Structure of the Flavocoenzyme of Two Homologous Amine Oxidases:  Monomeric Sarcosine Oxidase and <i>N</i>-Methyltryptophan Oxidase

Wagner, Mary Ann; Khanna, Peeyush; Jörns, Marilyn Schuman

doi:10.1021/bi982955o

Cited by 72 publications

(189 citation statements)

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“…Extinction coefficients were determined after enzyme denaturation with guanidine hydrochloride, as previously described (1). Spectral titrations with the Tyr317Phe mutant and methylthioacetate (MTA) or pyrrole-2-carboxylate (PCA) were conducted at 25 °C in 50 mM potassium phosphate buffer, pH 8.0.…”

Section: Spectroscopy and Data Analysismentioning

confidence: 99%

Ionization of Zwitterionic Amine Substrates Bound to Monomeric Sarcosine Oxidase

Zhao

Jörns

2005

Biochemistry

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Monomeric sarcosine oxidase (MSOX) binds the L-proline zwitterion (pK a = 10.6). The reactive substrate anion is generated by ionization of the ES complex (pK a = 8.0). Tyr317 was mutated to Phe to determine whether this step might involve proton transfer to an active site base. The mutation does not eliminate the ionizable group in the ES complex (pK a = 8.9) but does cause a 20-fold decrease in the maximum rate of the reductive half-reaction. Kinetically determined K d values for the ES complex formed with L-proline agree with results obtained in spectral titrations with wild type or mutant enzyme. Unlike wild type enzyme, K d values with the mutant enzyme are pH-dependent, suggesting that the mutation has perturbed the pK a of a group that affects the K d . As compared with wild type enzyme, an increase in charge transfer band energy is observed for mutant enzyme complexes with substrate analogs while a 10-fold decrease in the charge transfer band extinction coefficient is found for the complex with L-proline anion. The results eliminate Tyr317 as a possible acceptor of the proton released upon substrate ionization. Since previous studies rule out the only other nearby base, we conclude that L-proline is the ionizable group in the ES complex and that amino acids are activated for oxidation upon binding to MSOX by stabilization of the reactive substrate anion. Tyr317 may play a role in substrate activation and optimizing binding, as judged by the effects of its mutation on the observed pK a , reaction rates and charge transfer bands.Monomeric sarcosine oxidase (MSOX) 1 is a flavoprotein that contains covalently bound FAD [8a-(S-cysteinyl)FAD] (1). The enzyme catalyzes the oxidation of the methyl group in sarcosine (N-methylglycine) and the equivalent C-N bond in other secondary amino acids, such as L-proline. The crystal structure of free MSOX from Bacillus sp. B-0618 and complexes of the enzyme with various inhibitors have been determined (2-4). MSOX is a two-domain, 46 KDa protein with an overall topology similar to D-amino acid oxidase.MSOX is a member of a growing family of enzymes that contain covalently bound flavin and catalyze similar oxidation reactions with different amine substrates (5-8). Despite considerable attention, important questions regarding the mechanism of flavin-dependent amine oxidation reactions remain unresolved. Postulated mechanisms differ with respect to: 1) the requirement for an active site base; 2) the acceptor of the α-hydrogen removed from the carbon atom in the C-N bond undergoing oxidation; 3) geometric constraints on the orientation of flavin, substrate and a putative active site base (Scheme 1). In the hydride transfer mechanism, flavin N(5) acts as the acceptor of the α-hydrogen in a reaction that does not require an active site base. Single electron transfer (SET) mechanisms are initiated by electron transfer from substrate amino

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Section: Spectroscopy and Data Analysismentioning

confidence: 99%

Ionization of Zwitterionic Amine Substrates Bound to Monomeric Sarcosine Oxidase

Zhao

Jörns

2005

Biochemistry

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“…Control incubations showed that polyethylene glycol precipitation did not significantly decrease the catalytic efficiency or flavin content and recovered yields were 80 to 90%. Measurements showed that the enzyme-bound flavin absorbance coefficient for both FMO3 and FMO5 enzymes did not differ significantly from that of free FAD in the presence of guanidine hydrochloride (11,900 M Ϫ1 cm Ϫ1 ) (Wagner et al, 1999). This value was therefore used for determinations of the enzyme concentration under nondenaturing conditions.…”

Section: Methodsmentioning

confidence: 99%

“…The concentration of MBP-FMO-bound FAD was determined with absorbance at 450 nm as described previously for other flavin-containing enzymes (Wagner et al, 1999) with the following modification. To remove nonspecifically bound flavin from the samples, the MBP-FMO proteins were first precipitated using 20% PEG-8000 as described previously (Brunelle et al, 1997), and the supernatant was discarded.…”

Section: Methodsmentioning

confidence: 99%

Characterization of Human Flavin-Containing Monooxygenase (FMO) 3 and FMO5 Expressed as Maltose-Binding Protein Fusions

Reddy¹,

Ralph²,

Motika³

et al. 2010

Drug Metab Dispos

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ABSTRACT:The flavin-containing monooxygenase (FMO) family of enzymes oxygenates nucleophilic xenobiotics and endogenous substances. Human FMO3 and FMO5 are the predominant FMO forms in adult liver. These enzymes are naturally membrane-bound, and recombinant proteins are commercially available as microsomal preparations from insect cells (i.e., Supersome FMO). As an alternative, FMO3 has previously been expressed as a soluble protein, through use of an N-terminal maltosebinding protein (MBP) fusion. In the current study, MBP fusions of both human FMO3 and FMO5 were prepared to >90% purity in the presence of detergent and characterized for biochemical and kinetic parameters, and the parameters were compared with those of Supersome FMO samples. Although MBP-FMO enzymes afforded lower rates of turnover than the corresponding Supersome FMOs, both types of FMO showed identical substrate dependencies and similar responses to changes in assay conditions. Of interest, the FMO3 enzymes showed a 2-fold activation of k cat /K m in the presence of Triton X-100. Oligomeric analysis of MBP-FMO3 also showed disassociation from a high-order oligomeric form to a monomeric status in the presence of Triton X-100. This report serves as the first direct comparison between Supersome FMOs and the corresponding MBP fusions and the first report of a detergent-based activation of k cat /K m that corresponds to changes in oligomerization.

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“…This family is mainly composed of considerably smaller, monomeric proteins (~44 kDa) that contain covalently bound FAD as the only prosthetic group and exhibit modest sequence homology (~20% identity) with the β subunit of TSOX. Members of this family include monomeric sarcosine oxidase (MSOX), N-methyltryptophan oxidase (MTOX), pipecolate oxidase and nikD [11][12][13][14][15][16][17][18] . Crystal structures have been determined for MSOX 11, 14 , a monofunctional enzyme that exhibits sarcosine oxidase but not 5,10-CH 2 -H 4 folate synthase activity 5 .…”

Section: Introductionmentioning

confidence: 99%

Identification of a Stable Flavin-thiolate Adduct in Heterotetrameric Sarcosine Oxidase

Hynson

Mathews

Jörns

2006

Journal of Molecular Biology

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SummaryHeterotetrameric sarcosine oxidase (TSOX) is a complex bifunctional flavoenzyme that contains two flavins. Most of the FMN in recombinant TSOX is present as a covalent adduct with an endogenous ligand. Enzyme denaturation disrupts the adduct, accompanied by release of a stoichiometric amount of sulfide. Enzyme containing ≥ 90% unmodified FMN is prepared by displacement of the endogenous ligand with sulfite, a less tightly bound competing ligand. Reaction of adduct-depleted TSOX with sodium sulfide produces a stable complex that resembled the endogenous TSOX adduct and known 4a-S-cysteinyl flavin adducts. The results provide definitive evidence for sulfide as the endogenous TSOX ligand and strongly suggest that the modified FMN is a 4a-sulfide adduct. A comparable reaction with sodium sulfide is not detected with other flavoprotein oxidases. A model of the postulated TSOX adduct suggests that it is stabilized by nearby residues that may be important in the electron transferase/oxidase function of the coenzyme.

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Structure of the Flavocoenzyme of Two Homologous Amine Oxidases: Monomeric Sarcosine Oxidase and N-Methyltryptophan Oxidase

Cited by 72 publications

References 40 publications

Ionization of Zwitterionic Amine Substrates Bound to Monomeric Sarcosine Oxidase

Ionization of Zwitterionic Amine Substrates Bound to Monomeric Sarcosine Oxidase

Characterization of Human Flavin-Containing Monooxygenase (FMO) 3 and FMO5 Expressed as Maltose-Binding Protein Fusions

Identification of a Stable Flavin-thiolate Adduct in Heterotetrameric Sarcosine Oxidase

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