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J Biol Chem, Vol. 274, Issue 37, 26557-26562, September 10, 1999
From the Department of Biochemistry and Molecular Biology,
University of Oklahoma Health Sciences Center,
Oklahoma City, Oklahoma 73104
Hyaluronan (HA), a long linear polymer composed
of alternating glucuronic acid and N-acetylglucosamine
residues, is an essential polysaccharide in vertebrates and a putative
virulence factor in certain microbes. All known HA synthases utilize
UDP-sugar precursors. Previous reports describing the HA synthase
enzymes from Streptococcus bacteria and mammals, however,
did not agree on the molecular directionality of polymer elongation. We
show here that a HA synthase, PmHAS, from Gram-negative P. multocida bacteria polymerizes the HA chain by the addition of
sugar units to the nonreducing terminus. Recombinant PmHAS will
elongate exogenous HA oligosaccharide acceptors to form long polymers
in vitro; thus far no other HA synthase has displayed this
capability. The directionality of synthesis was established
definitively by testing the ability of PmHAS to elongate defined
oligosaccharide derivatives. Analysis of the initial stages of
synthesis demonstrated that PmHAS added single monosaccharide units
sequentially. Apparently the fidelity of the individual sugar transfer
reactions is sufficient to generate the authentic repeating structure
of HA. Therefore, simultaneous addition of disaccharide block units is
not required as hypothesized in some recent models of polysaccharide
biosynthesis. PmHAS appears distinct from other known HA synthases
based on differences in sequence, topology in the membrane, and
putative reaction mechanism.
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