Discovery of sarcosine dimethylglycine methyltransferase from Galdieria sulphuraria

Abstract

An enzyme with sarcosine dimethylglycine methyltransferase (SDMT) activity has been identified in the thermophilic eukaryote, Galdieria sulphuraria. The crystal structure of the enzyme, solved to a resolution of 1.95 A, revealed a fold highly similar to that of mycolic acid synthases. The kcat and apparent K(M) values were 64.3 min(-1) and 2.0 mM for sarcosine and 85.6 min(-1) and 2.8 mM for dimethylglycine, respectively. Apparent K(M) values of S-adenosylmethionine were 144 and 150 microM for sarcosine and dimethylglycine, respectively, and the enzyme melting temperature was 61.1 degrees C. Modeling of cofactor binding in the active site based on the structure of methoxy mycolic acid synthase 2 revealed a number of conserved interactions within the active site.

Figure 1

Betaine synthesis. Reactions carried out by sarcosine dimethylglycine methyltransferase are bordered by black lines.

Figure 2

A cartoon representation of the fold of a monomer of GsSDMT. The color scheme changes from blue to green to represent regions predicted to interact with the SAM cofactor. The helices are labeled H1 through H14, and the β-strands are labeled A through G.

Figure 3

An illustration of the GsSDMT dimer interface, with the subunits of the dimer colored green and blue.

Figure 4

Partial sequence alignment between GsSDMT and other experimentally verified SDMTs. Conserved residues are capitalized. Conserved residues near the predicted substrate-binding site are designated with an asterisk. The residues colored blue surround the predicted site of the cofactor.

Figure 5

A plot of the pH vs. % relative activity of SDMT. Reactions were performed from pH 5.5-8.5 in 100 mM MES, 100 mM HEPES, 100 mM TAPS and 100 μM MgCl₂ at 37 °C. Activity levels of dimethylglycine (•) and sarcosine (°) are normalized to the pH of maximal activity. Reactions at each pH were performed in triplicate.

Figure 6

Melting temperature profile for GsSDMT.

Figure 7

Stereoview of GsSDMT and MMAS2 structural alignment. GsSDMT (blue backbone) was aligned with MMAS2 (green backbone) with SAH cofactor in the active site. Conserved functional groups within the active site include 6 of 10 backbone nitrogen and oxygen atoms and Gln117 and Glu124. In addition, N112 and H46 appear to be potential residues capable of interaction with SAH.

Figure 8

Ribbon diagrams of GsSDMT (above) and GNMT (below) illustrating the difference in cofactor binding between the two enzymes. Color is used to define the structurally similar amino terminals of the proteins (green) as well as the structurally divergent carboxy terminals (blue in GsSDMT and red in GNMT). The placement of the cofactor (carbon:gray, sulfur:yellow, nitrogen:blue, oxygen:red) in GsSDMT is predicted based on the structure of MMAS2.