Purification and lipid dependence of the recombinant hyaluronan syntheses from Streptococcus pyogenes and Streptococcus equisimilis

The two hyaluronan synthases (HASs) from Streptococcus pyogenes (spHAS) and Streptococcus equisimilis (seHAS) were expressed in Escherichia coli as re combinant proteins containing His(6) tails. Both enzymes were expressed as major membrane proteins, accounting for similar to 5-8% of the total membra ne protein. Using nickel chelate affinity chromatography, the HASs were pur ified to homogeneity from n-dodecyl beta-D-maltoside extracts, High levels of HAS activity could be achieved only if the purified enzymes were supplem ented with either bovine or E. coli cardiolipin (CL), although bovine CL ga ve consistently greater activity. Mass spectroscopic analysis revealed that the fatty acid compositions of these two CL preparations did not overlap. The two HAS enzymes showed similar but distinct activation profiles with th e 10 other lipids tested. For example, phosphatidic acid and phosphatidylet hanolamine stimulated seHAS, but not spHAS, Phosphatidylserine stimulated b oth enzymes. spHAS appears to be more CL-specific than seHAS, although both purified enzymes still contain endogenous CL that can not easily be remove d. Both seHAS and spHAS were inhibited by phosphatidylcholine, sphingomyeli n, and sulfatides and were not substantially stimulated by cerebrosides, ph osphatidylglycerol, or phosphatidylinositol. With both HASs, CL increased t he K-m for UDP-GlcUA, but decreased the K-m for UDP-GlcNAc and gave an over all stimulation of V-max, A kinetic characterization of the two membrane-bo und and purified HASs is presented in the accompanying paper (Tlapak-Simmon s, V. L., Baggenstoss, B. A. Kumari, K., Heldermon, C., and Weigel, P. H. ( 1999) J. Biol. Chem, 274, 4246-4253), Both purified HASs became inactive af ter storage for similar to 5 days at 4 degrees C, Both purified enzymes als o lost activity over 4-5 days when stored at -80 degrees C in the presence of CL, but reached a level of activity that then slowly decreased over a pe riod of months. Although the purified enzymes stored in the absence of CL a t -80 degrees C were much less active, the enzymes retained this same low l evel of activity for at least 5 weeks. When both spHAS and seHAS were store d without CL at -80 degrees C, even after 2 months, they could be stimulate d by the addition of bovine CL to similar to 60% of the initial activity of the freshly purified enzyme.