Channelling and formation of 'active' formaldehyde in dimethylglycine oxidase

Abstract

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~40 A Ê 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.

Structure of arylO-demethylase offers molecular insight into a catalytic tyrosine-dependent mechanism

“…Tm_T shares 23% sequence identity with LigM, aligning with an rmsd of 2.4 Å over its 324-residue H 4 folate-binding fold. Similarly, the top 50 structural homologs align to LigM strictly through their respective folate-binding domains with rmsds ranging from 2.4 to 3.2 Å. Interestingly, LigM's aminomethyltransferase homologs catalyze a different reaction involving the production of 5,10-methylene-H 4 folate (20,21), and this difference in end products may explain why LigM shares such low overall sequence identity with its homologs (20-27%) despite sharing significant structural similarity.…”

Extensive Domain Motion and Electron Transfer in the Human Electron Transferring Flavoprotein·Medium Chain Acyl-CoA Dehydrogenase Complex

“…Although the primary dehydrogenases involved in fatty acid breakdown and amino acid catabolism share a common evolutionary origin and thereby retain highly similar structural features (4), the flavin dehydrogenases involved in 1-carbon metabolism are structurally and evolutionarily distinct enzymes (5). In addition, the membrane-bound ETF-ubiquinone oxidoreductase is unrelated to either of these dehydrogenase families.…”

Structures of dimethylsulfoniopropionate‐dependent demethylase from the marine organism Pelagabacter ubique

“…31,32 This further supports the conclusion that the reaction mechanism for DmdA is substantially different from that of other enzymes with a similar THF binding fold. 14,16 Considering the observations above, the accessibility water has to the active site, the number of acidic side chains in the active site (E63, D108, and E204), and the binding mode of DMSP relative to THF, all of the available evidence points to a concerted mechanism in which methyl transfer is coupled to proton transfer. This would be a concerted reaction, much like what has been proposed for S-adenosylmethionine (SAM)-dependent N-methyltransferases.…”

“…2). The arrangement of these domains and the identification of this overall fold as a prototype for THF binding has been uncovered within the last decade 14,28 and essentially comprises the cloverleaf-like positioning of these three domains. Despite the low sequence identity of 25% or less seen in Figure 1, the fold is conserved between these three enzymes.…”

“…6 Structures for most of the latter enzymes are available in the PDB, in both the apo form and with various ligands bound. [14][15][16][17] Although the residues of identity vary for each comparison, the sequence identity for the clycine cleavage system T-protein, dimethylglycine oxidase, and sarcosine oxidase is 26, 24, and 22%, respectively. As expected, many of the residues that are conserved are also involved in binding THF (see Fig.…”