title: Domain organization, catalysis and regulation of eukaryotic cystathionine beta-synthases.
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.
authors.affiliation: Department of Pediatrics, University of Colorado, School of Medicine, Aurora, Colorado, United States of America.
authors.forename: Tomas
authors.initials: T
authors.lastname: Majtan
authors.affiliation: Department of Physical Chemistry, Faculty of Sciences, University of Granada, Granada, Spain.
authors.forename: Angel L
authors.initials: AL
authors.lastname: Pey
authors.affiliation: Department of Physical Chemistry, Faculty of Science and Technology, University of the Basque Country (UPV/EHU), Leioa, Spain.
authors.forename: Roberto
authors.initials: R
authors.lastname: Fernández
authors.affiliation: Department of Physical Chemistry, Faculty of Science and Technology, University of the Basque Country (UPV/EHU), Leioa, Spain.
authors.forename: José A
authors.initials: JA
authors.lastname: Fernández
authors.affiliation: Structural Biology Unit, CIC bioGUNE, Derio, Bizkaia, Spain.
authors.forename: Luis A
authors.initials: LA
authors.lastname: Martínez-Cruz
authors.affiliation: Department of Pediatrics, University of Colorado, School of Medicine, Aurora, Colorado, United States of America.
authors.forename: Jan P
authors.initials: JP
authors.lastname: Kraus
mesh_terms: D000818:Animals; D002384:Catalysis; D020134:Catalytic Domain; D003541:Cystathionine beta-Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D015870:Gene Expression; D006801:Humans; D008958:Models, Molecular; D011487:Protein Conformation; D011489:Protein Denaturation; D054730:Protein Interaction Domains and Motifs; D055550:Protein Stability; D011994:Recombinant Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013816:Thermodynamics
chemical_list: D011994:Recombinant Proteins; D003541:Cystathionine beta-Synthase