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Glycobiology Advance Access published online on December 22, 2004
Glycobiology, doi:10.1093/glycob/cwi030
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© Oxford University Press 2004; all rights reserved.
Received October 22, 2004
Revised December 10, 2004
Accepted December 15, 2004

Article

Identification of a Membrane-Localized Cysteine Cluster near the Substrate Binding Sites of the Streptococcus Equisimilis Hyaluronan Synthase

Kshama Kumari 1 and Paul H. Weigel 1*

1 Department of Biochemistry & Molecular Biology and The Oklahoma Center for Medical Glycobiology University of Oklahoma Health Sciences Center Oklahoma City, OK 73190

* To whom correspondence should be addressed.
Paul H. Weigel, E-mail: paul-weigel{at}ouhsc.edu


  Abstract

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 wildtype enzyme (Kumari et al., J. Biol. Chem. 277, 13943, 2002). 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 that Cys367 is not. Substrate protection studies of wildtype 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 crosslink vicinyl sulfhydryl groups, also supported the conclusion that Cys262 and Cys281 are close enough to be crosslinked. 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.

Keywords: Sulfhydryl reagents, N-ethylmaleimide, enzyme inhibition, Cysteine modification, site directed mutagenesis.
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