Publications

• An internal reaction chamber in dimethylglycine oxidase provides efficient protection from exposure to toxic formaldehyde.

  Tralau T., Lafite P., Levy C., Combe J.P., Scrutton N.S., Leys D.

  We report a synthetic biology approach to demonstrate substrate channeling in an unusual bifunctional flavoprotein dimethylglycine oxidase. The catabolism of dimethylglycine through methyl group oxidation can potentially liberate toxic formaldehyde, a problem common to many amine oxidases and dehy ... >> More

  We report a synthetic biology approach to demonstrate substrate channeling in an unusual bifunctional flavoprotein dimethylglycine oxidase. The catabolism of dimethylglycine through methyl group oxidation can potentially liberate toxic formaldehyde, a problem common to many amine oxidases and dehydrogenases. Using a novel synthetic in vivo reporter system for cellular formaldehyde, we found that the oxidation of dimethylglycine is coupled to the synthesis of 5,10-methylenetetrahydrofolate through an unusual substrate channeling mechanism. We also showed that uncoupling of the active sites could be achieved by mutagenesis or deletion of the 5,10-methylenetetrahydrofolate synthase site and that this leads to accumulation of intracellular formaldehyde. Channeling occurs by nonbiased diffusion of the labile intermediate through a large solvent cavity connecting both active sites. This central "reaction chamber" is created by a modular protein architecture that appears primitive when compared with the sophisticated design of other paradigm substrate-channeling enzymes. The evolutionary origins of the latter were likely similar to dimethylglycine oxidase. This work demonstrates the utility of synthetic biology approaches to the study of enzyme mechanisms in vivo and points to novel channeling mechanisms that protect the cell milieu from potentially toxic reaction products. << Less

  J. Biol. Chem. 284:17826-17834(2009) [PubMed] [EuropePMC]

• Life support training in hospital dental residencies.

  Becker D.E.

  J Hosp Dent Pract 14:111-111(1980) [PubMed] [EuropePMC]

• Mechanistic aspects of the covalent flavoprotein dimethylglycine oxidase of Arthrobacter globiformis studied by stopped-flow spectrophotometry.

  Basran J., Bhanji N., Basran A., Nietlispach D., Mistry S., Meskys R., Scrutton N.S.

  Dimethylglycine oxidase (DMGO) is a covalent flavoenzyme from Arthrobacter globiformis that catalyzes the oxidative demethylation of dimethylglycine to yield sarcosine, formaldehyde, and hydrogen peroxide. Stopped-flow and steady-state kinetic studies have been used to study the reductive and oxid ... >> More

  Dimethylglycine oxidase (DMGO) is a covalent flavoenzyme from Arthrobacter globiformis that catalyzes the oxidative demethylation of dimethylglycine to yield sarcosine, formaldehyde, and hydrogen peroxide. Stopped-flow and steady-state kinetic studies have been used to study the reductive and oxidative half-reactions using dimethylglycine and O2 as substrates. The reductive half-reaction is triphasic. The rate of the fast phase is dependent on substrate concentration, involves flavin reduction, and has a limiting rate constant of 244 s(-1). This phase also displays a kinetic isotope effect of 2.9. Completion of the first kinetic phase generates an intermediate with broad spectral signature between 350 and 500 nm, which is attributed to a reduced enzyme-iminium charge-transfer species, similar to the purple intermediate that accumulates in reactions of D-amino acid oxidase (DAAO) with alanine. The second phase (16 s(-1)) is independent of substrate concentration and is attributed to iminium hydrolysis/deprotonation. The third phase (2 s(-1)) is attributed to product release, the rate of which is less than the steady-state turnover rate (10.6 s(-1)). Flavin oxidation of dithionite- and dimethylglycine-reduced enzyme by O2 occurs in a single phase, and the rate shows a linear dependence on oxygen concentration, giving bimolecular rate constants of 342 and 201 mM(-1) x s(-1), respectively. Enzyme-monitored turnover experiments indicate that decay of the reduced enzyme-iminium intermediate is rate-limiting, consistent with rate constants determined from single turnover studies. A minimal kinetic mechanism is presented, which establishes a close relationship to the mechanism of action of DAAO. The covalent flavin in dimethylglycine oxidase is identified as an alphaN1-histidyl48-FAD, and equilibrium titration studies establish a single redox center that displays typical flavoprotein 'oxidase' characteristics. << Less

  Biochemistry 41:4733-4743(2002) [PubMed] [EuropePMC]

• Studies of dimethylglycine oxidase isoenzymes in Arthrobacter globiformis cells.

  Casaite V., Poviloniene S., Meskiene R., Rutkiene R., Meskys R.

  Glycine betaine (GB) could be used by Arthrobacter globiformis cells as a sole carbon source. The cells took up this molecule in the low as well as in the high salinity medium. Addition of GB to the mineral medium with high salt concentration revealed that GB was also used as an osmoprotectant. Di ... >> More

  Glycine betaine (GB) could be used by Arthrobacter globiformis cells as a sole carbon source. The cells took up this molecule in the low as well as in the high salinity medium. Addition of GB to the mineral medium with high salt concentration revealed that GB was also used as an osmoprotectant. Dimethylglycine oxidase (DMGO) was involved in the catabolism of GB. Two genes for DMGO were detected in a cloned 26267 bp fragment of A. globiformis DNA. The genes involved in the tetrahydrofolate-dependent assimilation of methyl groups were located nearby the two of DMGO genes. Both cloned A. globiformis DMGO were active. The activity of DMGO was detected in A. globiformis cells and it depended on the addition of GB and the salinity of the medium. Reverse transcription-PCR demonstrated that the addition of GB influenced the transcription of dmg genes. << Less

  Curr Microbiol 62:1267-1273(2011) [PubMed] [EuropePMC]

• Channelling and formation of 'active' formaldehyde in dimethylglycine oxidase.

  Leys D., Basran J., Scrutton N.S.

  Here we report crystal structures of dimethylglycine oxidase (DMGO) from the bacterium Arthrobacter globiformis, a bifunctional enzyme that catalyzes the oxidation of N,N-dimethyl glycine and the formation of 5,10-methylene tetrahydrofolate. The N-terminal region binds FAD covalently and oxidizes ... >> More

  Here we report crystal structures of dimethylglycine oxidase (DMGO) from the bacterium Arthrobacter globiformis, a bifunctional enzyme that catalyzes the oxidation of N,N-dimethyl glycine and the formation of 5,10-methylene tetrahydrofolate. The N-terminal region binds FAD covalently and oxidizes dimethylglycine to a labile iminium intermediate. The C-terminal region binds tetrahydrofolate, comprises three domains arranged in a ring-like structure and is related to the T-protein of the glycine cleavage system. The complex with folinic acid indicates that this enzyme selectively activates the N10 amino group for initial attack on the substrate. Dead-end reactions with oxidized folate are avoided by the strict stereochemical constraints imposed by the folate-binding funnel. The active sites in DMGO are approximately 40 A apart, connected by a large irregular internal cavity. The tetrahydrofolate-binding funnel serves as a transient entry-exit port, and access to the internal cavity is controlled kinetically by tetrahydrofolate binding. The internal cavity enables sequestration of the reactive iminium intermediate prior to reaction with tetrahydrofolate and avoids formation of toxic formaldehyde. This mode of channelling in DMGO is distinct from other channelling mechanisms. << Less

  EMBO J. 22:4038-4048(2003) [PubMed] [EuropePMC]

• Organization of the genes involved in dimethylglycine and sarcosine degradation in Arthrobacter spp.: implications for glycine betaine catabolism.

  Meskys R., Harris R.J., Casaite V., Basran J., Scrutton N.S.

  The nucleotide sequences of two cloned DNA fragments containing the structural genes of heterotetrameric sarcosine oxidase (soxBDAG) and dimethylglycine dehydrogenase (dmg) from Arthrobater spp. 1-IN and Arthrobacter globiformis, respectively, have been determined. Open reading frames were identif ... >> More

  The nucleotide sequences of two cloned DNA fragments containing the structural genes of heterotetrameric sarcosine oxidase (soxBDAG) and dimethylglycine dehydrogenase (dmg) from Arthrobater spp. 1-IN and Arthrobacter globiformis, respectively, have been determined. Open reading frames were identified in the soxBDAG operon corresponding to the four subunits of heterotetrameric sarcosine oxidase by comparison with the N-terminal amino-acid sequences and the subunit relative molecular masses of the purified enzyme. Alignment of the deduced sarcosine oxidase amino-acid sequence with amino-acid sequences of functionally related proteins indicated that the arthrobacterial enzyme is highly homologous to sarcosine oxidase from Corynebacterium P-1. Deletion and expression analysis, and alignment of the deduced amino-acid sequence of the dmg gene, showed that dmg encodes a novel dimethylglycine oxidase, which is related to eukaryotic dimethylglycine dehydrogenase, and contains nucleotide-binding, flavinylation and folate-binding motifs. The recombinant dimethylglycine oxidase was purified to homogeneity and characterized. The DNA located upstream and downstream of both the soxBDAG and dmg genes is predicted to encode enzymes involved in the tetrahydrofolate-dependent assimilation of methyl groups. Based on the sequence analysis reported herein, pathways are proposed for glycine betaine catabolism in Arthrobacter species, which involve the identified folate-dependent enzymes. << Less

  Eur. J. Biochem. 268:3390-3398(2001) [PubMed] [EuropePMC]