Identification of a membrane-localized cysteine cluster near the substrate-binding sites of the Streptococcus equisimilis hyaluronan synthase.
The membrane-bound hyaluronan synthase (HAS) from Streptococcus equisimilis (seHAS), which is the smallest Class I HAS, has four cysteine residues (positions 226, 262, 281, and 367) that are generally conserved within this family. Although Cys-null seHAS is still active, chemical modification of cysteine residues causes inhibition of wild-type enzyme. Here we studied the effects of N-ethylmaleimide (NEM) treatment on a panel of seHAS Cys-mutants to examine the structural and functional roles of the four cysteine residues in the activity of the enzyme. We found that Cys226, Cys262, and Cys281 are reactive with NEM, but Cys367 is not. Substrate protection studies of wild-type seHAS and a variety of Cys-mutants revealed that binding of UDP-GlcUA, UDP-GlcNAc, or UDP can protect Cys226 and Cys262 from NEM inhibition. Inhibition of the six double Cys-mutants of seHAS by sodium arsenite, which can cross-link vicinyl sulfhydryl groups, also supported the conclusion that Cys262 and Cys281 are close enough to be cross-linked. Similar results indicated that Cys281 and Cys367 are also very close in the active enzyme. We conclude that three of the four Cys residues in seHAS (Cys262, Cys281, and Cys367) are clustered very close together, that these Cys residues and Cys226 are located at the inner surface of the cell membrane, and that Cys226 and Cys262 are located in or near a UDP binding site.
Kumari
Kshama
K
Department of Biochemistry and Molecular Biology and The Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center. Oklahoma City, OK 73190, USA.
Weigel
Paul H
PH
D000409:Alanine / Q000235:genetics; D001665:Binding Sites; D002247:Carbon Isotopes; D002462:Cell Membrane / Q000201:enzymology*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D014453:Glucuronosyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000076002:Hyaluronan Synthases; D007553:Isotope Labeling; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation / Q000235:genetics; D011485:Protein Binding; D012694:Serine / Q000235:genetics; D018502:Streptococcus equi / Q000201:enzymology*; D013329:Structure-Activity Relationship
D002247:Carbon Isotopes; D012694:Serine; D014453:Glucuronosyltransferase; D000076002:Hyaluronan Synthases; D003545:Cysteine; D000409:Alanine