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Volume 272, Number 51, Issue of December 19, 1997
pp. 32539-32546
(Received for publication, August 4, 1997, and in revised form, September 24, 1997)
From the Department of Biochemistry and Molecular Biology,
University of Oklahoma Health Sciences Center, Oklahoma City,
Oklahoma 73190
We previously reported the first
cloning of a functional glycosaminoglycan synthase, the hyaluronan
synthase (HAS) from Group A Streptococcus pyogenes (spHAS)
(DeAngelis, P. L., Papaconstantinou, J., and Weigel, P. H. (1993) J. Biol. Chem. 268, 19181-19184). Group A
spHAS was unrelated to a putative Group C HA synthase reported by
others (Lansing, M., Lellig, S., Mausolf, A., Martini, I., Crescenzi,
F., Oregon, M., and Prehm, P. (1993) Biochem. J. 289, 179-184). Here we report the isolation of a bona fide HA synthase gene
from a highly encapsulated strain of Group C Streptococcus equisimilis. The encoded protein, designated seHAS, is 417 amino acids long (calculated molecular weight, 47,778; calculated pI, 9.1)
and is the smallest member of the HAS family identified thus far. The
enzyme migrates anomalously fast in SDS-polyacrylamide gel
electrophoresis (~42,000 Da). The seHAS protein shows no similarity (<2% identity) to the previously reported Group C gene, which is not
an HA synthase. The seHAS and spHAS protein and coding sequences are 72 and 70% identical, respectively. seHAS is also similar to eukaryotic
HAS1 (~31% identical), HAS2 (~28% identical), and HAS3 (28%
identical). The deduced protein sequence of seHAS was confirmed by
reactivity with a synthetic peptide antibody. Recombinant seHAS
expressed in Escherichia coli was recovered in membranes as
a major protein (~10% of the total protein) and synthesized very
large HA (Mr >7 × 106) in
the presence of UDP-GlcNAc and UDP-GlcA. The product contained equimolar amounts of both sugars and was degraded by the specific Streptomyces hyaluronidase. Comparison of the two
recombinant streptococcal enzymes in isolated membranes showed that
seHAS and spHAS are essentially identical in the steady-state size
distribution of HA chains they synthesize, but seHAS has an intrinsic
2-fold faster rate of chain elongation (Vmax)
than spHAS. seHAS is the most active HA synthase identified thus far;
it polymerizes HA at an average rate of 160 monosaccharides/s. The two
bacterial HA synthase genes may have arisen from a common ancient gene
shared with the early evolving vertebrates.
Molecular Cloning, Expression, and Characterization of the
Authentic Hyaluronan Synthase from Group C Streptococcus
equisimilis
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