Amine oxidation by d-arginine dehydrogenase in Pseudomonas aeruginosa

Ouedraogo, Daniel; Ball, Jacob; Iyer, Archana; Reis, Renata A.G.; Vodovoz, Maria; Gadda, Giovanni

doi:10.1016/j.abb.2017.06.013

Cited by 14 publications

(18 citation statements)

References 61 publications

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“…A key mechanistic question that remains to be fully investigated is whether this step is generally coupled to dioxygen protonation as suggested by recent computational studies (see Scheme ). Is proton-coupled electron transfer a mechanistic feature shared by most oxygen-reacting flavoenzymes? Why do certain flavoenzymes react so poorly with dioxygen (e.g., flavocytochrome b 2 , d -arginine dehydrogenase, and cellobiose dehydrogenase)? Residues locally gating access to flavin N(5)–C(4a) locus might control the accessibility and, therefore, reactivity of the flavin with dioxygen, although other unidentified factors may contribute to the lack of reactivity in some instances .…”

Section: Discussionmentioning

confidence: 99%

“…Yet, more detailed studies have revealed that the enzyme operates optimally by using quinones as electron acceptor, while the reduced enzyme reacts poorly with molecular oxygen . Medium-chain acyl CoA dehydrogenase is another classic representative of flavoprotein dehydrogenases that show some sluggish activity with dioxygen. − An example of a flavoprotein dehydrogenase that is fully resilient toward dioxygen as electron acceptor is d -arginine dehydrogenase from Pseudomonas aeruginosa . The second and third groups of dioxygen-reactive flavoproteins, the oxidases and monooxygenases, instead react rapidly with molecular oxygen to yield the oxidized flavoprotein. ,− Flavin-dependent oxidases efficiently use O 2 as an electron acceptor to produce, in most cases, H 2 O 2 , whereas flavoprotein monooxygenases generally activate dioxygen by forming a C(4a)–(hydro)peroxide with the flavin, which is used to insert an oxygen atom into the substrate (Scheme ).…”

Section: Introductionmentioning

confidence: 99%

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Same Substrate, Many Reactions: Oxygen Activation in Flavoenzymes

et al. 2018

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Over time, organisms have evolved strategies to cope with the abundance of dioxygen on Earth. Oxygen-utilizing enzymes tightly control the reactions involving O mostly by modulating the reactivity of their cofactors. Flavins are extremely versatile cofactors that are capable of undergoing redox reactions by accepting either one electron or two electrons, alternating between the oxidized and the reduced states. The physical and chemical principles of flavin-based chemistry have been investigated widely. In the following pages we summarize the state of the art on a key area of research in flavin enzymology: the molecular basis for the activation of O by flavin-dependent oxidases and monooxygenases. In general terms, oxidases use O as an electron acceptor to produce HO, while monooxygenases activate O by forming a flavin intermediate and insert an oxygen atom into the substrate. First, we analyze how O reaches the flavin cofactor embedded in the protein matrix through dedicated access pathways. Then we approach O activation from the perspective of the monooxygenases, their preferred intermediate, the C(4a)-(hydro)peroxyflavin, and the cases in which other intermediates have been described. Finally, we focus on understanding how the architectures developed in the active sites of oxidases promote O activation and which other factors operate in its reactivity.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Same Substrate, Many Reactions: Oxygen Activation in Flavoenzymes

et al. 2018

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“…Because of the nature of electron transfer, the oxidizing substrates of flavoenzymes are not easily discerned from the structure, 72 so that the physiological oxidizing substrate can be difficult to identify, as in the case of arginine dehydrogenase. 73 Recently, a publication posited that NAD + is the second substrate for an ortholog of NicA2. 74 However, monitoring the reduction of NAD + in the presence of (S)-nicotine produced no reaction (Table S4).…”

Section: ■ Discussionmentioning

confidence: 99%

“…Well-studied examples include lactate oxidase, whose flavin reacts rapidly with O 2 , and the highly homologous flavocytochrome b 2 , whose flavin is oxidized by a cytochrome on a separate domain; and acyl CoA oxidase, whose FAD is oxidized by O 2 , and acyl CoA dehydrogenase, whose FAD is oxidized by electron-transfer flavoprotein. Because of the nature of electron transfer, the oxidizing substrates of flavoenzymes are not easily discerned from the structure, so that the physiological oxidizing substrate can be difficult to identify, as in the case of arginine dehydrogenase . Recently, a publication posited that NAD + is the second substrate for an ortholog of NicA2 .…”

Section: Discussionmentioning

confidence: 99%

Fast Kinetics Reveals Rate-Limiting Oxidation and the Role of the Aromatic Cage in the Mechanism of the Nicotine-Degrading Enzyme NicA2

et al. 2021

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In Pseudomonas putida, the flavoprotein nicotine oxidoreductase (NicA2) catalyzes the oxidation of (S)-nicotine to N-methyl-myosmine, which is nonenzymatically hydrolyzed to pseudooxynicotine. Structural analysis reveals a monoamine oxidase (MAO)-like fold with a conserved FAD-binding domain and variable substrate-binding domain. The flavoenzyme has a unique variation of the classic aromatic cage with flanking residue pair W427/N462. Previous mechanistic studies using O 2 as the oxidizing substrate show that NicA2 has a low apparent K m of 114 nM for (S)-nicotine with a very low apparent turnover number (k cat of 0.006 s −1 ). Herein, the mechanism of NicA2 was analyzed by transient kinetics. Single-site variants of W427 and N462 were used to probe the roles of these residues. Although several variants had moderately higher oxidase activity (7−12-fold), their reductive half-reactions using (S)-nicotine were generally significantly slower than that of wild-type NicA2. Notably, the reductive half-reaction of wild-type NicA2 is 5 orders of magnitude faster than the oxidative half-reaction with an apparent pseudo-first-order rate constant for the reaction of oxygen similar to k cat . X-ray crystal structures of the N462V and N462Y/W427Y variants complexed with (S)-nicotine (at 2.7 and 2.3 Å resolution, respectively) revealed no significant active-site rearrangements. A second substratebinding site was identified in N462Y/W427Y, consistent with observed substrate inhibition. Together, these findings elucidate the mechanism of a flavoenzyme that preferentially oxidizes tertiary amines with an efficient reductive half-reaction and a very slow oxidative half-reaction when O 2 is the oxidizing substrate, suggesting that the true oxidizing agent is unknown.

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“…Previous studies of the hD2HGDH and the P. putida D2HGDH showed that the enzyme could turn over with electron-transferring flavoprotein as an electron acceptor. 21,22,27 The lack of reactivity of PaD2HGDH with O 2 is shared only by a limited number of flavoprotein dehydrogenases and reductases that do not react or react very poorly with O 2 , including flavocytochrome b 2 , 87 medium chain acyl-CoA dehydrogenase, 88 D-arginine dehydrogenase, 89,90 cellobiose dehydrogenase, 91 and NADH:quinone oxidoreductase. 92 Residues locally gating access to the flavin C4a N5 atoms 93 or finely tuned electrostatics controlling flavin reactivity 94−96 might be responsible for the lack of O 2 reactivity in PaD2HGDH.…”

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confidence: 99%

Kinetic and Bioinformatic Characterization of d-2-Hydroxyglutarate Dehydrogenase from Pseudomonas aeruginosa PAO1

Quaye

Gadda

2020

Biochemistry

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D-2-Hydroxyglutarate dehydrogenase from Pseudomonas aeruginosa PAO1 (PaD2HGDH) catalyzes the oxidation of D-2-hydroxyglutarate to 2ketoglutarate, which is a necessary step in the serine biosynthetic pathway. The dependence of P. aeruginosa on PaD2HGDH makes the enzyme a potential therapeutic target against P. aeruginosa. In this study, recombinant His-tagged PaD2HGDH was expressed and purified to high levels from gene PA0317, which was previously annotated as an FAD-binding PCMH-type domain-containing protein. The enzyme cofactor was identified as FAD with fluorescence emission after phosphodiesterase treatment and with mass spectrometry analysis. PaD2HGDH had a k cat value of 11 s −1 and a K m value of 60 μM with D-2hydroxyglutarate at pH 7.4 and 25 °C. The enzyme was also active with D-malate but did not react with molecular oxygen. Steady-state kinetics with D-malate and phenazine methosulfate as an electron acceptor established a mechanism that was consistent with ping-pong bi-bi steady-state kinetics at pH 7.4. A comparison of the k cat /K m values with D-2-hydroxyglutarate and D-malate suggested that the C5 carboxylate of D-2-hydroxyglutarate is important for the substrate specificity of the enzyme. Other homologues of the enzyme have been previously grouped in the VAO/PMCH family of flavoproteins. PaD2HGDH shares fully conserved residues with other α-hydroxy acid oxidizing enzymes, and these conserved residues are found in the active site of the PaD2HDGH homology model. An Enzyme Function Initiative-Enzyme Similarity Tool Sequence Similarity Network analysis suggests a functional difference between PaD2HGDH and human D2HGDH, and no relationship with VAO. A phylogenetic tree analysis of PaD2HGDH, VAO, and human D2HGDH establishes genetic diversity among these enzymes.

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Amine oxidation by d-arginine dehydrogenase in Pseudomonas aeruginosa

Cited by 14 publications

References 61 publications

Same Substrate, Many Reactions: Oxygen Activation in Flavoenzymes

Same Substrate, Many Reactions: Oxygen Activation in Flavoenzymes

Fast Kinetics Reveals Rate-Limiting Oxidation and the Role of the Aromatic Cage in the Mechanism of the Nicotine-Degrading Enzyme NicA2

Kinetic and Bioinformatic Characterization of d-2-Hydroxyglutarate Dehydrogenase from Pseudomonas aeruginosa PAO1

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