RESUMEN
As part of a structural genomics project on bacterial gene products of unknown function, the crystal structures of YhdH, a putative quinone oxidoreductase, and its complex with NADP have been determined at 2.25 and 2.6 A resolution, respectively. The overall fold of YhdH is very similar to that of alcohol dehydrogenases and quinone reductases despite its low sequence identity. The absence of any Zn ion indicates that YdhH is a putative quinone oxidoreductase. YhdH forms a homodimer, with each subunit composed of two domains: a catalytic domain and a coenzyme-binding domain. NADP is bound in a deep cleft formed between the two domains. Large conformational changes occur upon NADP binding, with the two domains closing up to each other and narrowing the NADP-binding cleft. Comparisons of the YdhH active site with those of the quinone oxidoreductases from Escherichia coli and Thermus thermophilus made it possible to identify essential conserved residues as being Asn41, Asp43, Asp64 and Arg318. The active-site size is very narrow and unless an induced fit occurs is accessible only to reagents the size of benzoquinone.
Asunto(s)
Complejo I de Transporte de Electrón/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Quinona Reductasas/química , Secuencia de Aminoácidos , Arginina/química , Asparagina/química , Ácido Aspártico/química , Dominio Catalítico , Clonación Molecular , Cristalografía por Rayos X , Dimerización , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Thermus thermophilus/enzimologíaRESUMEN
In the course of a structural genomics program aiming at solving the structures of Escherichia coli open reading frame products of unknown function, we have determined the structure of YadB at 1.5A using molecular replacement. The YadB protein is 298 amino acid residues long and displays 34% sequence identity with E.coli glutamyl-tRNA synthetase (GluRS). It is much shorter than GluRS, which contains 468 residues, and lacks the complete domain interacting with the tRNA anticodon loop. As E.coli GluRS, YadB possesses a Zn2+ located in the putative tRNA acceptor stem-binding domain. The YadB cluster uses cysteine residues as the first three zinc ligands, but has a weaker tyrosine ligand at the fourth position. It shares with canonical amino acid RNA synthetases a major functional feature, namely activation of the amino acid (here glutamate). It differs, however, from GluRSs by the fact that the activation step is tRNA-independent and that it does not catalyze attachment of the activated glutamate to E.coli tRNAGlu, but to another, as yet unknown tRNA. These results suggest thus a novel function, distinct from that of GluRSs, for the yadB gene family.
Asunto(s)
Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Aminoacil-ARNt Sintetasas/química , Proteínas Portadoras/metabolismo , Cristalografía por Rayos X , Proteínas de Escherichia coli/química , Genes Bacterianos , Glutamato-ARNt Ligasa/química , Glutamato-ARNt Ligasa/genética , Glutamato-ARNt Ligasa/metabolismo , Ácido Glutámico/metabolismo , Cinética , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Conformación Proteica , ARN de Transferencia de Ácido Glutámico/metabolismo , Homología de Secuencia de Aminoácido , Thermus thermophilus/enzimología , Thermus thermophilus/genética , Zinc/metabolismoRESUMEN
The first results of a medium-scale structural genomics program clearly demonstrate the value of using a medium-throughput crystallization approach based on a two-step procedure: a large screening step employing robotics, followed by manual or automated optimization of the crystallization conditions. The structural genomics program was based on cloning in the Gateway vectors pDEST17, introducing a long 21-residue tail at the N-terminus. So far, this tail has not appeared to hamper crystallization. In ten months, 25 proteins were subjected to crystallization; 13 yielded crystals, of which ten led to usable data sets and five to structures. Furthermore, the results using a robot dispensing 50-200 nl drops indicate that smaller protein samples can be used for crystallization. These still partial results might indicate present and future directions for those who have to make crucial choices concerning their crystallization platform in structural genomics programs.