RESUMEN
S-Adenosylmethionine decarboxylase belongs to a small class of amino acid decarboxylases that use a covalently bound pyruvate as a prosthetic group. It is an essential enzyme for polyamine biosynthesis and provides an important target for the design of anti-parasitic and cancer chemotherapeutic agents. We have determined the structures of S-adenosylmethionine decarboxylase complexed with the competitive inhibitors methylglyoxal bis(guanylhydrazone) and 4-amidinoindan-1-one-2'-amidinohydrazone as well as the irreversible inhibitors 5'-deoxy-5'-[N-methyl-N-[(2-aminooxy)ethyl]amino]adenosine, 5'-deoxy-5'-[N-methyl-N-(3-hydrazinopropyl)amino]adenosine, and the methyl ester analogue of S-adenosylmethionine. These structures elucidate residues important for substrate binding and show how those residues interact with both covalently and noncovalently bound inhibitors. S-Adenosylmethionine decarboxylase has a four-layer alphabeta betaalpha sandwich fold with residues from both beta-sheets contributing to substrate and inhibitor binding. The side chains of conserved residues Phe7, Phe223, and Glu247 and the backbone carbonyl of Leu65 play important roles in binding and positioning the ligands. The catalytically important residues Cys82, Ser229, and His243 are positioned near the methionyl group of the substrate. One molecule of putrescine per monomer is observed between the two beta-sheets but far away from the active site. The activating effects of putrescine may be due to conformational changes in the enzyme, to electrostatic effects, or both. The adenosyl moiety of the bound ligand is observed in the unusual syn conformation. The five structures reported here provide a framework for interpretation of S-adenosylmethionine decarboxylase inhibition data and suggest strategies for the development of more potent and more specific inhibitors of S-adenosylmethionine decarboxylase.
Asunto(s)
Adenosilmetionina Descarboxilasa/química , Adenosilmetionina Descarboxilasa/metabolismo , Estructura Terciaria de Proteína , Adenosilmetionina Descarboxilasa/antagonistas & inhibidores , Adenosilmetionina Descarboxilasa/genética , Sitios de Unión , Cristalografía por Rayos X , Humanos , Ligandos , Modelos Moleculares , Estructura Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Pliegue de Proteína , Putrescina/química , Putrescina/metabolismo , Especificidad por SustratoRESUMEN
S-Adenosylmethionine decarboxylase (AdoMetDC) is synthesized as a proenzyme that cleaves itself in a putrescine-stimulated reaction via an N-->O acyl shift and beta-elimination to produce an active enzyme with a catalytically essential pyruvoyl residue at the new N-terminus. N-->O acyl shifts initiate the self-processing of other proteins such as inteins and amidohydrolases, but their mechanisms in such proteins are not well understood. We have solved the crystal structure of the H243A mutant of AdoMetDC to 1.5 A resolution. The mutant protein is trapped in the ester form, providing clear evidence for the structure of the ester intermediate in the processing of pyruvoyl enzymes. In addition, a putrescine molecule is bound in a charged region within the beta-sandwich, and cross-links the two beta-sheets through hydrogen bonds to several acidic residues and ordered water molecules. The high-resolution structure provides insight into the mechanism for the self-processing reaction and provides evidence for the mechanism for simulation of the self-processing reaction by putrescine. Studies of the effects of putrescine or 4-aminobutanol on the processing of mutant AdoMetDC proenzymes are consistent with a model in which a single activator molecule interacts with buried Asp174, Glu178, and Glu256, leading to an alteration in the position of Glu11, resulting in stimulation of self-processing.
Asunto(s)
Adenosilmetionina Descarboxilasa/química , Precursores de Proteínas/metabolismo , Estructura Terciaria de Proteína , Putrescina/metabolismo , Adenosilmetionina Descarboxilasa/antagonistas & inhibidores , Adenosilmetionina Descarboxilasa/genética , Adenosilmetionina Descarboxilasa/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Humanos , Cinética , Modelos Moleculares , Estructura Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Procesamiento Proteico-Postraduccional , Putrescina/química , Agua/químicaRESUMEN
The zinc metallopeptidase neurolysin is shown by x-ray crystallography to have large structural elements erected over the active site region that allow substrate access only through a deep narrow channel. This architecture accounts for specialization of this neuropeptidase to small bioactive peptide substrates without bulky secondary and tertiary structures. In addition, modeling studies indicate that the length of a substrate N-terminal to the site of hydrolysis is restricted to approximately 10 residues by the limited size of the active site cavity. Some structural elements of neurolysin, including a five-stranded beta-sheet and the two active site helices, are conserved with other metallopeptidases. The connecting loop regions of these elements, however, are much extended in neurolysin, and they, together with other open coil elements, line the active site cavity. These potentially flexible elements may account for the ability of the enzyme to cleave a variety of sequences.
Asunto(s)
Metaloendopeptidasas/química , Sitios de Unión , Cristalografía por Rayos X , Modelos Moleculares , Estructura Terciaria de Proteína , Especificidad por SustratoRESUMEN
The crystal structure of 3-deoxy-d-manno-octulosonate-8-phosphate synthase (KDOPS) from Escherichia coli was determined by molecular replacement using coordinates given to us by Radaev and co-workers prior to publication. The KDOPS crystals reported by Radaev et al. were grown in the presence of 1.4 M (NH(4))(2)SO(4) and 0.4 M (K/H)(3)PO(4). They are in the cubic space group I23 (a=228.6 A) with a tetramer in the asymmetric unit; the structure has been refined with data to 2.4 A. Our crystals of E. coli KDOPS, grown in 24 % (w/v) polyethylene glycol (PEG) 1500 in the presence of the substrates, 2-phosphoenolpyruvate (PEP) and d-arabinose-5-phosphate (A5P), are also in space group I23 (a=118.2 A), with one subunit in the asymmetric unit. The medium of crystallization, 1.8 M SO(4)/PO(4) versus 24 % PEG, does not significantly affect the conformation of KDOPS. The inter-monomer contacts in both structures are the same. The beta(8)/alpha(8) loop (residues 246 to 251) situated near the entrance to the active site is not seen in the 229 A structure but can be traced in the 118 A structure. Most significantly, Radaev et al. interpreted two SO(4)/PO(4) sites in the 229 A structure as marking the phosphate positions of the substrates, PEP and A5P, after the precedent of DAHPS. In the 118 A structure the inner of these two SO(4)/PO(4) peaks is present at the same position as in the 229 A structure of KDOPS. The outer phosphate peak in the 118 A KDOPS is 3.7 A from the outer SO(4)/PO(4) peak in the 229 A structure and is within hydrogen bonding distance of Arg63 of the same subunit and Arg120 of another subunit. Based on the precedent of the d-erythrose-4-phosphate (E4P) modeled in the active site of DAHPS, we have modeled PEP and A5P in KDOPS and compared the coordination of PEP and A5P in KDOPS with that of PEP and E4P in DAHPS.
Asunto(s)
Aldehído-Liasas/química , Escherichia coli/enzimología , Pentosafosfatos/química , Fosfoenolpiruvato/química , Aldehído-Liasas/metabolismo , Secuencia de Aminoácidos , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Pentosafosfatos/metabolismo , Fosfoenolpiruvato/metabolismoRESUMEN
Anthranilate synthase catalyzes the first step in the biosynthesis of tryptophan from chorismate. The anthranilate synthase partial complex from Salmonella typhimurium has been crystallized in space group P21212 with unit-cell dimensions a = 116.7, b = 101.2 and c = 66.8 A.
Asunto(s)
Antranilato Sintasa/química , Antranilato Sintasa/aislamiento & purificación , Salmonella typhimurium/enzimología , Antranilato Sintasa/genética , Cristalización , Cristalografía por Rayos X , Escherichia coli/genética , Sustancias Macromoleculares , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Salmonella typhimurium/genéticaRESUMEN
3-Deoxy-D-manno-octulosonate-8-phosphate (KDOP) synthase catalyzes the production of KDOP from phosphoenolpyruvate (PEP) and arabinose-5-phosphate (A5P). In gram-negative bacteria KDOP is subsequently dephosphorylated, cytidylylated, and linked to lipid A and is required for lipid A incorporation into the outer membrane (Raetz, Annu. Rev. Biochem. 59:129-170, 1990). We have crystallized two forms of KDOP synthase belonging to space groups I23 or I2(1)3, one with a = b = c = 118.0 A and the other with a = b = c = 233 A.