Publications

• N-methyltryptophan oxidase from Escherichia coli: reaction kinetics with N-methyl amino acid and carbinolamine substrates.

  Khanna P., Jorns M.S.

  N-Methyltryptophan oxidase (MTOX), a flavoenzyme from Escherichia coli, catalyzes the oxidative demethylation of N-methyl-L-tryptophan (k(cat) = 4600 min(-1)). Other secondary amino acids (e.g., sarcosine) are oxidized at a slower rate. We have identified carbinolamines as a new class of alternate ... >> More

  N-Methyltryptophan oxidase (MTOX), a flavoenzyme from Escherichia coli, catalyzes the oxidative demethylation of N-methyl-L-tryptophan (k(cat) = 4600 min(-1)). Other secondary amino acids (e.g., sarcosine) are oxidized at a slower rate. We have identified carbinolamines as a new class of alternate substrate. MTOX oxidation of the carbinolamine formed with L-tryptophan and formaldehyde yields N-formyl-L-tryptophan in a relatively slow reaction that does not compete with turnover of MTOX with N-methyl-L-tryptophan. Double reciprocal plots with N-methyl-L-tryptophan as the varied substrate are nearly parallel, but the slopes show a small, systematic variation depending on the oxygen concentration. N-Benzylglycine, a dead-end competitive inhibitor with respect to N-methyl-L-tryptophan, acts as a noncompetitive inhibitor with respect to oxygen. The results are consistent with a modified ping pong mechanism where oxygen binds to the reduced enzyme prior to dissociation of the imino acid product. MTOX is converted to a 2-electron reduced form upon anaerobic reaction with N-methyl-L-tryptophan, sarcosine, or the carbinolamine formed with L-tryptophan and formaldehyde. No evidence for a detectable intermediate was obtained by monitoring the spectral course of the latter two reactions. MTOX reduction with thioglycolate does, however, proceed via a readily detectable anionic, flavin radical intermediate. The reductive half-reaction with sarcosine at 4 degrees C exhibits saturation kinetics (k(lim) = 6.8 min(-1), K = 39 mM) and other features consistent with a mechanism in which a nearly irreversible reduction step (E(ox).S --> E(red).P) (k(lim)) is preceded by a rapidly attained equilibrium (K) between free E and the E.S complex. The 21 degrees C temperature difference can reasonably account for the 3.6-fold lower value obtained for k(lim) as compared with turnover at 25 degrees C (k(cat) = 24.5 min(-1)), suggesting that sarcosine is oxidized at a kinetically significant rate under anaerobic conditions and the reductive half-reaction is rate-limiting during turnover. These conclusions are, however, difficult to reconcile with steady-state kinetic patterns obtained with sarcosine that are consistent with a rapid equilibrium ordered mechanism with oxygen as the first substrate. The basis for the apparent stability of the MTOX.oxygen complex (K(d) = 72 microM) is unknown. << Less

  Biochemistry 40:1451-1459(2001) [PubMed] [EuropePMC]

• The X-ray structure of N-methyltryptophan oxidase reveals the structural determinants of substrate specificity.

  Ilari A., Bonamore A., Franceschini S., Fiorillo A., Boffi A., Colotti G.

  The X-ray structure of monomeric N-methyltryptophan oxidase from Escherichia coli (MTOX) has been solved at 3.2 A resolution by molecular replacement methods using Bacillus sp. sarcosine oxidase structure (MSOX, 43% sequence identity) as search model. The analysis of the substrate binding site hig ... >> More

  The X-ray structure of monomeric N-methyltryptophan oxidase from Escherichia coli (MTOX) has been solved at 3.2 A resolution by molecular replacement methods using Bacillus sp. sarcosine oxidase structure (MSOX, 43% sequence identity) as search model. The analysis of the substrate binding site highlights the structural determinants that favour the accommodation of the bulky N-methyltryptophan residue in MTOX. In fact, although the nature and geometry of the catalytic residues within the first contact shell of the FAD moiety appear to be virtually superposable in MTOX and MSOX, the presence of a Thr residue in position 239 in MTOX (Met245 in MSOX) located at the entrance of the active site appears to play a key role for the recognition of the amino acid substrate side chain. Accordingly, a 15 fold increase in k(cat) and 100 fold decrease in K(m) for sarcosine as substrate has been achieved in MTOX upon T239M mutation, with a concomitant three-fold decrease in activity towards N-methyltryptophan. These data provide clear evidence for the presence of a catalytic core, common to the members of the methylaminoacid oxidase subfamily, and of a side chain recognition pocket, located at the entrance of the active site, that can be adjusted to host diverse aminoacids in the different enzyme species. The site involved in the covalent attachment of flavin has also been addressed by screening degenerate mutants in the relevant positions around Cys308-FAD linkage. Lys341 appears to be the key residue involved in flavin incorporation and covalent linkage. << Less

  Proteins 71:2065-2075(2008) [PubMed] [EuropePMC]

• Pleiotropic impact of a single lysine mutation on biosynthesis of and catalysis by N-methyltryptophan oxidase.

  Bruckner R.C., Winans J., Jorns M.S.

  N-Methyltryptophan oxidase (MTOX) contains covalently bound FAD. N-Methyltryptophan binds in a cavity above the re face of the flavin ring. Lys259 is located above the opposite, si face. Replacement of Lys259 with Gln, Ala, or Met blocks (>95%) covalent flavin incorporation in vivo. The mutant apo ... >> More

  N-Methyltryptophan oxidase (MTOX) contains covalently bound FAD. N-Methyltryptophan binds in a cavity above the re face of the flavin ring. Lys259 is located above the opposite, si face. Replacement of Lys259 with Gln, Ala, or Met blocks (>95%) covalent flavin incorporation in vivo. The mutant apoproteins can be reconstituted with FAD. Apparent turnover rates (k(cat,app)) of the reconstituted enzymes are ~2500-fold slower than those of wild-type MTOX. Wild-type MTOX forms a charge-transfer E(ox)·S complex with the redox-active anionic form of NMT. The E(ox)·S complex formed with Lys259Gln does not exhibit a charge-transfer band and is converted to a reduced enzyme·imine complex (EH(2)·P) at a rate 60-fold slower than that of wild-type MTOX. The mutant EH(2)·P complex contains the imine zwitterion and exhibits a charge-transfer band, a feature not observed with the wild-type EH(2)·P complex. Reaction of reduced Lys259Gln with oxygen is 2500-fold slower than that of reduced wild-type MTOX. The latter reaction is unaffected by the presence of bound product. Dissociation of the wild-type EH(2)·P complex is 80-fold slower than k(cat). The mutant EH(2)·P complex dissociates 15-fold faster than k(cat,app). Consequently, EH(2)·P and free EH(2) are the species that react with oxygen during turnover of the wild-type and mutant enzyme, respectively. The results show that (i) Lys259 is the site of oxygen activation in MTOX and also plays a role in holoenzyme biosynthesis and N-methyltryptophan oxidation and (ii) MTOX contains separate active sites for N-methyltryptophan oxidation and oxygen reduction on opposite faces of the flavin ring. << Less

  Biochemistry 50:4949-4962(2011) [PubMed] [EuropePMC]

• Nucleotide sequence of the Escherichia coli solA gene encoding a sarcosine oxidase-like protein and characterization of its product.

  Koyama Y., Ohmori H.

  An Escherichia coli gene, designated solA, was identified between pyrC and htrB around 24.2 min of the genetic map. The solA gene product (SolA, 372 aa) shows strong similarities (at most 42% identities over the entire length) to monomeric sarcosine (N-methylglycine) oxidases from other bacteria. ... >> More

  An Escherichia coli gene, designated solA, was identified between pyrC and htrB around 24.2 min of the genetic map. The solA gene product (SolA, 372 aa) shows strong similarities (at most 42% identities over the entire length) to monomeric sarcosine (N-methylglycine) oxidases from other bacteria. However, only low levels of sarcosine oxidase activity were detected, even when SolA was overproduced. The SolA extracts exhibited much higher (about 200-fold) reactivity toward N-methyltryptophan than toward sarcosine. Thus, SolA appears to have a substrate specificity distinct from that of monomeric sarcosine oxidases, in spite of the strong similarities in the primary structure with such enzymes. << Less

  Gene 181:179-183(1996) [PubMed] [EuropePMC]