Oxidase Type Research Articles

Cytochrome <i>bd</i> as Antioxidant Redox Enzyme

One of the main functions of enzyme complexes that constitute electron transport (respiratory) chains of organisms is to maintain cellular redox homeostasis by oxidizing reducing equivalents, NADH and quinol. Cytochrome bd is a unique terminal oxidase of the chains of many bacteria including pathogenic species. This redox enzyme couples the oxidation of ubiquinol or menaquinol by molecular oxygen to the generation of proton motive force, a universal energy currency. The latter is used by the organism to produce ATP, another cellular energy currency, via oxidative phosphorylation. Escherichia coli contains two bd–type oxidases, bd-I and bd-II, encoded by the cydAB and appCB operons, respectively. Surprisingly, both bd enzymes make a further contribution to molecular mechanisms of maintaining the appropriate redox balance in the bacterial cell by means of elimination of reactive oxygen species, such as hydrogen peroxide. This review summarizes recent data on the redox-modulated H2O2-scavenging activities of cytochromes bd-I and bd-II from E. coli. The possibility of such antioxidant properties in cytochromes bd from other bacteria is also discussed.

Electrocatalytic and Spectroscopic Studies on Cytochrome Bd Oxidase, a Highly Diverse Bacterial Defense Factor

The selective reduction of oxygen to water is crucial to life and a central process in aerobic organisms. It is catalyzed by several different enzymes, including cytochrome bd oxidases that are solely present in prokaryotes, including several pathogens. In addition, these enzymes play a crucial role in protection against oxidative stress, in virulence, adaptability and antibiotics resistance. The reduction of O2 occurs at the high spin D-type heme in all cytochrome bd oxidases, that is also the binding site for several ligands from signaling processes, including NO, H2S and CO.Here we present the electrocatalytic study of the cytochrome bd I and bd II oxidases from Escherichia coli (1,2) as well on other related bd oxidases. Structural parameters that are crucial for the reactivity towards oxygen are analyzed. The pH dependency of the binding and release of NO, an important signaling factor is presented. The influence of mutants in the proton channel on the NO release is discussed.(1) Grauel, A. Kägi, J. Rasmussen, T. Makarchuk, I. Oppermann, S. Moumbock, A., Wohlwend, D., Müller, R., Melin, F., Günther, S., Hellwig, P., Böttcher, B., and Friedrich, T., ‘Structure of Escherichia coli cytochrome bd-II type oxidase with bound aurachin D’ (2021) Nat. Commun., 12:6498.(2) Nikolaev, A., Safarian, S., Thesseling, A., Wohlwend, D., Friedrich, T., Michel, H., Kusumoto, T., Sakamoto, J., Melin, F., Hellwig P. ‘Electrocatalytic evidence of the diversity of the oxygen reaction in the bacterial bd oxidase from different organisms’ (2021) Biochim. Biophys. Acta 1862, 148436.

Aircraft noise exposure induces pro-inflammatory vascular conditioning and amplifies vascular dysfunction and impairment of cardiac function after myocardial infarction.

Traffic noise may play an important role in the development and deterioration of ischaemic heart disease. Thus, we sought to determine the mechanisms of cardiovascular dysfunction and inflammation induced by aircraft noise in a mouse model of myocardial infarction (MI) and in humans with incident MI. C57BL/6J mice were exposed to noise alone (average sound pressure level 72 dB; peak level 85 dB) for up to 4 days, resulting in pro-inflammatory aortic gene expression in the myeloid cell adhesion/diapedesis pathways. The noise alone promoted adhesion and infiltration of inflammatory myeloid cells in vascular/cardiac tissue, paralleled by an increased percentage of leucocytes with a pro-inflammatory, reactive oxygen species (ROS)-producing phenotype and augmented expression of nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase type 2 (Nox2)/phosphorylation of nuclear factor 'kappa light chain enhancer' of activated B-cells (phospho-NFκB) in peripheral blood. Ligation of the left anterior descending artery resulted in worsening of cardiac function, pronounced cardiac infiltration of CD11b+ myeloid cells and Ly6Chigh monocytes, and induction of interleukin (IL) 6, IL-1β, CCL-2, and Nox2, being aggravated by noise exposure prior to MI. MI induced stronger endothelial dysfunction and more pronounced increases in vascular ROS in animals preconditioned with noise. Participants of the population-based Gutenberg Health Cohort Study (median follow-up:11.4 years) with incident MI revealed elevated C-reactive protein at baseline and worse left ventricular ejection fraction (LVEF) after MI in case of a history of noise exposure and subsequent annoyance development. Aircraft noise exposure before MI substantially amplifies subsequent cardiovascular inflammation and aggravates ischaemic heart failure, facilitated by a pro-inflammatory vascular conditioning. Our translational results suggest that measures to reduce environmental noise exposure will be helpful in improving the clinical outcome of subjects with MI.Key questionKey finding Take-home-MessageAircraft noise exposure before MI substantially amplifies cardiovascular inflammation and aggravates cardiac impairment after MI.

Structure of Escherichia coli cytochrome bd-II type oxidase with bound aurachin D

Structure of Escherichia coli cytochrome bd-II type oxidase with bound aurachin D

Structure of Escherichia coli cytochrome bd-II type oxidase with bound aurachin D

Cytochrome bd quinol:O2 oxidoreductases are respiratory terminal oxidases so far only identified in prokaryotes, including several pathogenic bacteria. Escherichia coli contains two bd oxidases of which only the bd-I type is structurally characterized. Here, we report the structure of the Escherichia coli cytochrome bd-II type oxidase with the bound inhibitor aurachin D as obtained by electron cryo-microscopy at 3 Å resolution. The oxidase consists of subunits AppB, C and X that show an architecture similar to that of bd-I. The three heme cofactors are found in AppC, while AppB is stabilized by a structural ubiquinone-8 at the homologous positions. A fourth subunit present in bd-I is lacking in bd-II. Accordingly, heme b595 is exposed to the membrane but heme d embedded within the protein and showing an unexpectedly high redox potential is the catalytically active centre. The structure of the Q-loop is fully resolved, revealing the specific aurachin binding.

Manganese impairs the QoxABCD terminal oxidase leading to respiration‐associated toxicity

Cell physiology relies on metalloenzymes and can be easily disrupted by imbalances in metal ion pools. Bacillus subtilis requires manganese for growth and has highly regulated mechanisms for import and efflux that help maintain homeostasis. Cells defective for manganese (Mn) efflux are highly sensitive to intoxication, but the processes impaired by Mn excess are often unknown. Here, we employed a forward genetics approach to identify pathways affected by manganese intoxication. Our results highlight a central role for the membrane-localized electron transport chain in metal intoxication during aerobic growth. In the presence of elevated manganese, there is an increased generation of reactive radical species associated with dysfunction of the major terminal oxidase, the cytochrome aa3 heme-copper menaquinol oxidase (QoxABCD). Intoxication is suppressed by diversion of menaquinol to alternative oxidases or by a mutation affecting heme A synthesis that is known to convert QoxABCD from an aa3 to a bo3 -type oxidase. Manganese sensitivity is also reduced by derepression of the MhqR regulon, which protects cells against reactive quinones. These results suggest that dysfunction of the cytochrome aa3 -type quinol oxidase contributes to metal-induced intoxication.

Inhibition of LPS-induced Nox2 activation by VAS2870 protects alveolar epithelial cells through eliminating ROS and restoring tight junctions

Inhibiting the production of reactive oxygen species (ROS) in alveolar epithelial cells (AECs) under oxidative distress becomes a new therapeutic strategy for acute respiratory distress syndrome (ARDS). Herein in the present study, we investigated effects of Nox2, the catalytic subunit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase type 2, on LPS-induced epithelium injury in ARDS mice and in human alveolar epithelial A549 cells. Severe lung injury, disruption of alveolar-capillary barrier with the loss of zonula occluden (ZO)-1 and up-regulated expression of Nox2 in AECs were exhibited in ARDS mice. In vitro, LPS induced decreased cell viability coupled with activated Nox2, high level of ROS, and destroyed ZO-1 distribution. Moreover, VAS2870 proved to inhibit Nox2 expression, reduce ROS generation, restore epithelium barrier integrity, and preserve cell viability in LPS-induced A549 cells. These data demonstrate that Nox2/ROS/ZO-1 axis is of great importance in AECs damage induced by LPS, and the utilization of VAS2870 to inhibit this pathway might lighten LPS-induced ARDS.

English

Ghana’s waters are biologically diverse with different fish species due to different ecological habitats and niches harboring several cryptic fish species. However, the fish production has reduced over the past decade; hence an urgent conservation and management strategies are required to save Ghanaian fishery such as accurate identification of species to formulate species-specific conservation and management strategies. Molecular method of fish identification, DNA barcoding, has proven its efficacy in species identification for both freshwater and marine species. In recent years, DNA barcoding has been accepted as a bio-identification system for living organisms globally. This system is fast and produces accurate species identification by using a short DNA sequence marker from a standard region of the DNA sequence of an organism’s genome to identify it as belonging to a particular individual or species based on Cytochrome C Oxidase type I (COI/ Cox 1) gene instead of the whole genome. Unfortunately, unlike the developed countries, this molecular method of fish identification is new in Ghana. This review article aims to examine the issues regarding fish identification and the need for DNA barcoding as a tool for taxonomic identification, grouping, and naming of fish species in Ghana. Also, this review takes a look at the current status and future direction of DNA barcoding fisheries in Ghana. In addition, the benefits of DNA Barcoding in fishery management and conservation are discussed.   Key words: DNA barcoding, Ghana, marine species, current status, management, conservation.

Abstract 4359: Oscillatory HIF-1α induction promotes proliferation of hypoxic cells through a lactate dependent quorum autophagy response

Abstract Intratumoral hypoxia is one of the most important factors present in the tumor microenvironment, regulating various steps in cancer growth and metastasis, including metabolism, vascularization, premetastatic niche formation, and dormancy. Cellular response to hypoxia is highly regulated, and is mediated by hypoxia-inducible factors (HIF), consisting of an oxygen (O2) regulated subunit, among which HIF-1α is the ubiquitously expressed homologue. HIF-1α stabilizes in hypoxia, and transcriptionally activates more than 1500 HIF identified target genes. Changes in HIF-1α activity, can, therefore result in large changes in gene expression, and phenotypic state of the cells. However, there are paradoxes in observations of HIF-1α responses. HIF-1α inhibits cell proliferation, but cells continue to divide in hypoxia. Similarly, HIF-1α induced indirect phosphorylation and inhibition of pyruvate dehydrogenase (PDH) diverts pyruvate away from acetyl-CoA toward lactate synthesis, decreasing respiration; but HIF-1α can also enhance mitochondrial respiration efficiency by controlling the expression cytochrome c oxidase type 4 (COX4) subunits. These paradoxes could potentially be addressed if individual cellular responses to hypoxia may be different than the bulk/average cell responses.Using fluorescent dynamic reporters of HIF-1α activity, we show that hypoxic responses in individual cells can be highly dynamic and variable across the population. These responses fall into three classes, including oscillatory activity. We identify a molecular mechanism that can account for all three response classes, demonstrating that the oscillations of HIF-1a activity and abundance are controlled by the reactive oxygen species-dependent chaperone-mediated autophagy in a subset of respiring cells. Furthermore, we find that the oscillatory response is modulated by the abundance of extracellular lactate in a quorum sensing-like mechanism. Importantly, we demonstrate that oscillatory HIF-1α activity promotes differential gene expression leading to enhanced cell division, which can help reduce the anti-proliferative effect of hypoxia. These results may help resolve the paradox of seemingly contradicting roles of HIF-1α in the control of metabolic cell states and cell proliferation. Further, specific transcription by oscillatory hypoxia suggest that gene regulation can specifically decode oscillatory signals, resulting in novel phenotypes in a subpopulation, with a selective advantage to survive and proliferate under hypoxic stress. Note: This abstract was not presented at the meeting. Citation Format: Kshitiz ., Junaid Afzal, Yasir Suhail, Hao Chang, Chi V. Dang, Andre Levchenko. Oscillatory HIF-1α induction promotes proliferation of hypoxic cells through a lactate dependent quorum autophagy response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4359.

Identification of Small Peptides that Inhibit NADPH Oxidase (Nox2) Activation.

Nicotinamide adenine phosphate (NADPH) oxidase type 2 (Nox2), a major source of reactive oxygen species in lungs, plays an important role in tissue damage associated with acute inflammatory diseases. The phospholipase A2 (PLA2) activity of peroxiredoxin 6 (Prdx6), called aiPLA2, is required for Nox2 activation through its role in the cellular generation of Rac, a key cytosolic component of the activation cascade. Lung surfactant protein A (SP-A) binds to Prdx6, inhibits its aiPLA2 activity, and prevents activation of Nox2. Based on protein docking software, we previously identified a 16 amino acid (aa) peptide derived from rat SP-A as the Prdx6 binding motif. We now identify the minimal effective sequences of rat/mouse and human SP-A as 9-aa sequences that we have called PLA2-inhibitory peptide (PIP).These sequences are PIP-1, rat/mouse; PIP-2, human; and PIP-3, a hybrid of PIPs 1&2. aiPLA2 activity in vitro was inhibited by 50% with ~7–10 µg PIP/µg Prdx6. Inhibition of the aiPLA2 activity and Nox2 activation of lungs in vivo was similar for intratracheal (IT) and intravenous (IV) administration of PIP-2, but required its incorporation into liposomes as a delivery vehicle; tissue ½ time for decrease of the in vivo inhibition of aiPLA2 activity after PIP-2 administration was ~50 h. These properties suggest that PIP-2 could be an effective therapeutic agent to prevent tissue injury associated with lung inflammation.

Offline encoding impaired by epigenetic regulations of monoamines in the guided propagation model of autism

BackgroundEnvironmental factors can modify the expression of genes, including those involved in the metabolism of neurotransmitters. Accounting for a control role of monoamine neurotransmitters, the guided propagation (GP) memory model may contribute to investigate the consequences of neuromodulation impairments on development disorders such as autism. A prenatal transient excess of ‘monoamine oxidase A’ enzyme is assumed here to trigger persistent epigenetic regulations that would induce imbalanced metabolisms of synaptic monoamines. When imported into the ‘offline’ encoding cycles of a GP model, the consequent ‘serotoninergic noise’ leads to aberrant memory structures that can be linked with autism symptoms.ResultsIn computer experiments, different levels of uncoupling between representations of monoamines correlate with the amount of impaired GP modules, the severity of irrelevant connections, as well as network overgrowth. Two types of faulty connections are respectively assumed to underlie autism traits, namely repetitive behavior and perceptual oversensitivity. Besides computational modelling, a genetic family-tree shows how the autism sex-ratio can result from combinations of pharmacological and epigenetic features.ConclusionsThese results suggest that the current rise of autism is favored by three possible sources of biological masking: (1) during sleep, when cyclic variations of monoamines may undergo disrupted enzymatic activities; (2) across generations of ‘healthy carriers’ protected by the X-chromosome silencing and a specific genetic variant; (3) early in life, as long as the brain development draws on pools of neurons born when the transient enzymatic excess and its persistent epigenetic regulation overlapped, and as long as the B type of monoamine oxidase does not significantly impact dopamine. A disease-modifying therapy can be derived from this study, which involves relevant biomarkers to be first monitored over several months of clinical trial.

The copper-deprivation stimulon of Corynebacterium glutamicum comprises proteins for biogenesis of the actinobacterial cytochrome bc1–aa3 supercomplex

Aerobic respiration in Corynebacterium glutamicum involves a cytochrome bc1-aa3 supercomplex with a diheme cytochrome c1, which is the only c-type cytochrome in this species. This organization is considered as typical for aerobic Actinobacteria. Whereas the biogenesis of heme-copper type oxidases like cytochrome aa3 has been studied extensively in α-proteobacteria, yeast, and mammals, nothing is known about this process in Actinobacteria. Here, we searched for assembly proteins of the supercomplex by identifying the copper-deprivation stimulon, which might include proteins that insert copper into cytochrome aa3 Using gene expression profiling, we found two copper starvation-induced proteins for supercomplex formation. The Cg2699 protein, named CtiP, contained 16 predicted transmembrane helices, and its sequence was similar to that of the copper importer CopD of Pseudomonas syringae in the N-terminal half and to the cytochrome oxidase maturation protein CtaG of Bacillus subtilis in its C-terminal half. CtiP deletion caused a growth defect similar to that produced by deletion of subunit I of cytochrome aa3, increased copper tolerance, triggered expression of the copper-deprivation stimulon under copper sufficiency, and prevented co-purification of the supercomplex subunits. The secreted Cg1884 protein, named CopC, had a C-terminal transmembrane helix and contained a Cu(II)-binding motif. Its absence caused a conditional growth defect, increased copper tolerance, and also prevented co-purification of the supercomplex subunits. CtiP and CopC are conserved among aerobic Actinobacteria, and we propose a model of their functions in cytochrome aa3 biogenesis. Furthermore, we found that the copper-deprivation response involves additional regulators besides the ECF sigma factor SigC.

Biphenyl Modulates the Expression and Function of Respiratory Oxidases in the Polychlorinated-Biphenyls Degrader Pseudomonas pseudoalcaligenes KF707.

Pseudomonas pseudoalcaligenes KF707 is a soil bacterium which is known for its capacity to aerobically degrade harmful organic compounds such as polychlorinated biphenyls (PCBs) using biphenyl as co-metabolite. Here we provide the first genetic and functional analysis of the KF707 respiratory terminal oxidases in cells grown with two different carbon sources: glucose and biphenyl. We identified five terminal oxidases in KF707: two c(c)aa3 type oxidases (Caa3 and Ccaa3), two cbb3 type oxidases (Cbb31 and Cbb32), and one bd type cyanide-insensitive quinol oxidase (CIO). While the activity and expression of both Cbb31 and Cbb32 oxidases was prevalent in glucose grown cells as compared to the other oxidases, the activity and expression of the Caa3 oxidase increased considerably only when biphenyl was used as carbon source in contrast to the Cbb32 oxidase which was repressed. Further, the respiratory activity and expression of CIO was up-regulated in a Cbb31 deletion strain as compared to W.T. whereas the CIO up-regulation was not present in Cbb32 and C(c)aa3 deletion mutants. These results, together, reveal that both function and expression of cbb3 and caa3 type oxidases in KF707 are modulated by biphenyl which is the co-metabolite needed for the activation of the PCBs-degradation pathway.

Exploiting the synthetic lethality between terminal respiratory oxidases to kill Mycobacterium tuberculosis and clear host infection

The recent discovery of small molecules targeting the cytochrome bc1 :aa3 in Mycobacterium tuberculosis triggered interest in the terminal respiratory oxidases for antituberculosis drug development. The mycobacterial cytochrome bc1 :aa3 consists of a menaquinone:cytochrome c reductase (bc1 ) and a cytochrome aa3 -type oxidase. The clinical-stage drug candidate Q203 interferes with the function of the subunit b of the menaquinone:cytochrome c reductase. Despite the affinity of Q203 for the bc1 :aa3 complex, the drug is only bacteriostatic and does not kill drug-tolerant persisters. This raises the possibility that the alternate terminal bd-type oxidase (cytochrome bd oxidase) is capable of maintaining a membrane potential and menaquinol oxidation in the presence of Q203. Here, we show that the electron flow through the cytochrome bd oxidase is sufficient to maintain respiration and ATP synthesis at a level high enough to protect M. tuberculosis from Q203-induced bacterial death. Upon genetic deletion of the cytochrome bd oxidase-encoding genes cydAB, Q203 inhibited mycobacterial respiration completely, became bactericidal, killed drug-tolerant mycobacterial persisters, and rapidly cleared M. tuberculosis infection in vivo. These results indicate a synthetic lethal interaction between the two terminal respiratory oxidases that can be exploited for anti-TB drug development. Our findings should be considered in the clinical development of drugs targeting the cytochrome bc1 :aa3 , as well as for the development of a drug combination targeting oxidative phosphorylation in M. tuberculosis.

Linezolid-Induced Serotonin Toxicity in a Patient Not Taking Monoamine Oxidase Inhibitors or Serotonin Receptor Antagonists

Linezolid is an oxazolidinone antibiotic with weak monoamine oxidase (MAO) type A and MAO type B inhibitory effects. Linezolid has been associated with serotonin toxicity when used concomitantly with multiple medications that are known to increase serotonin concentrations. We report the case of a 65-year-old woman with signs and symptoms of serotonin toxicity following administration of linezolid for treatment of methicillin-resistant Staphylococcus aureus pneumonia.

Distribution of Polyphenol Oxidase in some Cruciferae Vegetables

Enzymatic oxidation of polyphenolic compounds in damaged tissue of many fruits and vegetables induced undesirable browning as results in decreased marketability of products. A new type of polyphenol oxidase (PPO) that is “Phloroglucinol Oxidase (PhO)” have been reported in turnip and cabbage, and the purified enzymes also had strong peroxidase (POD) activity. Distribution of PPO from different plant source of cruciferae vegetables were observed. Cabbage, broccoli, turnip and cauliflower PPOs from Indonesia and Japan strongly oxidized 1,3,5-trihydroxybenzenes such as phloroglucinol (Phl) and phloroglucinol carboxylic acid, but not oxidized 1,2,3-trihydroxybenzenes and o-diphenol. On the other side, Chinese mustard, Chinese cabbage, shingensai, Japanese radish root, mizuna, takana, leaf mustard, katsuona and komatsuna also strongly oxidized 1,3,5-trihydroxybenzenes and little oxidized 1,2,3-trihydroxybenzenes such as pyrogallol and gallic acid, but not oxidized o-diphenol. These results indicated that the PPOs in all cruciferae vegetables are a group of a new type of PPO, however, the level activity of 7 varieties of vegetables from Indonesia and 15 varieties from Japan tested showed different activity level of PhO and POD.

Altering the phenolics profile of a green tea leaves extract using exogenous oxidases

Transformation from green tea leaves into black tea involves oxidation of catechins into theaflavins and other complex phenolics by endogenous enzymes in tea leaves. By employing tyrosinase and laccase, both from Agaricus bisporus, on green tea catechins, the oxidation process was directed towards a higher theaflavins content, which is considered an important quality parameter in tea. The main tea catechins were incubated with tyrosinase and laccase, and product formation was monitored by RP-UHPLC–PDA-ESI-MS. The kind of catechin, their substitution with a galloyl group, and the type of oxidase used were important factors determining theaflavin concentrations. In particular, incubation of epicatechin with epigallocatechin with tyrosinase gave a high, stable theaflavin content. In a green tea extract, tyrosinase increased the proportion of theaflavins by twofold compared to black tea. Laccase mainly formed insoluble complexes. Our results indicate that the phenolic profile of tea can be modulated by using commercially available exogenous oxidases.

Effect of denervation on the regulation of mitochondrial transcription factor A expression in skeletal muscle.

The purpose of this study was to determine how the expression of mitochondrial transcription factor A (Tfam), a protein that governs mitochondrial DNA (mtDNA) transcription and replication, is regulated during a state of reduced organelle content imposed by muscle disuse. We measured Tfam expression at 8 h, 16 h, 24 h, 3 days, or 7 days following denervation and hypothesized that decreases in Tfam expression would precede mitochondrial loss. Muscle mass was lowered by 13% and 38% at 3 and 7 days postdenervation, while cytochrome c oxidase activity fell by 33% and 39% at the same time points. Tfam promoter activation in vivo was reduced by 30-65% between 8 h and 3 days of denervation, while Tfam transcript half-life was increased following 8-24 h of denervation. Protein expression of RNA-binding proteins that promote mRNA degradation (CUG repeat-binding protein and K homology splicing regulator protein) was elevated at 3 and 7 days of denervation. Tfam localization within subsarcolemmal mitochondria was reduced after 3 and 7 days of denervation and was associated with suppression of the cytochrome c oxidase type I transcript at 3 days, indicating that denervation impairs both mitochondrial Tfam import and mtDNA transcription during an early period following denervation. These data suggest that putative signals downregulate Tfam transcription during the earliest stages following denervation but are counteracted by increases in Tfam mRNA stability. Import of Tfam into the mitochondrion seems to be the most critical point of regulation of this protein during the early onset of denervation, an impairment of which is coincident with the loss of mitochondria during muscle disuse.

Novel alcohol oxidase with glycolate oxidase activity from Ochrobactrum sp. AIU 033

Abstract We revealed that Ochrobactrum sp. AIU 033, which accumulated a high concentration of glyoxylate from glycolate, produced an enzyme catalyzing oxidation of glycolate to glyoxylate. The enzyme further oxidized lactate and primary alcohols (C2–C10), but did not oxidize glyoxylate, ethylene glycol, glycerol, or methanol. The K m value for glycolate (167 mM) was higher than that for primary alcohols. The glycolate oxidase activity was optimum at pH 5.5, and more than 80% of the enzyme activity remained in the pH range from 5.5 to 6.5 and at below 35 °C. The enzyme had a molecular mass of 130 kDa and was composed of an α 2 β 2 structure, in which the α subunit was 52 kDa and the β subunit was 14 kDa. The enzyme was a flavoprotein and contained two iron atoms. The N-terminal sequences of the 52 kDa subunit and 14 kDa subunit had high similarity to those of putative glucose–methanol–choline oxidoreductases and putative 2-keto-gluconate dehydrogenase. These findings implied that the enzyme was a novel type of alcohol oxidase exhibiting glycolate oxidase activity. The enzyme accumulated glyoxylate with time, but oxalate, which is the oxidation product of glyoxylate, was not detected. This result also indicated that the enzyme catalyzed the formation of glyoxylate in the resting cell-reaction and thus could be useful in the enzymatic production of glyoxylate.

A Biochemical Approach to Study the Role of the Terminal Oxidases in Aerobic Respiration in Shewanella oneidensis MR-1

The genome of the facultative anaerobic γ-proteobacterium Shewanella oneidensis MR-1 encodes for three terminal oxidases: a bd-type quinol oxidase and two heme-copper oxidases, a A-type cytochrome c oxidase and a cbb 3-type oxidase. In this study, we used a biochemical approach and directly measured oxidase activities coupled to mass-spectrometry analysis to investigate the physiological role of the three terminal oxidases under aerobic and microaerobic conditions. Our data revealed that the cbb 3-type oxidase is the major terminal oxidase under aerobic conditions while both cbb 3-type and bd-type oxidases are involved in respiration at low-O2 tensions. On the contrary, the low O2-affinity A-type cytochrome c oxidase was not detected in our experimental conditions even under aerobic conditions and would therefore not be required for aerobic respiration in S. oneidensis MR-1. In addition, the deduced amino acid sequence suggests that the A-type cytochrome c oxidase is a ccaa 3-type oxidase since an uncommon extra-C terminal domain contains two c-type heme binding motifs. The particularity of the aerobic respiratory pathway and the physiological implication of the presence of a ccaa 3-type oxidase in S. oneidensis MR-1 are discussed.