dehydrogenase enzyme activity levels were unaffected by aeration.     

Formation of diacetyl by cell-free extracts of Leuconostoc lactis

Abstract Diacetyl formation was linear with time and with protein concentration when a cell-free extract of Leuconostoc lactis NCW1 was added to a buffer system containing pyruvate, thiamine pyrophosphate and MgS₄ (final concentrations 60 mM, 0.11 mM and 0.22 mM, respectively). No diacetyl was detected in the absence of pyruvate or cell-free extract and no increase in diacetyl formation was detected on the addition of acetyl-CoA. When 2-acetolactate (1.6 mM) was the substrate, autodecarboxylation to diacetyl and acetoin occurred under aerobic and anaerobic conditions. When cell-free extract was added, decarboxylation of 2-acetolactate to acetoin and diacetyl increased 4–6-fold, under aerobic and anaerobic conditions. When the cell-free extract was boiled, diacetyl formation from 2-acetolactate was reduced to the level of autodecarboxylation. The results suggest that diacetyl is formed enzymatically in the presence and absence of oxygen, as well as spontaneously, from 2-acetolactate.     

Kinetics of the reaction of superoxide anion with ferric horseradish peroxidase

Reaction of horseradish peroxidase A2 and C with superoxide anion (O2-) has been studied using pulse radiolysis technique. Peroxidase C formed Compound I and an oxy form of the enzyme due to reaction of ferric enzyme with hydrogen peroxide (H2O2) and O2-, respectively. At low concentrations of O2- (less than 1 mM), O2- reacted with ferric peroxidase C nearly quantitatively and formation of H2O2 was negligible. The rate constant for the reaction was found to be increased below pH 6 and this phenomenon can be explained by assuming that HO2 reacts with peroxidase C more rapidly than O2-. In contrast the formation of oxyperoxidase could not be detected in the case of peroxidase A2 after the pulse, and only Compound I of the enzyme was formed. Peroxidase A2, however, produced the oxy form upon aerobic addition of NADH, suggesting that O2- can also react with peroxidase A2 to form the oxy form. The results at present indicate that the rate constant for the reaction of O2- with peroxidase A2 is smaller than 103 M-1.s-1.     

Purification and characterization of two phosphoglucomutases from Lactococcus lactis subsp. lactis and their regulation in maltose- and glucose-utilizing cells.

Two distinct forms of phosphoglucomutase were found in Lactococcus lactis subsp. lactis, strains 19435 and 65.1, growing on maltose: beta-phosphoglucomutase (beta-PGM), which catalyzes the reversible conversion of beta-glucose 1-phosphate to glucose 6-phosphate in the maltose catabolism, and alpha-phosphoglucomutase (alpha-PGM). beta-PGM was purified to more than 90% homogeneity in crude cell extract from maltose-grown lactococci, and polyclonal antisera to the enzyme were prepared. The molecular mass of beta-PGM was estimated by gel filtration to be 28 kDa; its isoelectric point was 4.8. The corresponding values for alpha-PGM were 65 kDa and 4.4, respectively. The expression of both PGM enzymes was investigated under different growth conditions. The specific activity and amount of beta-PGM per milliliter of cell extract increased with time in lactococci grown on maltose, but the enzyme was absent in lactococci grown on glucose, indicating enzyme synthesis to be induced by maltose in the growth medium. When glucose was added to maltose-grown lactococci, both the specific activity and amount of beta-PGM per milliliter of cell extract decreased rapidly. This suggests that synthesis of beta-PGM is repressed by glucose in the medium. Although the specific activity of alpha-PGM did not change during growth on maltose or glucose, lactococcal strain 19435 showed a much higher specific activity of both alpha- and beta-PGM than strain 65.1 when grown on maltose.     

Properties and function of fumarate reductase (NADH) in Streptococcus lactis.

The fumarate reductase (NADH) present in cell-free extracts of S. lactis C10 was purified approximately 100-fed by chromatography on DEAE-cellulose in the presence of the non-ionic detergent Teric X-10, and some of the properties of this partially purified enzyme were characterized. Fumarate was able to act as a terminal electron acceptor and decreased the amount of lactate formed and oxygen used during the metabolism of pyruvate by resting cells of S. lactis. Anaerobic growth of S. lactis on glycerol was not observed and fumarate reduction was not coupled with glycerol-3-phosphate oxidation.     

Isolation, characterization, and physiological role of the pyruvate dehydrogenase complex and alpha-acetolactate synthase of Lactococcus lactis subsp. lactis bv. diacetylactis.

The pyruvate dehydrogenase complex of Lactococcus lactis subsp. lactis bv. diacetylactis has a specific activity of 6.6 U/mg and a Km of 1 mM for pyruvate. The specific activities of E2 and E3 in the complex are 30 and 0.36 U/mg, respectively. The complex is very sensitive to NADH inhibition and consists of four subunits: E1 alpha (44 kDa), E1 beta (35 kDa), E2 (73 kDa), and E3 (60 kDa). The L. lactis alpha-acetolactate synthase has a specific activity of 103 U/mg and a Km of 50 mM for pyruvate. Thiamine pyrophosphate (Km = 3.2 microM) and divalent cations are essential for activity. The native enzyme measures 172 kDa and consists of 62-kDa monomers. The role of both enzymes in product formation is discussed in view of NADH inhibition and competition for pyruvate.     

Glucose metabolism and NADH recycling by Treponema hyodysenteriae, the agent of swine dysentery.

Glucose metabolism and the mechanisms of NADH oxidation by Treponema hyodysenteriae were studied. Under an N2 atmosphere, washed cell suspensions of the spirochete consumed glucose and produced acetate, butyrate, H2, and CO2. Approximately twice as much H2 as CO2 was produced. Determinations of radioactivity in products of [14C]glucose and [14C]pyruvate metabolism and analyses of enzyme activities in cell lysates revealed that glucose was catabolized to pyruvate via the Embden-Meyerhof-Parnas pathway. The results of pyruvate exchange reactions with NaH14CO3 and Na14COOH demonstrated that pyruvate was converted to acetyl coenzyme A (acetyl-CoA), H2, and CO2 by a clostridium-type phosphoroclastic mechanism. NADH:ferredoxin oxidoreductase and hydrogenase activities were present in cell lysates and produced H2 from NADH oxidation. Phosphotransacetylase and acetate kinase catalyzed the formation of acetate from acetyl-CoA. Butyrate was formed from acetyl-CoA via a pathway that involved 3-hydroxybutyryl-coenzyme A (CoA) dehydrogenase, butyryl-CoA dehydrogenase, and butyryl-CoA transferase. T. hyodysenteriae cell suspensions generated less H2 and butyrate under 10% O2-90% N2 than under 100% N2. Cell lysates contained NADH oxidase, NADH peroxidase, and superoxide dismutase activities. These findings indicated there are three major mechanisms that T. hyodysenteriae cells use to recycle NADH generated from the Embden-Meyerhof-Parnas pathway--enzymes in the pathway from acetyl-CoA to butyrate, NADH:ferredoxin oxidoreductase, and NADH oxidase. Versatility in methods of NADH oxidation and an ability to metabolize oxygen could benefit T. hyodysenteriae cells in the colonization of tissues of the swine large bowel.     

Glucose metabolism and NADH recycling by Treponema hyodysenteriae, the agent of swine dysentery

Glucose metabolism and the mechanisms of NADH oxidation by Treponema hyodysenteriae were studied. Under an N2 atmosphere, washed cell suspensions of the spirochete consumed glucose and produced acetate, butyrate, H2, and CO2. Approximately twice as much H2 as CO2 was produced. Determinations of radioactivity in products of [14C]glucose and [14C]pyruvate metabolism and analyses of enzyme activities in cell lysates revealed that glucose was catabolized to pyruvate via the Embden-Meyerhof-Parnas pathway. The results of pyruvate exchange reactions with NaH14CO3 and Na14COOH demonstrated that pyruvate was converted to acetyl coenzyme A (acetyl-CoA), H2, and CO2 by a clostridium-type phosphoroclastic mechanism. NADH:ferredoxin oxidoreductase and hydrogenase activities were present in cell lysates and produced H2 from NADH oxidation. Phosphotransacetylase and acetate kinase catalyzed the formation of acetate from acetyl-CoA. Butyrate was formed from acetyl-CoA via a pathway that involved 3-hydroxybutyryl-coenzyme A (CoA) dehydrogenase, butyryl-CoA dehydrogenase, and butyryl-CoA transferase. T. hyodysenteriae cell suspensions generated less H2 and butyrate under 10% O2-90% N2 than under 100% N2. Cell lysates contained NADH oxidase, NADH peroxidase, and superoxide dismutase activities. These findings indicated there are three major mechanisms that T. hyodysenteriae cells use to recycle NADH generated from the Embden-Meyerhof-Parnas pathway--enzymes in the pathway from acetyl-CoA to butyrate, NADH:ferredoxin oxidoreductase, and NADH oxidase. Versatility in methods of NADH oxidation and an ability to metabolize oxygen could benefit T. hyodysenteriae cells in the colonization of tissues of the swine large bowel.     

Production of a heterologous nonheme catalase by Lactobacillus casei: an efficient tool for removal of H2O2 and protection of Lactobacillus bulgaricus from oxidative stress in milk

Lactic acid bacteria (LAB) are generally sensitive to H2O2, a compound that they can paradoxically produce themselves, as is the case for Lactobacillus bulgaricus. Lactobacillus plantarum ATCC 14431 is one of the very few LAB strains able to degrade H2O2 through the action of a nonheme, manganese-dependent catalase (hereafter called MnKat). The MnKat gene was expressed in three catalase-deficient LAB species: L. bulgaricus ATCC 11842, Lactobacillus casei BL23, and Lactococcus lactis MG1363. While the protein could be detected in all heterologous hosts, enzyme activity was observed only in L. casei. This is probably due to the differences in the Mn contents of the cells, which are reportedly similar in L. plantarum and L. casei but at least 10- and 100-fold lower in Lactococcus lactis and L. bulgaricus, respectively. The expression of the MnKat gene in L. casei conferred enhanced oxidative stress resistance, as measured by an increase in the survival rate after exposure to H2O2, and improved long-term survival in aerated cultures. In mixtures of L. casei producing MnKat and L. bulgaricus, L. casei can eliminate H2O2 from the culture medium, thereby protecting both L. casei and L. bulgaricus from its deleterious effects.     

Identification of a conserved sequence in flavoproteins essential for the correct conformation and activity of the NADH oxidase NoxE of Lactococcus lactis

Water-forming NADH oxidases (encoded by noxE, nox2, or nox) are flavoproteins generally implicated in the aerobic survival of microaerophilic bacteria, such as lactic acid bacteria. However, some natural Lactococcus lactis strains produce an inactive NoxE. We examined the role of NoxE in the oxygen tolerance of L. lactis in the rich synthetic medium GM17. Inactivation of noxE suppressed 95% of NADH oxidase activity but only slightly affected aerobic growth, oxidative stress resistance, and NAD regeneration. However, noxE inactivation strongly impaired oxygen consumption and mixed-acid fermentation. We found that the A303T mutation is responsible for the loss of activity of a naturally occurring variant of NoxE. Replacement of A303 with T or G or of G307 with S or A by site-directed mutagenesis led to NoxE aggregation and the total loss of activity. We demonstrated that L299 is involved in NoxE activity, probably contributing to positioning flavin adenine dinucleotide (FAD) in the active site. These residues are part of the strongly conserved sequence LA(T)XXAXXXG included in an alpha helix that is present in other flavoprotein disulfide reductase (FDR) family flavoproteins that display very similar three-dimensional structures.