Abstract
Hyaluronan (HA) synthase (HAS) is a membrane-bound enzyme that utilizes UDP-glucuronic acid (GlcUA) and UDP-GlcNAc to synthesize HA. The HAS from Streptococcus pyogenes (spHAS, 419 amino acids) contains six Cys residues, whereas the enzyme from Streptococcus equisimilis (seHAS, 417 amino acids) contains four Cys residues. These Cys residues of seHAS are highly conserved in all Class I HAS family members. Here we investigated the structural and functional roles of these conserved cysteines in seHAS by using site-directed mutagenesis and sensitivity to sulfhydryl modifying reagents. Both seHAS and spHAS were inhibited by sulfhydryl reagents such as N-ethylmaleimide (NEM) and iodoacetamide in a dose-dependent and time-dependent manner. These inhibition curves were biphasic, indicating the presence of sensitive and insensitive components. After treatment of seHAS with NEM, the V(max) value was decreased approximately 50%, and the K(m) values changed only slightly. All the Cys-to-Ala mutants of seHAS were partially active. The least active single (C226A), double (C226A,C262A), or triple (C226A,C262A,C367A) Cys mutants retained 24, 3.2, and 1.4% activity, respectively, compared with wild-type enzyme. Surprisingly, the V(max) value of the seHAS(cys-null) mutant was approximately 17% of wild-type, although the K(m) values for both substrates were increased 3-6-fold. Cys residues, therefore, are not involved in a critical interaction necessary for either substrate binding or catalysis. However, the distribution of HA products was shifted to a smaller size in approximately 25% of the seHAS Cys mutants, particularly the triple mutants. Mass spectroscopic analysis of wild-type and Cys-null seHAS as well as the labeling of all double Cys-to-Ala mutants with [(14)C]NEM demonstrated that seHAS contains no disulfide bonds. We conclude that the four Cys residues in seHAS are not directly involved in catalysis, but that one or more of these Cys residues are located in or near substrate binding or glycosyltransferase active sites, so that their modification hinders the functions of HAS.
Highlights
HAS1 is a membrane-bound enzyme that catalyzes the synthesis of HA in both eukaryotes and prokaryotes
We conclude that the four Cys residues in seHAS are not directly involved in catalysis, but that one or more of these Cys residues are located in or near substrate binding or glycosyltransferase active sites, so that their modification hinders the functions of HAS
Sodium arsenite and 5,5Ј-dithiobis-(2-nitrobenzoic acid) inhibited each HAS activity. These results indicate that one or more Cys residues are important for the overall HA synthesis activity of the seHAS and spHAS proteins
Summary
Primers, and Reagents—The expression vector pKK223 was from Amersham Biosciences, Inc. Determination of HA Size Produced by seHAS Variants—The relative Mr of the HA synthesized by wild-type seHAS or the Cys mutants was determined by agarose gel electrophoresis [32] of 14C-labeled HA products synthesized under the assay conditions described above. The integrated density value values for seHAS bands in membranes containing wild-type or mutant proteins were compared with the standard to estimate the seHAS protein content per mg of membrane protein These data were used to normalize the seHAS enzyme activity in the membrane preparations for wild-type and each variant seHAS. Suspensions of membranes containing seHAS or spHAS were incubated with 0 –5 mM sulfhydryl reagent at 4 °C, and the reactions were stopped by adding DTE to a final concentration of 10 mM. Spectra were an average of 80 –120 scans and were processed using the 19-point Savitsky-Golay smoothing option included in the software provided by the manufacturer
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