DMGO

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Abstract

Mammalian sarcosine dehydrogenase (SDH) and dimethylglycine dehydrogenase (DMGDH) are mitochondrial enzymes involved in choline and 1-carbon metabolism. SDH plays an important role in the glycine/sarcosine regulatory cycle, and its deficiency results in sarcosinemia, a rare autosomal metabolic defect manifested by elevated blood and urine sarcosine levels. DMGDH deficiency leads to increased levels of N, N-dimethylglycine in the blood. These highly related enzymes bind covalently to FAD and catalyze the oxidation of sarcosine and dimethylglycine to the corresponding products glycine and sarcosine, respectively, in the reductive half-reaction. In the absence of tetrahydrofolate (THF), formaldehyde is the second product catalyzed, formed by hydrolysis of a labile imine intermediate. Electron-transferring flavoprotein (ETF) links the activities of SDH and DMGDH to the respiratory redox chain in an oxidative half-reaction that completes the catalytic cycle.

The primary structures of these enzymes show the presence of two active sites located in distinct regions of the protein. A nucleotide-binding motif at the N-terminus of SDH and DMGDH suggests that they are more distantly related to bacterial monomeric sarcosine oxidase (MSOX). The C-terminal sequence is highly similar to that of the T protein (an aminomethyltransferase) of the glycine cleavage system. Sarcosine or betaine is a common source of carbon and energy for many microorganisms, and glycine betaine is also an osmoprotectant.

Dimethylglycine oxidase (DMGO) from Arthrobacter globiformis is part of the betaine catabolic pathway and is 30% identical to mammalian SDH and DMGDH. DMGO uses molecular oxygen instead of ETF in the oxidative half-reaction, but like SDH and DMGDH, substrate oxidation is associated with the formation of 5,10-methylene-THF or formaldehyde, depending on the availability of THF.

Overall structure of DMGO

The 1.60 Å crystal structure of DMGO was determined by MIRAS approach. The enzyme was observed to be a dimer of tightly packed dimers, with the four monomers related by 222 crystallographic point symmetry. Consistent with protein sequence analysis, each monomer contained two functionally distinct regions. The N-terminal region responsible for dimethylglycine oxidation contains residues 1-420. The C-terminal region (residues 421-830) contains the second active site responsible for the formation of 5,10-methylene-THF.

Figure 1. Stereo view of a DMGO monomer (Leys, D.; et al. 2003)

Quaternary structure

The 222 point symmetry of the crystal is consistent with the internal symmetry of the tetramer. The entire structure resembles a flattened tetrahedron with dimensions of about 120×120×70Å. The FAD domain occupies the four corners and the C-terminal domain is located in the center of the tetrahedron. The interfaces between a monomer and two crystallographically related molecules are very different. The strongest interaction occurs with the -x, y, -z symmetry-related monomers and consists almost entirely of the folate-binding domain. The shape complementarity (SC) value obtained with the 1.7 Å probe is 0.76. Analytical gel filtration experiments indicated that the size of the protein was slightly larger than predicted for a homodimer, possibly due to the dissociation of the protein into dimers at low protein concentrations. There is currently no indication that DMGO has any allosteric effect, and tetramerization may be associated with improved enzyme stability. Despite high overall sequence identity, none of the interface contact residues in DMGO is conserved among mammalian dehydrogenases, suggesting that quaternary organization may differ among mammalian enzymes.

Figure 2. Structure of the DMGO tetramer (Leys, D.; et al. 2003)

Folate-binding region

The three domains contained in the C-terminal folate-binding region are located around the central hole in a cloverleaf-like arrangement. No significant structural homology with known structures was found. Domain 1 (residues 469-507 and 600-689) is a six-stranded antiparallel β-sheet that is packed on one side by several α-helices. It is preceded by an irregular structure consisting of several small secondary elements.  Domain 1 is interrupted by a single excursion of the polypeptide chain, forming a five-stranded antiparallel β-sheet, domain 2 (residues 508-599). Domains 1 and 2 have similar topology and folds. C-terminal domain 3 (residues 743-830) is a distorted six-stranded jelly roll, packed vertically with the β-sheets of domains 1 and 2.

Figure 3. Stereo view of the N5, N10-methylene-THF synthase domain (Leys, D.; et al. 2003)