Secondary Amine Mono-oxygenase Research Articles

Heme-heme oxygenase complex. Structure of the catalytic site and its implication for oxygen activation.

Heme oxygenase, a central monooxygenase enzyme of the heme catabolism and the associated generation of carbon monoxide, forms a 1:1 stoichiometric complex with iron protoporphyrin IX, which is a prosthetic active center and at the same time the substrate of the enzyme. By using EPR, resonance Raman, and optical absorption spectroscopic techniques, we have determined the axial ligand coordination of the enzyme-heme complex. The ferric heme iron in the heme-enzyme complex at neutral pH is six-coordinate high spin, while at alkaline pH (pKa 7.6), the complex becomes low spin. Spectra of ferrous forms of the complex indicate that histidine serves as the iron proximal axial ligand and that the residue is in its neutral imidazole rather than its imidazolate protonation state. Thus, the active site of the heme-heme oxygenase complex has a myoglobin-like structure rather than an active site similar to the large cytochrome P-450 class of monooxygenases. As a consequence, the activated form of the heme-heme oxygenase complex, a peroxo intermediate, is different from that of the cytochrome P-450 monooxygenases, in which the activated form is an oxo intermediate. The overall catalytic mechanism is probably more closely related to that of other monooxygenases with myoglobin-like active sites, such as secondary amine monooxygenase.

Intrasubstituent isotope effect studies of oxidative N-demethylations catalyzed by secondary amine monooxygenase. Comparison to cytochrome P-450

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTIntrasubstituent isotope effect studies of oxidative N-demethylations catalyzed by secondary amine monooxygenase. Comparison to cytochrome P-450Barton K. Hawkins and John H. DawsonCite this: J. Am. Chem. Soc. 1992, 114, 9, 3547–3549Publication Date (Print):April 1, 1992Publication History Published online1 May 2002Published inissue 1 April 1992https://doi.org/10.1021/ja00035a059RIGHTS & PERMISSIONSArticle Views144Altmetric-Citations11LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (423 KB) Get e-Alerts Get e-Alerts

Secondary amine mono-oxygenase: Looks like myoglobin, acts like cytochrome P-450. Mechanistic studies of oxidative N-demethylation.

Secondary amine mono-oxygenase: Looks like myoglobin, acts like cytochrome P-450. Mechanistic studies of oxidative N-demethylation.

Spectroscopic Characterization of Secondary Amine Mono-oxygenase: Comparison to Cytochrome P-450 and Myoglobin

Abstract Secondary amine mono-oxygenase from Pseudomonas aminovorans catalyzes the NAD(P)H- and dioxygen-dependent N-dealkylation of secondary amines to yield a primary amine and an aldehyde. Heme iron, flavin, and non-heme iron prosthetic groups are known to be present in the oligomeric enzyme. The N-dealkylation reaction is also catalyzed by the only other heme-containing mono-oxygenase, cytochrome P-450. In order to identify the heme iron axial ligands of secondary amine mono-oxygenase so as to better define the structural requirements for oxygen activation by heme enzymes, we have investigated the spectroscopic properties of the enzyme. The application of three different spectroscopic techniques, UV-visible absorption, magnetic circular dichroism and electron paramagnetic resonance, to study eight separate enzyme derivatives has provided extensive and convincing evidence for the presence of a proximal histidine ligand. This conclusion is based primarily on comparisons of the spectral properties of the enzyme with those of parallel derivatives of myoglobin (histidine proximal ligand) and P-450 (cysteinate proximal ligand). Spectral studies of ferric secondary amine mono-oxygenase as a function of pH have led to the proposal that the distal ligand is water. Deprotonation of the distal water ligand occurs upon either raising the pH to 9.0 or substrate (dimethylamine) binding. In contrast, the deoxyferrous enzyme appears to have a weakly bound nitrogen donor distal ligand. Initial spectroscopic studies of the iron-sulfur units in the enzyme are interpreted in terms of a pair of Fe2S2 clusters. Secondary amine mono-oxygenase is unique in its ability to function as cytochrome P-450 in activating molecular oxygen but to do so with a myoglobin-like active site. As such, it provides an important system with which to probe structure-function relations in heme-containing oxygenases.

Secondary amine mono-oxygenase: Specificity and kinetic isotope effects

Secondary amine mono-oxygenase: Specificity and kinetic isotope effects

Probing Structure-Function Relations in Heme-Containing Oxygenases and Peroxidases

Structural factors that influence functional properties are examined in the case of four heme enzymes: cytochrome P-450, chloroperoxidase, horseradish peroxidase, and secondary amine mono-oxygenase. The identity of the axial ligand, the nature of the heme environment, and the steric accessibility of the heme iron and heme edge combine to play major roles in determining the reactivity of each enzyme. The importance of synthetic porphyrin models in understanding the properties of the protein-free metal center is emphasized. The conclusions described herein have been derived from studies at the interface between biological and inorganic chemistry.

Purification to homogeneity and initial physical characterization of secondary amine monooxygenase.

Secondary amine monooxygenase from Pseudomonas aminovorans grown on trimethylamine has been purified 265-fold to apparent homogeneity. The purified enzyme exhibits a specific activity of 14.7 mumol of NADPH oxidized per min per mg of protein, a native molecular weight of 210,000, and nondisulfide-linked subunits of molecular weight 42,000, 36,000, and 24,000, each of which is required for activity. The enzyme is extremely labile during purification; rapid handling and the presence of 5% ethanol are essential to enzyme stability. Storage at 77 K in the presence of NADH (1 mM) also confers considerable stability to the purified enzyme. The heme prosthetic group in the enzyme has been identified as protoporphyrin IX. The quantification of prosthetic group components reveals the presence of 1.6 mol of flavin as FMN, 2.0 mol of heme iron, 4.0 mol of acid-soluble (nonheme) iron, and 3.6 mol of free sulfide/210,000 molecular weight enzyme. Ferric and ferrous-CO secondary amine monooxygenase exhibit electronic absorption spectra that are very similar to those of analogous myoglobin derivatives and, therefore, quite distinct from parallel forms of cytochrome P-450, the most extensively studied heme iron-containing monooxygenase. Like myoglobin and, again, in contrast to P-450, this enzyme forms a very stable dioxygen complex. In fact, it is this oxygen-bound form of the enzyme that is obtained from the purification procedure. Once again, the absorption spectrum of oxygenated secondary amine monooxygenase is nearly identical to that of oxymyoglobin. The spectroscopic similarities between secondary amine monooxygenase and myoglobin suggest the presence of an endogenous histidine fifth ligand to the heme iron of the enzymes.

A steady-state kinetic study of the reaction catalysed by the secondary-amine mono-oxygenase of Pseudomonas aminovorans.

1. Secondary-amine mono-oxygenase (proposed EC group 1.14.99.-) was partially purified from trimethylamine-grown Pseudomonas aminovorans by (NH4)2SO4 fractionation, gel filtration, hydrophobic chromatography on 5-aminopentylamino-Sepharose, and affinity chromatography on Sepharose-bound NADH. 2. Some problems in the affinity-chromatography step are discussed. 3. A steady-state kinetic analysis varying substrate, oxygen and electron-donor concentrations was performed, which, over the concentration range studied, gave a series of families of approximately parallel double-reciprocal plots. From secondary and tertiary plots, Michaelis constants of 0.160 mM, 0.086 mM and 0.121 mM were obtained for dimethylamine, NADPH and oxygen respectively. 4. Product-inhibition studies supported the postulated Hexa Uni Ping Pong (triple-transfer) reaction mechanism.

Inhibition by carbon monoxide of the secondary-amine mono-oxygenase of Pseudomonas aminovorans and the photochemical action spectrum for its reversal.

The secondary-amine mono-oxygenase (EC 1.14.99.--) of Pseudomonas aminovorans is potently inhibited by carbon monoxide. The degree of inhibition of the purified enzyme was determined by the CO:O2 ratio rather than by the absolute concentration of carbon monoxide. The partition constant (the CO:O2 ratio causing 50% inhibition of activity) was 9.2 X 10(-4). The inhibition could be reversed by light, and the extent of reversal was proportional to the light intensity. With monochromatic light of wavelength 417 nm, the light sensitivity, L, was determined to be 2.5 X 10(8) cm2 min/mol quantum. The photochemical action spectrum for the light reversal of inhibition showed a single maximum of effectiveness at about 420 nm. The difference spectrum of the enzyme (reduced by NADH) on bubbling with CO (compared with an NADH-reduced reference sample) showed a peak at 426 nm. The preparations showed none of the spectral properties to cytochrome P-450 mono-oxygenase preparations, and was much more sensitive to carbon monoxide. The enzyme behaves as a typical o-type cytochrome (i.e. a carbon-monoxide-reactive b-type cytochrome), and its sensitivity to carbon monoxide as well as in its spectral properties, shows close resemblances to haemoglobin.

Microbial oxidation of amines. Partial purification of a trimethylamine mono-oxygenase from Pseudomonas aminovorans and its role in growth on trimethylamine.

1. A mono-oxygenase, which oxidizes trimethylamine and other tertiary amines bearing methyl or ethyl groups, was partially purified sixfold from Pseudomonas aminovorans grown on trimethylamine as sole carbon source. 2. The preferred electron donor was NADPH. The enzyme had a pH optimum of 8.0-9.4 for trimethylamine oxidation, and 8.8-9.2 for dimethylamine oxidation. 3. The oxidation product of trimethylamine was shown to be trimethylamine N-oxide. Other tertiary amines were probably also converted into N-oxides. 4. The enzyme also oxidized secondary amines. 5. The oxidation of trimethylamine was only slightly inhibited by CO and not at all by KCN or proadifen hydrochloride (SKF 525-A), but was inhibited by trimethylsulphonium chloride, tetramethylammonium chloride, 2,4-dichloro-6-phenylphenoxyethylamine (Lilly 53325) and its NN-diethyl derivative (Lilly 18947). 6. The oxidation of dimethylamine showed a similar response to inhibitors and a parallel loss in activity on heating at 35 degrees C. 7. The activities of the trimethylamine mono-oxygenase, trimethylamine N-oxide demethylase and the secondary-amine mono-oxygenase increased severalfold during adaptation of succinate-grown bacteria to growth on trimethylamine, and the trimethylamine mono-oxygenase was the first enzyme to show an increase in activity. It is concluded that all three enzymes are involved in growth on trimethylamine by this organism.

Primary amines as uncouplers of electron transport from hydroxylation in the secondary-amine mono-oxygenase system of Pseudomonas aminovorans

Methylamine, ethylamine and n-propylamine caused an oxidation of NADH in the presence of the partially-purified secondary-amine mono-oxygenase system of Pseudomonas aminovorans. The primary amine did not disappear and no hydroxylation product was formed, so it was concluded that primary amines were non-substrate effectors uncoupling NADH oxidation from hydroxylation. K m and V max values were determined for the three primary amines and compared with the values for the corresponding secondary amine substrates. Methylamine competitively inhibited the oxidation of dimethylamine to formaldehyde. It was not possible to establish whether H 2O 2 was formed since the enzyme preparation contained catalase. Substrates and non-substrate effectors were equally effective in causing conversion of a reduced-oxygenated intermediate form of the enzyme back to the original oxidized form.