The two hyaluronan synthases (HASs) from Streptococcus pyogenes (spHAS) and Streptococcus equisimilis (seHAS) were expressed in Escherichia coli as recombinant proteins containing His 6 tails. Both enzymes were expressed as major membrane proteins, accounting for ϳ5-8% of the total membrane protein. Using nickel chelate affinity chromatography, the HASs were purified to homogeneity from n-dodecyl ␤-D-maltoside extracts. High levels of HAS activity could be achieved only if the purified enzymes were supplemented with either bovine or E. coli cardiolipin (CL), although bovine CL gave 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 the 10 other lipids tested. For example, phosphatidic acid and phosphatidylethanolamine stimulated seHAS, but not spHAS. Phosphatidylserine stimulated both enzymes. spHAS appears to be more CL-specific than se-HAS, although both purified enzymes still contain endogenous CL that can not easily be removed. Both seHAS and spHAS were inhibited by phosphatidylcholine, sphingomyelin, and sulfatides and were not substantially stimulated by cerebrosides, phosphatidylglycerol, or phosphatidylinositol. With both HASs, CL increased the K m for UDP-GlcUA, but decreased the K m for UDP-GlcNAc and gave an overall stimulation of V max . A kinetic characterization of the two membrane-bound and purified HASs is presented in the accompanying paper (Tlapak-Simmons, 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 after storage for ϳ5 days at 4°C. Both purified enzymes also lost activity over 4 -5 days when stored at -80°C in the presence of CL, but reached a level of activity that then slowly decreased over a period of months. Although the purified enzymes stored in the absence of CL at ؊80°C were much less active, the enzymes retained this same low level of activity for at least 5 weeks. When both spHAS and seHAS were stored without CL at ؊80°C, even after 2 months, they could be stimulated by the addition of bovine CL to ϳ60% of the initial activity of the freshly purified enzyme.Since the discovery of HA 1 over 60 years ago (1), this saccharide polymer, which contains repeating disaccharide units of GlcUA␤(1,3)GlcNAc␤(1,4), has been shown to have numerous biological functions. For example, HA provides the viscous lubrication of synovial fluid in joints and provides cartilage with its viscoelastic properties. HA is involved in a wide variety of cellular functions and behaviors, including cell migration (2, 3) development (4 -6), differentiation (7-9), phagocytosis (6), and proteoglycan synthesis (2, 4). As well as being a major structural component of the matrix, HA has wound healing, pharmaceutical, and analgesic effects (10 -14) and is also being used as a vehicle for drug delivery (15,16).Although cell-free HA biosynthesis was achieved ...

S-Adenosylmethionine decarboxylase (AdoMetDC) is a pyruvoyl-dependent enzyme that is processed from a single polypeptide into two subunits creating the cofactor. In the human enzyme, both the proenzyme processing reaction and enzyme activity are stimulated by the polyamine putrescine. The processing reaction of Trypanosoma cruzi AdoMetDC was studied in an in vitro translation system. The enzyme was fully processed in the absence of putrescine, and the rate of this reaction was not stimulated by addition of the polyamine. Residues in the putrescine binding site of the human enzyme were evaluated for their role in processing of the T. cruzi enzyme. The E15A, I80K/S178E, D174A, and E256A mutant T. cruzi enzymes were fully processed. In contrast, mutation of R13 to Leu (the equivalent residue in the human enzyme) abolished processing of the T. cruzi enzyme, demonstrating that Arg at position 13 is a major determinant for proenzyme processing in the parasite enzyme. This amino acid change is a key structural difference that is likely to be a factor in the finding that putrescine has no role in processing of the T. cruzi enzyme. In contrast, the activity of T. cruzi AdoMetDC is stimulated by putrescine. Equilibrium sedimentation experiments demonstrated that putrescine does not alter the oligomeric state of the enzyme. The putrescine binding constant for binding to the T. cruzi enzyme (K(d) = 150 microM) was measured by a fluorescence assay and by ultrafiltration with a radiolabeled ligand. The mutant T. cruzi enzyme D174V no longer binds putrescine, and is not activated by the diamine. In contrast, mutation of E15, S178, E256, and I80 had no effect on putrescine binding. The k(cat)/K(m) values for E15A and E256A mutants were stimulated by putrescine to a smaller extent than the wild-type enzyme (2- and 4-fold vs 11-fold, respectively). These data suggest that the putrescine binding site on the T. cruzi enzyme contains only limited elements (D174) in common with the human enzyme and that the diamine plays different roles in the function of the mammalian and parasite enzymes.