A single-step pathway for the synthesis of glucosylglycerate in Persephonella marina

Centro de Neurociências e Biologia Celular, Universidade de Coimbra, 3004-517 Coimbra, Portugal and; Departamento de Bioquímica, Universidade de Coimbra, 3001-401 Coimbra, Portugal

^* To whom correspondence should be addressed. Email: milton{at}ci.uc.pt.

	Abstract

A single-step pathway for the synthesis of the compatible solute glucosylglycerate (GG) is proposed based on the activity of a recombinant glucosylglycerate synthase (Ggs) from Persephonella marina. The corresponding gene encoded a putative glycosyltransferase that was part of an operon-like structure which also contained the genes for glucosyl-3-phosphoglycerate synthase (GpgS) and glucosyl-3-phosphoglycerate phosphatase (GpgP), the enzymes that lead to the synthesis of GG through the formation of glucosyl-3-phosphoglycerate. The putative glucosyltransferase gene was expressed in Escherichia coli and the recombinant product catalyzed the synthesis of GG in one-step from ADP-glucose and D-glycerate, with Km values at 70°C of 1.5 and 2.1 mM, respectively. This glucosylglycerate synthase (Ggs) was also able to use GDP- and UDP-glucose as donors to form GG, with lower efficiencies. Maximal activity was reached between 80 and 85°C and Mg²⁺ or Ca²⁺ were required for catalysis. Ggs activity was maximal and remained nearly constant between pH 5.5 and 8.0 and the half-life for inactivation was 74 h at 85°C and 8 min at 100°C. This is the first report of an enzyme catalyzing the synthesis of GG in one step and of the existence of two pathways for GG synthesis in the same organism.