Publications

• Crystal Structures of Cystathionine beta-Synthase from Saccharomyces cerevisiae: One Enzymatic Step at a Time.

  Tu Y., Kreinbring C.A., Hill M., Liu C., Petsko G.A., McCune C.D., Berkowitz D.B., Liu D., Ringe D.

  Cystathionine β-synthase (CBS) is a key regulator of sulfur amino acid metabolism, taking homocysteine from the methionine cycle to the biosynthesis of cysteine via the trans-sulfuration pathway. CBS is also a predominant source of H₂S biogenesis. Roles for CBS have been reported for ne ... >> More

  Cystathionine β-synthase (CBS) is a key regulator of sulfur amino acid metabolism, taking homocysteine from the methionine cycle to the biosynthesis of cysteine via the trans-sulfuration pathway. CBS is also a predominant source of H₂S biogenesis. Roles for CBS have been reported for neuronal death pursuant to cerebral ischemia, promoting ovarian tumor growth, and maintaining drug-resistant phenotype by controlling redox behavior and regulating mitochondrial bioenergetics. The trans-sulfuration pathway is well-conserved in eukaryotes, but the analogous enzymes have different enzymatic behavior in different organisms. CBSs from the higher organisms contain a heme in an N-terminal domain. Though the presence of the heme, whose functions in CBSs have yet to be elucidated, is biochemically interesting, it hampers UV-vis absorption spectroscopy investigations of pyridoxal 5'-phosphate (PLP) species. CBS from Saccharomyces cerevisiae (yCBS) naturally lacks the heme-containing N-terminal domain, which makes it an ideal model for spectroscopic studies of the enzymological reaction catalyzed and allows structural studies of the basic yCBS catalytic core (yCBS-cc). Here we present the crystal structure of yCBS-cc, solved to 1.5 Å. Crystal structures of yCBS-cc in complex with enzymatic reaction intermediates have been captured, providing a structural basis for residues involved in catalysis. Finally, the structure of the yCBS-cc cofactor complex generated by incubation with an inhibitor shows apparent off-pathway chemistry not normally seen with CBS. << Less

  Biochemistry 57:3134-3145(2018) [PubMed] [EuropePMC]

• Purification, crystallization and preliminary crystallographic analysis of human cystathionine beta-synthase.

  Oyenarte I., Majtan T., Ereno J., Corral-Rodriguez M.A., Kraus J.P., Martinez-Cruz L.A.

  Human cystathionine β-synthase (CBS) is a pyridoxal-5'-phosphate-dependent hemeprotein, whose catalytic activity is regulated by S-adenosylmethionine. CBS catalyzes the β-replacement reaction of homocysteine (Hcy) with serine to yield cystathionine. CBS is a key regulator of plasma levels of the t ... >> More

  Human cystathionine β-synthase (CBS) is a pyridoxal-5'-phosphate-dependent hemeprotein, whose catalytic activity is regulated by S-adenosylmethionine. CBS catalyzes the β-replacement reaction of homocysteine (Hcy) with serine to yield cystathionine. CBS is a key regulator of plasma levels of the thrombogenic Hcy and deficiency in CBS is the single most common cause of homocystinuria, an inherited metabolic disorder of sulfur amino acids. The properties of CBS enzymes, such as domain organization, oligomerization degree or regulatory mechanisms, are not conserved across the eukaryotes. The current body of knowledge is insufficient to understand these differences and their impact on CBS function and physiology. To overcome this deficiency, we have addressed the crystallization and preliminary crystallographic analysis of a protein construct (hCBS516-525) that contains the full-length CBS from Homo sapiens (hCBS) and just lacks amino-acid residues 516-525, which are located in a disordered loop. The human enzyme yielded crystals belonging to space group I222, with unit-cell parameters a=124.98, b=136.33, c=169.83 Å and diffracting X-rays to a resolution of 3.0 Å. The crystal structure appears to contain two molecules in the asymmetric unit which presumably correspond to a dimeric form of the enzyme. << Less

  Acta Crystallogr Sect F Struct Biol Cryst Commun 68:1318-1322(2012) [PubMed] [EuropePMC]

• Cystathionine beta-synthase from human liver: improved purification scheme and additional characterization of the enzyme in crude and pure form.

  Kraus J.P., Rosenberg L.E.

  The previously published procedure (Kraus et al. (1978) J. Biol. Chem. 253, 6523-6528) for the purification of cystathionine beta-synthase [L-serine hydro-lyase (adding homocysteine) EC 4.2.1.22], a pyridoxal 5'-phosphate-dependent enzyme from human liver has been modified. The new procedure, star ... >> More

  The previously published procedure (Kraus et al. (1978) J. Biol. Chem. 253, 6523-6528) for the purification of cystathionine beta-synthase [L-serine hydro-lyase (adding homocysteine) EC 4.2.1.22], a pyridoxal 5'-phosphate-dependent enzyme from human liver has been modified. The new procedure, starting with a liver homogenate "aged" for 7 days at 4 degrees C, yielded homogeneous enzyme purified over 3000-fold with a much improved yield. "Aging" of the enzyme in crude homogenates yields a form apparently smaller by gel electrophoresis and with significantly increased activity and antigenicity. This species of cystathionine beta-synthase does not form stable complexes with other proteins during purification as does the previously employed, freshly used species. An absorption spectrum and an amino acid composition of the pure enzyme were determined; the amino-terminal residue was shown to be methionine. The isoelectric points of holosynthase and aposynthase were estimated to be 5.2 and 5.6, respectively. Rabbit antiserum raised against the pure cystathionine beta-synthase was characterized using as antigen crude synthase from five different mammalian species as well as the pure human enzyme. << Less

  Arch Biochem Biophys 222:44-52(1983) [PubMed] [EuropePMC]

• Purification, crystallization and preliminary crystallographic analysis of the full-length cystathionine beta-synthase from Apis mellifera.

  Oyenarte I., Majtan T., Ereno J., Corral-Rodriguez M.A., Klaudiny J., Majtan J., Kraus J.P., Martinez-Cruz L.A.

  Cystathionine β-synthase (CBS) is a pyridoxal-5'-phosphate-dependent enzyme that catalyzes the first step of the transsulfuration pathway, namely the condensation of serine with homocysteine to form cystathionine. Mutations in the CBS gene are the single most common cause of hereditary homocystinu ... >> More

  Cystathionine β-synthase (CBS) is a pyridoxal-5'-phosphate-dependent enzyme that catalyzes the first step of the transsulfuration pathway, namely the condensation of serine with homocysteine to form cystathionine. Mutations in the CBS gene are the single most common cause of hereditary homocystinuria, a multisystemic disease affecting to various extents the vasculature, connective tissues and central nervous system. At present, the crystal structure of CBS from Drosophila melanogaster is the only available structure of the full-length enzyme. Here we describe a cloning, overexpression, purification and preliminary crystallographic analysis of a full-length CBS from Apis mellifera (AmCBS) which maintains 51 and 46% sequence identity with its Drosophila and human homologs, respectively. The AmCBS yielded crystals belonging to space group P2(1)2(1)2(1), with unit-cell parameters a=85.90, b=95.87, c=180.33 Å. Diffraction data were collected to a resolution of 3.0 Å. The crystal structure contained two molecules in the asymmetric unit which presumably correspond to the dimeric species observed in solution. << Less

  Acta Crystallogr Sect F Struct Biol Cryst Commun 68:1323-1328(2012) [PubMed] [EuropePMC]