| Summary: | Lipopolysaccharyl α-1,4-galactosyltransferase C (EC 2.4.1.x, LgtC) from
Neisseria meningitidis is an important enzyme in the biosynthesis of lipooligosaccharides
in this bacterium. The reaction catalyzed by this enzyme is the transfer of a galactosyl
moiety from uridine diphosphogalactose (UDPGal) to a lipooligosaccharide acceptor
containing a galactose residue at the nonreducing terminus, forming a Gal-α-1,4-Gal
linkage in the process. Because the reaction proceeds with net retention of
stereochemistry at the centre undergoing substitution, LgtC is classified as a "retaining"
glycosyltransferase. By analogy with the well studied retaining glycosidases, LgtC is
therefore believed to follow a double displacement mechanism involving the formation
and subsequent breakdown of a covalent glycosyl-enzyme intermediate.
A detailed kinetic and mechanistic characterization of LgtC was carried out. This
enzyme was found to be highly specific for the donor substrate, UDPGal, binding this
compound with micromolar affinity. As for the acceptor substrate, the enzyme is far less
selective, as a range of compounds was found to be capable of fulfilling this role. In the
absence of suitable glycosyl acceptors, water was also found to be capable of functioning
in such a capacity, with the result being hydrolysis of UDPGal to yield UDP and
galactose. Since the reaction catalyzed by LgtC involves the binding of two substrates
and the release of two products, an understanding of the order in which these events
occur is also important to the characterization of this enzyme. On the basis of kinetic
studies together with a UDPGal/UDP exchange study, LgtC was deduced to follow a
sequential kinetic mechanism, which requires that all substrates be bound to the enzyme
before any chemical catalysis and release of products can occur. Further clarification of the order of substrate binding and product release was then achieved through a series of
inhibition studies with both substrate analogues and product. On the basis of these
studies, LgtC was concluded to follow an ordered Bl Bl kinetic mechanism in which
UDPGal binds first, followed by lactose. After catalysis, the trisaccharide is the first
product to be released, followed by UDP.
In our quest to understand the chemical mechanism of LgtC, much effort was
directed towards the attempted trapping of the putative covalent glycosyl-enzyme
intermediate and identification of the nucleophile involved in its formation. When these
endeavours were unsuccessful, alternatives to the double displacement mechanism,
wherein the nucleophile is not enzymic were then sought. With the aid of the threedimensional
structure of this enzyme, all candidate nucleophiles were identified and
assessed for their ability to fulfill such a role. Unfortunately, none appeared to be likely
to function in such a capacity. Another possibility is that LgtC does not employ a double
displacement mechanism, but rather proceeds through an Swi-like mechanism involving
the approach of the nucleophile from the same side of the reaction centre as the departing
leaving group. However, such a mechanism is not well precedented and no concrete
evidence is available to support its occurrence in LgtC. Therefore, at this point in time,
the mechanism by which LgtC catalyzes its glycosyl transfer reaction is still a mystery.
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