Characterization of the FAD-Containing N-Methyltryptophan Oxidase from Escherichia coli

Abstract

N-Methyltryptophan oxidase (MTOX) is a flavoenzyme that catalyzes the oxidative demethylation of N-methyl-L-tryptophan and other N-methyl amino acids, including sarcosine, which is a poor substrate. The Escherichia coli gene encoding MTOX (solA) was isolated on the basis of its sequence homology with monomeric sarcosine oxidase, a sarcosine-inducible enzyme found in many bacteria. These studies show that MTOX is expressed as a constitutive enzyme in a wild-type E. coli K-12 strain, providing the first evidence that solA is a functional gene. MTOX expression is enhanced 3-fold by growth on minimal media but not induced by N-methyl-L-tryptophan, L-tryptophan, or 3-indoleacrylate. MTOX forms an anionic flavin semiquinone and a reversible, covalent flavin-sulfite complex (K(d) = 1.7 mM), properties characteristic of flavoprotein oxidases. Rates of formation (k(on) = 5.4 x 10(-3) M(-1) s(-1)) and dissociation (k(off) = 1.3 x 10(-5) s(-1)) of the MTOX-sulfite complex are orders of magnitude slower than observed with most other flavoprotein oxidases. The pK(a) for ionization of oxidized FAD at N(3)H in MTOX (8.36) is two pH units lower than that observed for free FAD. The MTOX active site was probed by characterization of various substrate analogues that act as competitive inhibitors with respect to N-methyl-L-tryptophan. Qualitatively similar perturbations of the MTOX visible absorption spectrum are observed for complexes formed with various aromatic carboxylates, including benzoate, 3-indole-(CH(2))(n)-CO(2)(-) and 2-indole-CO(2)(-). The most stable complex with 3-indole-(CH(2))(n)-CO(2)(-) is formed with 3-indolepropionate (K(d) = 0.79 mM), a derivative with the same side chain length as N-methyl-L-tryptophan. Benzoate binding is enhanced upon protonation of a group in the enzyme-benzoate complex (pK(EL) = 6.87) but blocked by ionization of a group in the free enzyme (pK(E) = 8.41), which is attributed to N(3)H of FAD. Difference spectra observed for the aromatic carboxylate complexes are virtually mirror images of those observed with sarcosine analogues (N,N'-dimethylglycine, N-benzylglycine). Charge-transfer complexes are formed with 3-indoleacrylate, pyrrole-2-carboxylate, and CH(3)XCH(2)CO(2)(-) (X = S, Se, Te).

An Internal Reaction Chamber in Dimethylglycine Oxidase Provides Efficient Protection from Exposure to Toxic Formaldehyde

“…Direct intracellular enzymatic formaldehyde release can be induced in E. coli by providing N-MeTrp in the growth medium. This serves as substrate for MTOX, a constitutively expressed E. coli enzyme homologous to the N-terminal amine oxidase domain of DMGO (41,42). Unlike DMGO, MTOX does not contain (or is not associated with) 5,10-CH 2 -THF synthase activity, and oxygen-dependent N-MeTrp oxidation results in the formation of Trp and formaldehyde.…”

“…3B). The expression of MTOX is enhanced by the presence of N-MeTrp (42), and the fluorescence values we obtained were therefore normalized versus total MTOX enzyme activity in the corresponding cellular extracts. We demonstrated that a response to intracellular formaldehyde formation is obtained from levels as low as 0.1 M N-MeTrp with a nonlinear increase in the fluorescence per unit of enzyme activity obtained within the range of N-MeTrp tested.…”

Characterization and Metabolic Function of a Peroxisomal Sarcosine and Pipecolate Oxidase from Arabidopsis

“…Use of Sarcosine As Sole Nitrogen Source by E. coli Expressing AtSOX-Wild type E. coli uses glycine as the sole nitrogen source (34) but is not expected to use sarcosine as efficiently because of low endogenous SOX activity (35). Consistent with this prediction, growth of wild type cells on plates was poor when sarcosine replaced glycine as the nitrogen source ( Fig.…”

“…Import Reactions-In vitro protein import reactions (final volume 200 l) were initiated by adding 35 S-labeled glycolate oxidase or AtSOX (5 ϫ 10 5 cpm trichloroacetic acid-precipitable protein) to 350 g of glyoxysomes in the presence of import buffer (25 mM Mes-KOH, pH 6.0, 0.5 M sucrose, 10 mM KCl, 1 mM MgCl 2 , and 5 mM MgATP). The reaction temperature was 26°C unless otherwise noted.…”

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

“…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 .…”