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Publication Details
AFRICAN RESEARCH NEXUS
SHINING A SPOTLIGHT ON AFRICAN RESEARCH
biochemistry, genetics and molecular biology
The N domain of human angiotensin-I-converting enzyme: The role of N-glycosylation and the crystal structure in complex with an N domain-specific phosphinic inhibitor, RXP407
Journal of Biological Chemistry, Volume 285, No. 46, Year 2010
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Description
Angiotensin-I-converting enzyme (ACE) plays a critical role in the regulation of blood pressure through its central role in the renin-angiotensin and kallikrein-kinin systems. ACE contains two domains, the N and C domains, both of which are heavily glycosylated. Structural studies of ACE have been fraught with severe difficulties because of surface glycosylation of the protein. In order to investigate the role of glycosylation in the N domain and to create suitable forms for crystallization, we have investigated the importance of the 10 potential N-linked glycan sites using enzymatic deglycosylation, limited proteolysis, and mass spectrometry. A number of glycosylation mutants were generated via site-directed mutagenesis, expressed in CHO cells, and analyzed for enzymatic activity and thermal stability. At least eight of 10 of the potential glycan sites are glycosylated; three C-terminal sites were sufficient for expression of active N domain, whereas two N-terminal sites are important for its thermal stability. The minimally glycosylated Ndom389 construct was highly suitable for crystallization studies. The structure in the presence of an N domain-selective phosphinic inhibitor RXP407 was determined to 2.0 Å resolution. The Ndom389 structure revealed a hinge region that may contribute to the breathing motion proposed for substrate binding. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.
Authors & Co-Authors
Anthony, Colin S.
South Africa, Cape Town
University of Cape Town
Corradi, Hazel Ruth
United Kingdom, Bath
University of Bath
Schwager, Sylva L.U.
South Africa, Cape Town
University of Cape Town
Redelinghuys, Pierre
United Kingdom, Aberdeen
University of Aberdeen School of Medicine, Medical Sciences and Nutrition
Georgiadis, Dimitris
Greece, Athens
National and Kapodistrian University of Athens
Dive, Vincent
France, Gif-sur-yvette
Institut de Biologie et de Technologies de Saclay
Ravi Acharya, K.
United Kingdom, Bath
University of Bath
Sturrock, Edward D.
South Africa, Cape Town
University of Cape Town
Statistics
Citations: 78
Authors: 8
Affiliations: 5
Identifiers
Doi:
10.1074/jbc.M110.167866
ISSN:
00219258
e-ISSN:
1083351X
Research Areas
Noncommunicable Diseases