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J. Biol. Chem., Vol. 281, Issue 17, 11755-11760, April 28, 2006

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Mutation of Two Intramembrane Polar Residues Conserved within the Hyaluronan Synthase Family Alters Hyaluronan Product Size^*

Kshama Kumari, Bruce A. Baggenstoss, Andria L. Parker, and Paul H. Weigel¹

From the Department of Biochemistry and Molecular Biology and The Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104

We identified two conserved polar amino acids within different membrane domains (MD) of Streptococcus equisimilis hyaluronan synthase (seHAS), Lys⁴⁸ in MD2 and Glu³²⁷ in MD4. In eukaryotic HASs, the position of the Glu is very similar and the Lys is replaced by a conserved polar Gln. To assess whether Lys⁴⁸ and Glu³²⁷ interact or influence seHAS activity, we investigated the effects of changing Lys⁴⁸ to Arg or Glu and Glu³²⁷ to Lys, Asp, or Gln. Mutants, including a double switch variant with Lys⁴⁸ and Glu³²⁷ exchanged, were expressed and assayed in Escherichia coli membranes. SeHAS(E327Q) and seHAS(E327K) were expressed at low levels, whereas seHAS(E327D) and the Lys⁴⁸ mutants were expressed well. The specific enzyme activities (relative to wild type) were 17 and 7% for the K48R and K48E mutants and 26 and 38% for the E327Q and E327D mutants, respectively. In contrast, seHAS(E327K) showed only 0.16% of wild-type activity but was rescued over 46-fold by changing Lys⁴⁸ to Glu. Expression of the seHAS(E327K,K48E) protein was also rescued to near wild-type levels. Based on size exclusion chromatography coupled to multiangle laser light scattering analysis, all the variants synthesized hyaluronan (HA) of smaller weight-average molar mass than wild-type enzyme (3.6 MDa); the smallest HA (0.6 MDa) was made by seHAS(E327K,K48E) and seHAS(K48E). The results indicate that Glu³²⁷ within MD4 is a critical residue for the stability of seHAS, that it may interact with Lys⁴⁸ within MD2, and that these residues are involved in the ability of HAS to synthesize very large HA.

Received for publication, January 24, 2006

^* This work was supported by NIGMS, National Institutes of Health Grant GM35978. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 405-271-1288; Fax: 405-271-3092; E-mail: paul-weigel{at}ouhsc.edu.

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