L. pneumophila CMP-5,7-di-N-acetyllegionaminic acid synthetase (LpCLS)-involved chemoenzymatic synthesis of sialosides and analogues

Abstract

5,7-Di-N-acetyllegionaminic acid (Leg5,7Ac₂) is a bacterial nonulosonic acid (NulO) analogue of sialic acids, an important class of monosaccharides in mammals and in some bacteria. To develop efficient one-pot multienzyme (OPME) glycosylation systems for synthesizing Leg5,7Ac₂-glycosides, Legionella pneumophila cytidine 5′-monophosphate (CMP)-Leg5,7Ac₂ synthetase (LpCLS) was cloned and characterized. It was successfully used in producing Leg5,7Ac₂-glycosides from chemoenzymatically synthesized Leg5,7Ac₂ using a one-pot two-enzyme system or from its chemically synthesized six-carbon monosaccharide precursor 2,4-diacetamido-2,4,6-trideoxymannose (6deoxyMan2,4diNAc) in a one-pot three-enzyme system. In addition, LpCLS was shown to tolerate Neu5Ac7NAc, a C9-hydroxyl analogue of Leg5,7Ac₂ and also a stable analogue of 7-O-acetylneuraminic acid (Neu5,7Ac₂), to allow OPME synthesis of the corresponding α2–3-linked sialosides, from chemically synthesized six-carbon monosaccharide precursor 4-N-acetyl-4-deoxy-N-acetylmannosamine (ManNAc7NAc).