Sialyltransferases with enhanced legionaminic acid transferase activity for the preparation of analogs of sialoglycoconjugates.

Legionaminic acids (Leg) are bacterial analogs of neuraminic acid, with the same stereochemistry but different substituents at C5, C7 and C9. Hence they may be incorporated into useful analogs of sialoglycoconjugates, and we previously reported two sialyltransferases that could utilize cytidine monophosphate (CMP)-Leg5Ac7Ac for preparation of Leg glycoconjugates, which were resistant to sialidases [Watson DC, Leclerc S, Wakarchuk WW, Young NM. 2011. Enzymatic synthesis and properties of glycoconjugates with legionaminic acid as a replacement for neuraminic acid. Glycobiology. 21:99-108.]. These were the porcine ST3Gal1 and Pasteurella multocida sialyltransferases. We now report two additional sialyltransferases with superior Leg-transferase properties to the previous two. These are (i) a truncated form of a Photobacterium α2,6-sialyltransferase with an Ala-Met mutation in its active site, and (ii) an α2,3-sialyltransferase from Neisseria meningitidis MC58 with a higher transferase activity than the P. multocida enzyme, with either CMP-Neu5Ac or CMP-Leg5Ac7Ac as the donor. These enzymes will enable the production of useful Leg5Ac7Ac glycoconjugate derivatives with either α2,6 or α2,3 linkages and unique biological properties.

Watson DC ，Wakarchuk WW ，Leclerc S ，Schur MJ ，Schoenhofen IC ，Young NM ，Gilbert M ... -  《-》

Enzymatic synthesis and properties of glycoconjugates with legionaminic acid as a replacement for neuraminic acid.

Watson DC ，Leclerc S ，Wakarchuk WW ，Young NM ... -  《-》

Biosynthesis of Legionaminic Acid and Its Incorporation Into Glycoconjugates.

Legionaminic acids are analogs of sialic acid that occur in cell surface glycoconjugates of several bacteria. Because legionaminic acids share the same stereochemistry as sialic acid but differ at C7 and C9, they are interesting analogs to probe the impact of varying exocyclic moieties (C7-C9) on biological activities such as susceptibilities to sialidases, interactions with Siglecs and immunogenicity. There are currently no reports on the bacterial enzymes that transfer legionaminic acids to these cell surface glycoconjugates, but some mammalian and bacterial sialyltransferases display donor promiscuity and can use CMP-Leg5,7Ac2 efficiently enough to transfer Leg5,7Ac2 to their natural acceptor glycans. When the natural activity with CMP-Leg5,7Ac2 is significant but relatively low, an alternate strategy has been to engineer versions with improved activity to transfer Leg5,7Ac2. Importantly, we have found that some bacterial sialyltransferases are very efficient for transferring Leg5,7Ac2 to small synthetic glycans with various aglycones. The two mammalian sialyltransferases that have been tested so far (porcine ST3Gal1 and human ST6Gal1) were found to be more efficient than the bacterial sialyltransferases for the modification of glycoproteins. We provide a review of the sialyltransferases selected to modify different types of glycoconjugates with Leg5,7Ac2, including small synthetic acceptors, glycolipids, and glycoproteins. In the first part, we also propose an optimized biosynthetic pathway for in vitro preparation of the donor CMP-Leg5,7Ac2, based on enzymes selected from two bacteria that naturally produce legionaminic acid.

Schoenhofen IC ，Young NM ，Gilbert M 《-》

Preparation of legionaminic acid analogs of sialo-glycoconjugates by means of mammalian sialyltransferases.

Watson DC ，Wakarchuk WW ，Gervais C ，Durocher Y ，Robotham A ，Fernandes SM ，Schnaar RL ，Young NM ，Gilbert M ... -  《-》

A Pasteurella multocida sialyltransferase displaying dual trans-sialidase activities for production of 3'-sialyl and 6'-sialyl glycans.

This study examined a recombinant Pasteurella multocida sialyltransferase exhibiting dual trans-sialidase activities. The enzyme catalyzed trans-sialylation using either 2-O-(p-nitrophenyl)-α-d-N-acetylneuraminic acid or casein glycomacropeptide (whey protein) as the sialyl donor and lactose as the acceptor, resulting in production of both 3'-sialyllactose and 6'-sialyllactose. This is the first study reporting α-2,6-trans-sialidase activity of this sialyltransferase (EC 2.4.99.1 and 2.4.99.4). A response surface design was used to evaluate the effects of three reaction parameters (pH, temperature, and lactose concentration) on enzymatic production of 3'- and 6'-sialyllactoses using 5% (w/v) casein glycomacropeptide (equivalent to 9mM bound sialic acid) as the donor. The maximum yield of 3'-sialyllactose (2.75±0.35mM) was achieved at a reaction condition with pH 6.4, 40°C, 100mM lactose after 6h; and the largest concentration of 6'-sialyllactose (3.33±0.38mM) was achieved under a condition with pH 5.4, 40°C, 100mM lactose after 8h. 6'-sialyllactose was presumably formed from α-2,3 bound sialic acid in the casein glycomacropeptide as well as from 3'-sialyllactose produced in the reaction. The kcat/Km value for the enzyme using 3'-sialyllactose as the donor for 6'-sialyllactose synthesis at pH 5.4 and 40°C was determined to be 23.22±0.7M(-1)s(-1). Moreover, the enzyme was capable of catalyzing the synthesis of both 3'- and 6'-sialylated galactooligosaccharides, when galactooligosaccharides served as acceptors.

Guo Y ，Jers C ，Meyer AS ，Arnous A ，Li H ，Kirpekar F ，Mikkelsen JD ... -  《-》