Structure of Escherichia coli YhdH, a putative quinone oxidoreductase.

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.

Crystal structure and kinetics identify Escherichia coli YdcW gene product as a medium-chain aldehyde dehydrogenase.

In the context of a medium-scaled structural genomics program aiming at solving the structures of as many as possible bacterial unknown open reading frame products from Escherichia coli (Y prefix), we have solved the structure of YdcW at 2.1A resolution, using molecular replacement. According to its sequence identity, YdcW has been classified into the betaine aldehyde dehydrogenases family (EC 1.2.1.8), catalysing the oxidation of betaine aldehyde into glycine betaine. The structure of YdcW resembles that of other aldehyde dehydrogenases: it is tetrameric and binds a NADH molecule in each monomer. The NADH molecules, bound in the active site by soaking, are revealed to be in the "hydrolysis position". Activities experiments demonstrate that YdcW is more active on medium-chains aldehyde than on betaine aldehyde. However, soaking of betaine into YdcW crystals revealed its presence in one of the subunits, in two positions, a putative resting position and a hydride transfer ready position. Analysis of kinetics data and of the active site shape suggest an optimum binding of n-alkyl aldehydes up to seven to eight carbon atoms, possibly followed by a bulky cyclic or aromatic group.

The Escherichia coli YadB gene product reveals a novel aminoacyl-tRNA synthetase like activity.

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.

Structural genomics of the SARS coronavirus: cloning, expression, crystallization and preliminary crystallographic study of the Nsp9 protein.

The aetiologic agent of the recent epidemics of Severe Acute Respiratory Syndrome (SARS) is a positive-stranded RNA virus (SARS-CoV) belonging to the Coronaviridae family and its genome differs substantially from those of other known coronaviruses. SARS-CoV is transmissible mainly by the respiratory route and to date there is no vaccine and no prophylactic or therapeutic treatments against this agent. A SARS-CoV whole-genome approach has been developed aimed at determining the crystal structure of all of its proteins or domains. These studies are expected to greatly facilitate drug design. The genomes of coronaviruses are between 27 and 31.5 kbp in length, the largest of the known RNA viruses, and encode 20-30 mature proteins. The functions of many of these polypeptides, including the Nsp9-Nsp10 replicase-cleavage products, are still unknown. Here, the cloning, Escherichia coli expression, purification and crystallization of the SARS-CoV Nsp9 protein, the first SARS-CoV protein to be crystallized, are reported. Nsp9 crystals diffract to 2.8 A resolution and belong to space group P6(1/5)22, with unit-cell parameters a = b = 89.7, c = 136.7 A. With two molecules in the asymmetric unit, the solvent content is 60% (V(M) = 3.1 A(3) Da(-1)).

A medium-throughput crystallization approach.

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.