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Please use this identifier to cite or link to this item: http://hdl.handle.net/10481/32857

Title: Domain Organization, Catalysis and Regulation of Eukaryotic Cystathionine Beta-Synthases
Authors: Majtan, Tomas
Pey, Ángel L.
Fernández, Roberto
Fernández, José A.
Martínez-Cruz, Luis A.
Kraus, Jan P.
Issue Date: 2014
Abstract: Cystathionine beta-synthase (CBS) is a key regulator of sulfur amino acid metabolism diverting homocysteine, a toxic intermediate of the methionine cycle, via the transsulfuration pathway to the biosynthesis of cysteine. Although the pathway itself is well conserved among eukaryotes, properties of eukaryotic CBS enzymes vary greatly. Here we present a side-by-side biochemical and biophysical comparison of human (hCBS), fruit fly (dCBS) and yeast (yCBS) enzymes. Preparation and characterization of the full-length and truncated enzymes, lacking the regulatory domains, suggested that eukaryotic CBS exists in one of at least two significantly different conformations impacting the enzyme’s catalytic activity, oligomeric status and regulation. Truncation of hCBS and yCBS, but not dCBS, resulted in enzyme activation and formation of dimers compared to native tetramers. The dCBS and yCBS are not regulated by the allosteric activator of hCBS, S-adenosylmethionine (AdoMet); however, they have significantly higher specific activities in the canonical as well as alternative reactions compared to hCBS. Unlike yCBS, the heme-containing dCBS and hCBS showed increased thermal stability and retention of the enzyme’s catalytic activity. The mass-spectrometry analysis and isothermal titration calorimetry showed clear presence and binding of AdoMet to yCBS and hCBS, but not dCBS. However, the role of AdoMet binding to yCBS remains unclear, unlike its role in hCBS. This study provides valuable information for understanding the complexity of the domain organization, catalytic specificity and regulation among eukaryotic CBS enzymes.
Sponsorship: This work was supported by Postdoctoral Fellowship 0920079G from the American Heart Association (to TM), by National Institutes of Health Grant HL065217, by American Heart Association Grant In-Aid 09GRNT2110159, by a grant from the Jerome Lejeune Foundation (all to JPK) and by a research contract RYC2009-04147 (to ALP). In addition, grant support (P11-CTS-07187, CSD2009-00088 and BIO2012-34937) to Dr. Jose M. Sanchez-Ruiz (University of Granada) and SGIker technical and human support (UPV/EHU, MICINN, GV/EJ, ESF) are gratefully acknowledged.
Publisher: Public Library of Science (PLOS)
Keywords: Cofactors (biochemistry)
Crystal structure
Enzyme purification
Enzyme regulation
Enzymes
Heme
Matrix-assisted laser desoprtion ionization mass spectrometry
Polymerase chain reaction
URI: http://hdl.handle.net/10481/32857
ISSN: 1932-6203
Rights : Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License
Citation: Majtan, T.; et al. Domain Organization, Catalysis and Regulation of Eukaryotic Cystathionine Beta-Synthases. Plos One, 9(8): e105290 (2014). [http://hdl.handle.net/10481/32857]
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