Structural basis for the regulatory interaction of the methylglyoxal synthase MgsA with the carbon flux regulator Crh in

Abstract

Utilization of energy-rich carbon sources such as glucose is fundamental to the evolutionary success of bacteria. Glucose can be catabolized via glycolysis for feeding the intermediary metabolism. The methylglyoxal synthase MgsA produces methylglyoxal from the glycolytic intermediate dihydroxyacetone phosphate. Methylglyoxal is toxic, requiring stringent regulation of MgsA activity. In the Gram-positive bacterium Bacillus subtilis, an interaction with the phosphoprotein Crh controls MgsA activity. In the absence of preferred carbon sources, Crh is present in the nonphosphorylated state and binds to and thereby inhibits MgsA. To better understand the mechanism of regulation of MgsA, here we performed biochemical and structural analyses of B. subtilis MgsA and of its interaction with Crh. Our results indicated that MgsA forms a hexamer (i.e. a trimer of dimers) in the crystal structure, whereas it seems to exist in an equilibrium between a dimer and hexamer in solution. In the hexamer, two alternative dimers could be distinguished, but only one appeared to prevail in solution. Further analysis strongly suggested that the hexamer is the biologically active form. In vitro cross-linking studies revealed that Crh interacts with the Nterminal helices of MgsA and that the Crh-MgsA binding inactivates MgsA by distorting and thereby blocking its active site. In summary, our results http://www.jbc.org/cgi

A novel hyperthermophilic methylglyoxal synthase: molecular dynamic analysis on the regional fluctuations

“…Figure 1 shows the specific activities of the purified MGSs measured at 30 °C and pH 7.5, along with bsMGS, a mesophilic MGS used for 1,2-propanediol synthesis with the highest known specific activity described previously 4 . The specific activities of cdMGS and opMGS were 0.21 and 0.07 mol min −1 g −1 , respectively.…”

“…Each value and error bar represents the mean and the standard deviation of at least three independent trials, respectively. The specific activity of bsMGS from Bacillus subtilis has been reported previously and presented here for comparison 4 .…”

“…The thermophiles, especially the hyperthermophilic opMGS, are shorter toward the C-terminal than the mesophilic enzymes, generating more compact structures. The active site residues identified from ecMGS and bsMGS are highly conserved among all five MGSs 3,4 . D71 (numbering www.nature.com/scientificreports/ in ecMGS) is known to play a key catalytic role as a base to withdraw a pro-S proton from the C 3 atom of DHAP, while H19 can stabilize the O 3 hydroxyl group via hydrogen bonding.…”

“…Key interactions including specific salt bridges and hydrogen bonds between a rigid beta-sheet core and surrounding alpha helices were found to contribute to the stabilisation of the hyperthermophilic enzyme by reducing the regional fluctuations in the protein structure. The structural information and analysis approach in this study can be further exploited for the engineering and industrial application of the enzyme.Methylglyoxal synthase (MGS, EC number 4.2.3.3) is a lyase for an efficient removal of a phosphate moiety from dihydroxyacetone phosphate (DHAP) to form pyruvaldehyde, an enol precursor that can be tautomerized spontaneously to methylglyoxal (MG) [1][2][3][4] . DHAP and MG are intermediates in a bypass of the cellular glycolytic pathway, where MG is converted to lactate and subsequently oxidized to pyruvate.…”

“…Methylglyoxal synthase (MGS, EC number 4.2.3.3) is a lyase for an efficient removal of a phosphate moiety from dihydroxyacetone phosphate (DHAP) to form pyruvaldehyde, an enol precursor that can be tautomerized spontaneously to methylglyoxal (MG) [1][2][3][4] . DHAP and MG are intermediates in a bypass of the cellular glycolytic pathway, where MG is converted to lactate and subsequently oxidized to pyruvate.…”