The crystallographic structure of Mannosyl-3-phosphoglycerate Synthase from Thermus thermophilus HB27 provides new insights into the GT-A retaining mechanism.

ITQB PhD Seminar

Title: The crystallographic structure of Mannosyl-3-phosphoglycerate Synthase from Thermus thermophilus HB27 provides new insights into the GT-A retaining mechanism.

Speaker: Susana Gonçalves

Laboratory: Protein Crystallography

Abstract
Interest in the regulatory mechanisms for thermo-osmotic adaptation, based on the de novo synthesis of compatible solutes (Brown, 1976), which are mostly present in (hyper)thermophilic organisms, arises from their versatility for multi-functional applications in the biotechnological field (da Costa et al, 1998). The compound α-D-mannosylglycerate (MG) is commonly found in marine hydrothermal eco-systems, and has been classified primarily as an osmolyte. In addition, it plays a broader role in cell survival at supra-optimal growth temperatures (Silva et al., 1999). In fact, MG possesses a superior ability to protect enzymes against thermal denaturation in vitro, and its potential usefulness in biotechnological and pharmaceutical applications has been often claimed (Borges et al., 2002; Faria et al., 2008).
In most organisms, MG synthesis proceeds via a two-reaction pathway: in the first step GDP α D mannose (GDP-man) and D-3 phosphoglycerate are converted into 2-(-D-mannosyl)-3-phosphoglycerate by the action of Mannosyl-3-phosphoglycerate Synthase (MpgS; EC 2.4.1.217). The phosphorylated intermediate is subsequently dephosphorylated by a specific phosphatase (MpgP; EC 3.1.5.70) to yield MG (Borges et al, 2004). The biochemical characterization of MpgS lead to its classification into the retaining GlycosylTransferase family 55 (GT55; www.cazy.org).
In this presentation, the crystallographic structure of MpgS from Thermus thermophilus HB27 in its apo-form and binary complex with bound GDP--D-mannose (GDP-man) and Mg2+ is described and analysed. Evidence for a second co-catalytic metal ion was obtained from combined structural, kinetic and mutagenic data. Additionally, the structural details of the MpgS:GDP-man:Mg2+ binary complex, provide us with insights into the mechanisms governing substrate specificity, as well as into the dynamics that accompanies catalysis.
In light of the two mechanisms currently proposed for the glycosyl-transfer catalysis with a retention stereochemical outcome (Lairson et al, 2008), we describe the likeliest catalytic nucleophile and propose it to be representative for the GT55 family. Finally, the gathered structural information lead us to propose a more complete structural signature in the rational identification of a common scaffold between the inverting and retaining GTs.
References:
1. Brown, A.D. (1976). Bacteriol. Rev. 40: 803-846.
2. da Costa, M., Santos, H. &Galinski E.A., (1998). Adv Biochem Eng Biotechnol, 61: 117-53.
3. Faria, T. Q., Mingote, A., Siopa, F., Ventura, R., Maycock, C., & Santos, H. (2008). Carbohydr. Res. 343(18): 3025-3033.
4. Borges, N., Marugg, J. D., Empadinhas, N., da Costa, M. S. & Santos, H. (2004). J. Biol. Chem. 279, 9892-9898.
5. Lairson, L.L., Henrissat, B., Davies,G.J. & Withers,S.G. (2008). Ann. Rev. Biochem. 77: 521-555.