RESUMO
Aurodox is a member of the family of kirromycin antibiotics, which inhibit protein biosynthesis by binding to elongation factor Tu (EF-Tu). We have determined the crystal structure of the 1:1:1 complex of Thermus thermophilus EF-Tu with GDP and aurodox to 2.0-A resolution. During its catalytic cycle, EF-Tu adopts two strikingly different conformations depending on the nucleotide bound: the GDP form and the GTP form. In the present structure, a GTP complex-like conformation of EF-Tu is observed, although GDP is bound to the nucleotide-binding site. This is consistent with previous proposals that aurodox fixes EF-Tu on the ribosome by locking it in its GTP form. Binding of EF-Tu.GDP to aminoacyl-tRNA and mutually exclusive binding of kirromycin and elongation factor Ts to EF-Tu can be explained on the basis of the structure. For many previously observed mutations that provide resistance to kirromycin, it can now be understood how they prevent interaction with the antibiotic. An unexpected feature of the structure is the reorientation of the His-85 side chain toward the nucleotide-binding site. We propose that this residue stabilizes the transition state of GTP hydrolysis, explaining the acceleration of the reaction by kirromycin-type antibiotics.
Assuntos
Antibacterianos/metabolismo , Aurodox/química , Aurodox/metabolismo , Guanosina Difosfato/metabolismo , Fator Tu de Elongação de Peptídeos/química , Fator Tu de Elongação de Peptídeos/metabolismo , Antibacterianos/química , Sítios de Ligação , Guanosina Trifosfato/metabolismo , Guanilil Imidodifosfato/metabolismo , Leucina , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/metabolismo , Thermus thermophilus/metabolismo , TirosinaRESUMO
Hen egg-white lysozyme has been crystallized at slightly alkaline pH using 2-methyl-2,4-pentanediol (MPD) as the precipitant. The crystals are nearly isomorphous to crystals grown at acidic pH using sodium chloride as the precipitant. However, the growth kinetics differ markedly between the two conditions. The major reason for this is a molecule of MPD that binds tightly in between two lysozyme molecules and favors the growth of the crystals along the crystallographic c direction over growth perpendicular to it.
Assuntos
Muramidase/química , Animais , Precipitação Química , Galinhas , Cristalização , Cristalografia por Raios X , Feminino , Glicóis , Concentração de Íons de Hidrogênio , Conformação Proteica , Eletricidade EstáticaRESUMO
BACKGROUND: RNA cyclases are a family of RNA-modifying enzymes that are conserved in eucarya, bacteria and archaea. They catalyze the ATP-dependent conversion of the 3'-phosphate to the 2',3'-cyclic phosphodiester at the end of RNA, in a reaction involving formation of the covalent AMP-cyclase intermediate. These enzymes might be responsible for production of the cyclic phosphate RNA ends that are known to be required by many RNA ligases in both prokaryotes and eukaryotes. RESULTS: The high-resolution structure of the Escherichia coli RNA 3'-terminal phosphate cyclase was determined using multiwavelength anomalous diffraction. Two orthorhombic crystal forms of E. coli cyclase (space group P2(1)2(1)2(1) and P2(1)2(1)2) were used to solve and refine the structure to 2.1 A resolution (R factor 20.4%; R(free) 27.6%). Each molecule of RNA cyclase consists of two domains. The larger domain contains three repeats of a folding unit comprising two parallel alpha helices and a four-stranded beta sheet; this fold was previously identified in translation initiation factor 3 (IF3). The large domain is similar to one of the two domains of 5-enolpyruvylshikimate-3-phosphate synthase and UDP-N-acetylglucosamine enolpyruvyl transferase. The smaller domain uses a similar secondary structure element with different topology, observed in many other proteins such as thioredoxin. CONCLUSIONS: The fold of RNA cyclase consists of known elements connected in a new and unique manner. Although the active site of this enzyme could not be unambiguously assigned, it can be mapped to a region surrounding His309, an adenylate acceptor, in which a number of amino acids are highly conserved in the enzyme from different sources. The structure of E. coli cyclase will be useful for interpretation of structural and mechanistic features of this and other related enzymes.
Assuntos
Ligases/química , Sequência de Aminoácidos , Animais , Domínio Catalítico , Cristalografia por Raios X , Escherichia coli/enzimologia , Escherichia coli/genética , Humanos , Ligases/genética , Modelos Moleculares , Dados de Sequência Molecular , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Eletricidade EstáticaAssuntos
Proteínas Luminescentes/química , Proteínas de Fluorescência Verde , Histidina/química , Concentração de Íons de Hidrogênio , Íons , Luz , Modelos Químicos , Modelos Moleculares , Mutação , Fenilalanina/química , Conformação Proteica , Estrutura Quaternária de Proteína , Temperatura , Triptofano/química , Tirosina/químicaRESUMO
The amino acid sequence and tertiary structure of Wolinella succinogenes L-asparaginase were determined, and were compared with the structures of other type-II bacterial L-asparaginases. Each chain of this homotetrameric enzyme consists of 330 residues. The amino acid sequence is 40-50% identical to the sequences of related proteins from other bacterial sources, and all residues previously shown to be crucial for the catalytic action of these enzymes are identical. Differences between the amino acid sequence of W. succinogenes L-asparaginase and that of related enzymes are discussed in terms of the possible influence on the substrate specificity. The overall fold of the protein subunit is almost identical to that observed for other L-asparaginases. Two fragments in each subunit, a very highly flexible loop (approximately 20 amino acids) that forms part of the active site, and the N-terminus (two amino acids), are not defined in the structure. The orientation of Thr14, a residue probably involved in the catalytic activity, indicates the absence of ligand in the active-site pocket. The rigid part of the active site, which includes the asparaginase triad Thr93-Lys 166-Asp94, is structurally very highly conserved with equivalent regions found in other type-II bacterial L-asparaginases.
Assuntos
Asparaginase/química , Wolinella/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/química , Sítios de Ligação/genética , Clonagem Molecular , Sequência Conservada/genética , Cristalografia por Raios X , Escherichia coli , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alinhamento de Sequência , Análise de Sequência , Especificidade por SubstratoRESUMO
Escherichia coli asparaginase II catalyzes the hydrolysis of L-asparagine to L-aspartate via a threonine-bound acyl-enzyme intermediate. A nearly inactive mutant in which one of the active site threonines, Thr-89, was replaced by valine was constructed, expressed, and crystallized. Its structure, solved at 2.2 A resolution, shows high overall similarity to the wild-type enzyme, but an aspartyl moiety is covalently bound to Thr-12, resembling a reaction intermediate. Kinetic analysis confirms the deacylation deficiency, which is also explained on a structural basis. The previously identified oxyanion hole is described in more detail.
Assuntos
Asparaginase/química , Escherichia coli/enzimologia , Asparaginase/genética , Asparaginase/metabolismo , Ácido Aspártico/química , Sequência de Bases , Sítios de Ligação/genética , Cristalografia por Raios X , Escherichia coli/genética , Ligação de Hidrogênio , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos/genética , Mutação PuntualRESUMO
A thermostable aminoacylase (N-acylamino acid amidohydrolase, EC 3.5.1.14) from Bacillus stearothermophilus was overexpressed in E. coli and characterized with respect to metal content, metal dependence, heat stability, and quaternary structure. Like other enzymes of the aminoacylase family, native aminoacylase contains one Zn2+ ion per subunit. Several other transition metal ions (Co2+, Mn2+ and Cd2+) also sustain aminoacylase activity toward N-acetyl L-alanine with Cd2+ giving the highest turnover number. The stability constants of the respective metal complexes were estimated by activity measurements in metal buffer systems. Co2+ also acts as an activator mainly by lowering the Km for the substrate. These data and CD spectra obtained with the native and the metal-free enzyme suggest a predominantly structural role for the intrinsic metal ion of thermostable aminoacylase. In contrast to previous reports the enzyme behaved as a dimer in analytical gel filtration.
Assuntos
Amidoidrolases/metabolismo , Geobacillus stearothermophilus/enzimologia , Metais/química , Amidoidrolases/biossíntese , Amidoidrolases/química , Catálise , Cromatografia em Gel , Dicroísmo Circular , Cobalto/química , Cobalto/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli/enzimologia , Geobacillus stearothermophilus/genética , Concentração de Íons de Hidrogênio , Cinética , Plasmídeos , Estrutura Secundária de Proteína , Espectrofotometria Ultravioleta , Zinco/química , Zinco/metabolismoRESUMO
The domain structure of hog-kidney aminoacylase I was studied by limited proteolytic digestion with trypsin and characterization of the resulting fragments. In the native enzyme, the sequences from residue 6 to 196 and 307 to 406 are resistant to trypsin and remain tightly bound in nondenaturing solvents, while the intervening sequence (197-306) is efficiently degraded by trypsin. We conclude that the N-terminal half of the molecule and its C-terminal fourth form two independently folded domains. Both contain a peculiar PWW(A,L) sequence motif preceded by several strongly polar residues. We propose that these sequences form surface loops that mediate the membrane association of aminoacyclase I. We further show that the three free cysteine residues and the essential Zn2+ ion reside in the trypsin-resistant domains, while the intervening sequence contains the only disulfide H bond of the protein.