Domain Organization, Catalysis and Regulation of Eukaryotic Cystathionine Beta-Synthases Tomas Majtan 1 , Angel L. Pey 2 , Roberto Ferna ´ ndez 3 , Jose ´ A. Ferna ´ ndez 3 , Luis A. Martı´nez-Cruz 4 , Jan P. Kraus 1 * 1 Department of Pediatrics, University of Colorado, School of Medicine, Aurora, Colorado, United States of America, 2 Department of Physical Chemistry, Faculty of Sciences, University of Granada, Granada, Spain, 3 Department of Physical Chemistry, Faculty of Science and Technology, University of the Basque Country (UPV/EHU), Leioa, Spain, 4 Structural Biology Unit, CIC bioGUNE, Derio, Bizkaia, Spain 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. Citation: Majtan T, Pey AL, Ferna ´ndez R, Ferna ´ndez JA, Martı ´nez-Cruz LA, et al. (2014) Domain Organization, Catalysis and Regulation of Eukaryotic Cystathionine Beta-Synthases. PLoS ONE 9(8): e105290. doi:10.1371/journal.pone.0105290 Editor: Vladimir N. Uversky, University of South Florida College of Medicine, United States of America Received May 30, 2014; Accepted July 23, 2014; Published August 14, 2014 Copyright: ß 2014 Majtan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: 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. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: jan.kraus@ucdenver.edu Introduction Methionine (Met) is an essential sulfur amino acid for mammals and its metabolism comprises two intersecting metabolic pathways: the methionine cycle, found in all tissues, and the transsulfuration pathway, which occurs in a limited number of tissues, but mainly in liver and kidney [1]. Both pathways compete for homocysteine (Hcy), a central intermediate that has been formed from Met. While in the methionine cycle Hcy is converted back to Met by either methionine synthase or betaine-homocysteine methyltrans- ferase, in the transsulfuration pathway Hcy is irreversibly converted to cysteine (Cys). The transsulfuration pathway is believed to be the sole route for Cys synthesis in vertebrates [2]. Thus, Hcy formation and its distribution between these two pathways represents an occasion for regulatory intervention. Cystathionine beta-synthase (CBS) is the enzyme, which regulates the flux of Hcy through the transsulfuration pathway and thus commits Hcy to the synthesis of Cys [3]. Deficiency in CBS results in a serious metabolic disorder, homocystinuria, clinically mani- fested chiefly by connective tissue defects, mental retardation and thromboembolism [4]. Considering the importance of CBS in sulfur amino acid metabolism, it is interesting that domain organization, quaternary structure and regulatory mechanism of CBS enzymes are not conserved across phyla (Fig. 1A–B). The extensively studied human CBS (hCBS) is a homotetrameric enzyme of 63 kDa polypeptides, each consisting of three distinct domains (reviewed in [3,5]). The N-terminal domain of hCBS binds heme, which binds via a Cys/His ligation [6]. The origin and role of the heme in CBS is still an enigma and it is believed to function as a redox sensor [5] and/or to play a structural role facilitating a correct folding [7,8]. The highly conserved central region forms a catalytic domain containing the PLP cofactor. The C-terminal domain houses a tandem of CBS domains, a structural motif known to bind adenosine nucleotides and to regulate protein function [9]. Indeed, the catalytic activity as well as kinetic stability of hCBS is increased upon interaction of S-adenosyl-L-methionine (AdoMet) with the CBS domains [10,11]. In comparison to hCBS, the variability in PLOS ONE | www.plosone.org 1 August 2014 | Volume 9 | Issue 8 | e105290