Mutagenesis was used to probe the interface between the small GTPase Cdc42p and the CRIB domain motif of Ste20p. Members of a cluster of hydrophobic residues of Cdc42p were changed to alanine and/or arginine. The interaction of the wild-type and mutant proteins was measured using the two-hybrid assay; many, but not all, changes reduced interaction between Cdc42p and the target CRIB domain. Mutations in conserved residues in the CRIB domain were also tested for their importance in the association with Cdc42p. Two conserved CRIB domain histidines were changed to aspartic acid. These mutants reduced mating, as well as responsiveness to pheromone-induced gene expression and cell cycle arrest, but did not reduce in vitro the kinase activity of Ste20p. GFP-tagged mutant proteins were unable to localize to sites of polarized growth. In addition, these point mutants were synthetically lethal with disruption of CLA4 and blocked the Ste20p-Cdc42p two-hybrid interaction. Compensatory mutations in Cdc42p that reestablished the two-hybrid association with the mutant Ste20p CRIB domain baits were identified. These mutations improved the pheromone responsiveness of cells containing the CRIB mutations, but did not rescue the lethality associated with the CRIB mutant CLA4 deletion interaction. These results suggest that the Ste20p-Cdc42p interaction plays a direct role in Ste20p kinase function and that this interaction is required for efficient activity of the pheromone response pathway.

The histidine biosynthetic pathway is an ancient one found in bacteria, archaebacteria, fungi, and plants that converts 5-phosphoribosyl 1-pyrophosphate to l -histidine in 10 enzymatic reactions. This pathway provided a paradigm for the operon, transcriptional regulation of gene expression, and feedback inhibition of a pathway. l -histidinol dehydrogenase (HisD, EC 1.1.1.23 ) catalyzes the last two steps in the biosynthesis of l -histidine: sequential NAD-dependent oxidations of l -histidinol to l -histidinaldehyde and then to l -histidine. HisD functions as a homodimer and requires the presence of one Zn 2+ cation per monomer. We have determined the three-dimensional structure of Escherichia coli HisD in the apo state as well as complexes with substrate, Zn 2+ , and NAD + (best resolution is 1.7 Å). Each monomer is made of four domains, whereas the intertwined dimer possibly results from domain swapping. Two domains display a very similar incomplete Rossmann fold that suggests an ancient event of gene duplication. Residues from both monomers form the active site. Zn 2+ plays a crucial role in substrate binding but is not directly involved in catalysis. The active site residue His-327 participates in acid-base catalysis, whereas Glu-326 activates a water molecule. NAD + binds weakly to one of the Rossmann fold domains in a manner different from that previously observed for other proteins having a Rossmann fold.

In Escherichia coli, RlmB catalyzes the methylation of guanosine 2251, a modification conserved in the peptidyltransferase domain of 23S rRNA. The crystal structure of this 2'O-methyltransferase has been determined at 2.5 A resolution. RlmB consists of an N-terminal domain connected by a flexible extended linker to a catalytic C-terminal domain and forms a dimer in solution. The C-terminal domain displays a divergent methyltransferase fold with a unique knotted region, and lacks the classic AdoMet binding site features. The N-terminal domain is similar to ribosomal proteins L7 and L30, suggesting a role in 23S rRNA recognition. The conserved residues in this novel family of 2'O-methyltransferases cluster in the knotted region, suggesting the location of the catalytic and AdoMet binding sites.

We have initiated a structural genomics project based on selected protein families, with the representatives for structural studies obtained from the genome of E. coli.A total of 160 genes have been cloned to date, with 126 successfully overexpressing soluble protein with at least one fusion system.We are currently applying robotics methods allowing cloning in an automated manner.Target genes have been cloned as N-terminal fusions with GST, (His)6, or (His)8 affinity tags.Over 60 proteins have so far been purified to homogeneity.Purified proteins are further characterized for homogeneity and suitable solution properties using a combination of dynamic light scattering and electrophoretic methods.Purified protein samples are screened for initial crystallization conditions in 96-well format using a sparse-matrix approach.A specialized database has been developed to follow the progress of various proteins and to store all relevant experimental data (http://sgen.bri.nrc.ca/bsgi).Web-based software for displaying and searching the information from this local database and for tracking deposited structures in the PDB as well as the progress of other structural genomics projects has been developed.To date, crystals have been obtained for 37 of the purified proteins.of these, diffraction quality crystals were obtained for 16 proteins.Using SeMet-substituted proteins we have determined the structures of 13 of these proteins by MAD phasing.Structures determined include 2amino-3-ketobutyrate CoA ligase, part of the threonine salvage pathway, MoeA protein, involved in molydopterin biosynthesis, histidinol phosphate aminotransferase and L-histidinol dehydrogenase, two enzymes associated with histidine biosynthesis, and RsuA, a 16S rRNA psuedouridine synthase.

2-Amino-3-ketobutyrate CoA ligase (KBL, EC 2.3.1.29) is a pyridoxal phosphate (PLP) dependent enzyme, which catalyzes the second reaction step on the main metabolic degradation pathway for threonine. It acts in concert with threonine dehydrogenase and converts 2-amino-3-ketobutyrate, the product of threonine dehydrogenation by the latter enzyme, with the participation of cofactor CoA, to glycine and acetyl-CoA. The enzyme has been well conserved during evolution, with 54% amino acid sequence identity between the Escherichia coli and human enzymes. We present the three-dimensional structure of E. coli KBL determined at 2.0 Angstrom resolution. KBL belongs to the alpha family of PLP-dependent enzymes, for which the prototypic member is aspartate aminotransferase. Its closest structural homologue is E. coli 8-amino-7-oxononanoate synthase. Like many other members of the ct family, the functional form of KBL is a dimer, and one such dimer is found in the asymmetric unit in the crystal. There are two active sites per dimer, located at the dimer interface. Both monomers contribute side chains to each active/ substrate binding site. Electron density maps indicated the presence in the crystal of the Schiff base intermediate of 2-amino-3-ketobutyrate and PLP, an external aldimine, which remained bound to KBL throughout the protein purification procedure. The observed interactions between the aldimine and the side chains in the substrate binding site explain the specificity for the substrate and provide the basis for a detailed proposal of the reaction mechanism of KBL. A putative binding site of the CoA cofactor was assigned, and implications for the cooperation with threonine dehydrogenase were considered.

A pilot project based on gene targets selected from the genome of E. coli has been initiated with 38 genes for initial cloning. Of these, 18 proteins have been purified to date and some crystals were obtained for twelve of them. Of these, four proteins yielded crystals diffracting to a sufficiently high resolution to warrant structural investigation. We have determined 3-D structures of three of these proteins using Se-Met labeling and MAD methods, while the structure of the fourth one was simultaneously determined by another group. To manage the parallel work on many proteins by several researchers it became necessary to create a searchable database containing the pertinent information about every stage of the work.

MoeA is involved in synthesis of the molybdopterin cofactor, although its function is not yet clearly defined. The three-dimensional structure of the Escherichia coli protein was solved at 2.2 A resolution. The locations of highly conserved residues among the prokaryotic and eukaryotic MoeA homologs identifies a cleft in the dimer interface as the likely functional site. Of the four domains of MoeA, domain 2 displays a novel fold and domains 1 and 4 each have only one known structural homolog. Domain 3, in contrast, is structurally similar to many other proteins. The protein that resembles domain 3 most closely is MogA, another protein required for molybdopterin cofactor synthesis. The overall similarity between MoeA and MogA, and the similarities in a constellation of residues that are strongly conserved in MoeA, suggests that these proteins bind similar ligands or substrates and may have similar functions.

A microbial community of a compost biofilter treating toluene vapors was investigated using serum-bottle assays and mineral-agar plates. Toluene was not consumed in the absence of oxygen. However, filter-bed extracts exposed to toluene vapor as the only carbon source produced distinct colony types (phenotypic groups) that were counted separately. Strains from each group were isolated and checked for toluene-degradation activity in serum bottles. Only 15% of colonies were true toluene degraders. This population was divided into 11 genotypic groups based on DNA fingerprints. Identification of a member of each group using 16S rRNA gene-sequence comparison showed that they belonged to seven genera: Acinetobacter, Azoarcus, Mycobacterium, Nevskia, Pseudomonas, Pseudonocardia and Rhodococcus. Together, members of the genera Pseudonocardia and Rhodococcus were 34 times more numerous than all the others. We hypothesized that these two organisms are K-strategists (adapted to a resource-restricted and crowded environment) and that the compost biofilter is a K-environment. This would explain why they are not outnumbered by faster growers like Pseudomonas or Acinetobacter species, which would be r-strategists (adapted to a resource-abundant and uncrowded environment).