A common catalytic mechanism for proteins of the HutI family

Imidazolonepropionase (HutI) (imidazolone-5-propanote hydrolase, EC 3.5.2.7) is a member of the amidohydrolase superfamily and catalyzes the conversion of imidazolone-5-propanoate to N-formimino-L-glutamate in the histidine degradation pathway. We have determined the three-dimensional crystal structures of HutI from Agrobacterium tumefaciens (At-HutI) and an environmental sample from the Sargasso Sea Ocean Going Survey (Es-HutI) bound to the product [ N-formimino-L-glutamate (NIG)] and an inhibitor [3-(2,5-dioxoimidazolidin-4-yl)propionic acid (DIP)], respectively. In both structures, the active site is contained within each monomer, and its organization displays the landmark feature of the amidohydrolase superfamily, showing a metal ligand (iron), four histidines, and one aspartic acid. A catalytic mechanism involving His265 is proposed on the basis of the inhibitor-bound structure. This mechanism is applicable to all HutI forms.     

Mutability of an HNH nuclease imidazole general base and exchange of a deprotonation mechanism

Several unique protein folds that catalyze the hydrolysis of phosphodiester bonds have arisen independently in nature, including the PD(D/E)XK superfamily (typified by type II restriction endonucleases and many recombination and repair enzymes) and the HNH superfamily (found in an equally wide array of enzymes, including bacterial colicins and homing endonucleases). Whereas the identity and position of catalytic residues within the PD(D/E)XK superfamily are highly variable, the active sites of HNH nucleases are much more strongly conserved. In this study, the ability of an HNH nuclease to tolerate a mutation of its most conserved catalytic residue (its histidine general base), and the mechanism of the most active enzyme variant, were characterized. Conversion of this residue into several altered chemistries, glutamine, lysine, or glutamate, resulted in measurable activity. The histidine to glutamine mutant displays the highest residual activity and a pH profile similar to that of the wild-type enzyme. This activity is dependent on the presence of a neighboring imidazole ring, which has taken over as a less efficient general base for the reaction. This result implies that mutational pathways to alternative HNH-derived catalytic sites do exist but are not as extensively or successfully diverged or reoptimized in nature as variants of the PD(D/E)XK nuclease superfamily. This is possibly due to multiple steric constraints placed on the compact HNH motif, which is simultaneously involved in protein folding, DNA binding, and catalysis, as well as the use of a planar, aromatic imidazole group as a general base.     

Crystal structures of Pseudomonas aeruginosa guanidinobutyrase and guanidinopropionase, members of the ureohydrolase superfamily

Pseudomonas aeruginosa guanidinobutyrase (GbuA) and guanidinopropionase (GpuA) catalyze the hydrolysis of 4-guanidinobutyrate and 3-guanidinopropionate, respectively. They belong to the ureohydrolase superfamily, which includes arginase, agmatinase, proclavaminate amidinohydrolase, and formiminoglutamase. In this study, we have determined the crystal structures of GbuA and GpuA from P. aeruginosa to provide a structural insight into their substrate specificity. Although GbuA and GpuA share a common structural fold of the typical ureohydrolase superfamily, they exhibit significant variations in two active site loops. Mutagenesis of Met161 of GbuA and Tyr157 of GpuA, both of which are located in the active site loop 1 and predicted to be involved in substrate recognition, significantly affected their enzymatic properties, implying their important roles in catalysis.     

The core dimerization domains of histidine kinases contain recognition specificity for the cognate response regulator

Histidine kinases DivJ and PleC initiate signal transduction pathways that regulate an early cell division cycle step and the gain of motility later in the Caulobacter crescentus cell cycle, respectively. The essential single-domain response regulator DivK functions downstream of these kinases to catalyze phosphotransfer from DivJ and PleC. We have used a yeast two-hybrid screen to investigate the molecular basis of DivJ and PleC interaction with DivK and to identify other His-Asp signal transduction proteins that interact with DivK. The only His-Asp proteins identified in the two-hybrid screen were five members of the histidine kinase superfamily. The finding that most of the kinase clones isolated correspond to either DivJ or PleC supports the previous conclusion that DivJ and PleC are cognate DivK kinases. A 66-amino-acid sequence common to all cloned DivJ and PleC fragments contains the conserved helix 1, helix 2 sequence that forms a four-helix bundle in histidine kinases required for dimerization, autophosphorylation and phosphotransfer. We present results that indicate that the four-helix bundle subdomain is not only necessary for binding of the response regulator but also sufficient for in vivo recognition specificity between DivK and its cognate histidine kinases. The other three kinases identified in this study correspond to DivL, an essential tyrosine kinase belonging to the same kinase subfamily as DivJ and PleC, and the two previously uncharacterized, soluble histidine kinases CckN and CckO. We discuss the significance of these results as they relate to kinase response regulator recognition specificity and the fidelity of phosphotransfer in signal transduction pathways.     

Purification and properties of formylglutamate amidohydrolase from Pseudomonas putida.

Formylglutamate amidohydrolase (FGase) catalyzes the terminal reaction in the five-step pathway for histidine utilization in Pseudomonas putida. By this action, N-formyl-L-glutamate (FG) is hydrolyzed to produce L-glutamate plus formate. Urocanate, the first product in the pathway, induced all five enzymes, but FG was able to induce FGase alone, although less efficiently than urocanate did. This induction by FG resulted in the formation of an FGase with electrophoretic mobility identical to that of the FGase induced by urocanate. A 9.6-kilobase-pair HindIII DNA fragment containing the P. putida FGase gene was cloned into the corresponding site on plasmid pBEU1 maintained in Escherichia coli. Insertion of the fragment in either orientation on the vector resulted in expression, but a higher level was noted in one direction, suggesting that the FGase gene can be expressed from either of two vector promoters with different efficiencies or from a single vector promoter in addition to a less efficient Pseudomonas promoter. FGase was purified 1,110-fold from the higher-expression clone in a yield of 10% through six steps. Divalent metal ions stimulated activity, and among those tested (Co, Fe, Zn, Ca, Ni, Cd, Mn, and Mg), Co(II) was the best activator, followed by Fe(II). FGase exhibited a Km of 14 mM for FG and a specific activity of 100 mumol/min per mg of protein in the presence of 5 mM substrate and 0.8 mM CoCl2 at 30 degrees C. The enzyme was maximally active in the range of pH 7 to 8. FGase was found to be a monomer of molecular weight 50,000. N-Acetyl-L-glutamate was not a substrate for the enzyme, but both it and N-formyl-L-aspartate were competitive inhibitors of formylglutamate hydrolysis, exhibiting Ki values of 6 and 9 mM, respectively. The absence of FGase activity as an integral part of histidine breakdown in most other organisms and the somewhat uncoordinated regulation of FGase synthesis with that of the other hut enzymes in Pseudomonas suggest that the gene encoding its synthesis may have evolved separately from the remaining hut genes.     

The origin and ecological significance of multiple branches for histidine utilization in Pseudomonas aeruginosa PAO1

Pseudomonas proliferate in a wide spectrum of harsh and variable environments. In many of these environments, amino acids, such as histidine, are a valuable source of carbon, nitrogen and energy. Here, we demonstrate that the histidine uptake and utilization (hut) pathway of Pseudomonas aeruginosa PAO1 contains two branches from the intermediate formiminoglutamate to the product glutamate. Genetic analysis revealed that the four-step route is dispensable as long as the five-step route is present (and vice versa). Mutants with deletions of either the four-step (HutE) or five-step (HutFG) branches were competed against each other and the wild-type strain to test the hypothesis of ecological redundancy; that is, that the presence of two pathways confers no benefit beyond that delivered by the individual pathways. Fitness assays performed under several environmental conditions led us to reject this hypothesis; the four-step pathway can provide an advantage when histidine is the sole carbon source. An IclR-type regulator (HutR) was identified that regulates the four-step pathway. Comparison of sequenced genomes revealed that P.aeruginosa strains and P.fluorescens Pf-5 have branched hut pathways. Phylogenetic analyses suggests that the gene encoding formiminoglutamase (hutE) was acquired by horizontal gene transfer from a Ralstonia-like ancestor. Potential barriers to inter-species transfer of the hutRE module were explored by transferring it from P.aeruginosa PAO1 to P.fluorescens SBW25. Transfer of the operon conferred the ability to utilize histidine via the four-step pathway in a single step, but the fitness cost of acquiring this new operon was found to be environment dependent.     

Pseudomonas aeruginosa D-arabinofuranose biosynthetic pathway and its role in type IV pilus assembly

Pseudomonas aeruginosa strains PA7 and Pa5196 glycosylate their type IVa pilins with α1,5-linked D-arabinofuranose (d-Araf), a rare sugar configuration identical to that found in cell wall polymers of the Corynebacterineae. Despite this chemical identity, the pathway for biosynthesis of α1,5-D-Araf in Gram-negative bacteria is unknown. Bioinformatics analyses pointed to a cluster of seven P. aeruginosa genes, including homologues of the Mycobacterium tuberculosis genes Rv3806c, Rv3790, and Rv3791, required for synthesis of a polyprenyl-linked d-ribose precursor and its epimerization to D-Araf. Pa5196 mutants lacking the orthologues of those genes had non-arabinosylated pilins, poor twitching motility, and significantly fewer surface pili than the wild type even in a retraction-deficient (pilT) background. The Pa5196 pilus system assembled heterologous non-glycosylated pilins efficiently, demonstrating that it does not require post-translationally modified subunits. Together the data suggest that pilins of group IV strains need to be glycosylated for productive subunit-subunit interactions. A recombinant P. aeruginosa PAO1 strain co-expressing the genes for d-Araf biosynthesis, the pilin modification enzyme TfpW, and the acceptor PilA(IV) produced arabinosylated pili, confirming that the Pa5196 genes identified are both necessary and sufficient. A P. aeruginosa epimerase knock-out could be complemented with the corresponding Mycobacterium smegmatis gene, demonstrating conservation between the systems of the Corynebacterineae and Pseudomonas. This work describes a novel Gram-negative pathway for biosynthesis of d-Araf, a key therapeutic target in Corynebacterineae.     

Regulation of P311 expression by Met-hepatocyte growth factor/scatter factor and the ubiquitin/proteasome system

P311 is a mouse cDNA originally identified for its high expression in late-stage embryonic brain and adult cerebellum, hippocampus, and olfactory bulb. The protein product of P311, however, had not been identified previously, and its function remains unknown. We report here that P311 expression is regulated at multiple levels by pathways that control cellular transformation. P311 mRNA expression was decreased sharply in both neural and smooth muscle cells when the cells were transformed by coexpression of the oncogenic tyrosine kinase receptor Met and its ligand hepatocyte growth factor/scatter factor. The P311 mRNA was found to encode an 8-kDa polypeptide that was subject to rapid degradation by the lactacystin-sensitive ubiquitin/proteasome system and an unidentified metalloprotease, resulting in a protein half-life of about 5 min. These data suggest that P311 expression is dramatically decreased by several pathways that regulate cellular growth.     

Rad51 protein from the thermotolerant yeast Pichia angusta as a typical but thermodependent member of the Rad51 family

The Rad51 protein from the methylotrophic yeast Pichia angusta (Rad51(Pa)) of the taxonomic complex Hansenula polymorpha is a homolog of the RecA-RadA-Rad51 protein superfamily, which promotes homologous recombination and recombination repair in prokaryotes and eukaryotes. We cloned the RAD51 gene from the cDNA library of the thermotolerant P. angusta strain BKM Y1397. Induction of this gene in a rad51-deficient Saccharomyces cerevisiae strain partially complemented the survival rate after ionizing radiation. Purified Rad51(Pa) protein exhibited properties typical of the superfamily, including the stoichiometry of binding to single-stranded DNA (ssDNA) (one protomer of Rad51(Pa) per 3 nucleotides) and DNA specificity for ssDNA-dependent ATP hydrolysis [poly(dC) > poly(dT) > phiX174 ssDNA > poly(dA) > double-stranded M13 DNA]. An inefficient ATPase and very low cooperativity for ATP interaction position Rad51(Pa) closer to Rad51 than to RecA. Judging by thermoinactivation, Rad51(Pa) alone was 20-fold more thermostable at 37 degrees C than its S. cerevisiae homolog (Rad51(Sc)). Moreover, it maintained ssDNA-dependent ATPase and DNA transferase activities up to 52 to 54 degrees C, whereas Rad51(Sc) was completely inactive at 47 degrees C. A quick nucleation and an efficient final-product formation in the strand exchange reaction promoted by Rad51(Pa) occurred only at temperatures above 42 degrees C. These reaction characteristics suggest that Rad51(Pa) is dependent on high temperatures for activity.     

ADP-ribosylation of cyclophilin A by Pseudomonas aeruginosa exoenzyme S

The virulence of the opportunistic pathogen Pseudomonas aeruginosa (Pa) is in part mediated by the type III secretion (TTS) of bacterial proteins into eukaryotic hosts. Exoenzyme S (ExoS) is a bifunctional Pa TTS effector protein, with GTPase-activating (GAP) and ADP-ribosyltransferase (ADPRT) activities. Known cellular substrates of TTS-translocated ExoS (TTS-ExoS) ADPRT activity include proteins in the Ras superfamily and ERM family proteins. This study describes the ADP-ribosylation of a non-G-protein substrate of TTS-ExoS, cyclophilin A (CpA), a peptidyl-prolyl isomerase (PPIase). Four novel 17 kDa proteins (pI 6.5-6.8) were recognized in a proteomic screen of lysates of human epithelial cells that had been exposed to ExoS-producing Pa, but not an isogenic non-ExoS producing strain. The proteins were identified as isoforms of CpA using MALDI-TOF mass spectrometry and confirmed by Western blotting. Mutagenesis analysis identified arginine 55 and 69 of CpA as sites of ExoS ADP-ribosylation. Examination of the effect of ExoS ADP-ribosylation on CpA function found a moderate (19%) decrease in prolyl isomerization of a Xaa-Pro containing peptides. In comparison, GST-CpA co-immunoprecipitation studies found ExoS ADP-ribosylation of CpA to efficiently inhibit CpA binding to calcineurin/PP2B phosphatase. Our results support that ExoS ADP-ribosylates and affects the function of the cytosolic protein, CpA, with the predominant functional effect relating to interference of CpA-cellular protein interactions.     

Cloning, structure, and expression of the Escherichia coli K-12 hisC gene.

We used an expression vector plasmid containing the Escherichia coli K-12 histidine operon regulatory region to subclone the E. coli hisC gene. Analysis of plasmid-coded proteins showed that hisC was expressed in minicells. A protein with an apparent molecular weight of 38,500 was identified as the primary product of the hisC gene. Expression was under control of the hisGp promoter and resulted in very efficient synthesis (over 100-fold above the wild-type levels) of imidazolylacetolphosphate:L-glutamate aminotransferase, the hisC gene product. The complete nucleotide sequence of the hisC gene has been determined. The gene is 1,071 nucleotides long and codes for a protein of 356 amino acids with only one histidine residue.     

Evolution of function in the crotonase superfamily: (3S)-methylglutaconyl-CoA hydratase from Pseudomonas putida

Members of the enoyl-CoA hydratase (crotonase) superfamily catalyze different overall reactions that utilize a common catalytic strategy delivered by a shared structural scaffold; the substrates are usually acyl esters of coenzyme A, and the intermediates are usually thioester enolate anions stabilized by a conserved oxyanion hole. In many bacterial genomes, orthologous members that contain homologues of acid/base catalyst Glu164 but not of Glu144 in rat mitochondrial crotonase are encoded by operons of which the functions have not been assigned. Focusing on the orthologues from Pseudomonas aeruginosa and P. putida, we have determined that these operons encode enzymes in leucine catabolism with the unknown enzyme assigned as (3S)-methylglutaconyl-CoA hydratase (MGCH), which catalyzes the syn-hydration of (E)-3-methylglutaconyl-CoA to (3S)-hydroxymethylglutaryl-CoA. The discovery that bacterial MGCHs catalyze hydration of enoyl-CoAs utilizing a single active-site residue contrasts with the paradigm crotonases as well as with the recently identified mammalian MGCHs that use homologues of both Glu144 and Glu164 in crotonase. Substrate analogues lacking a gamma-carboxylate have been shown to be competitive inhibitors of the enzyme, and installation of a glutamate for the "missing" homologue of Glu144 fails to introduce hydratase activity with the substrate analogues. Thus, bacterial MGCHs may provide an example of opportunistic evolution in which a carboxylate group of the substrate functionally replaces one of the active site glutamate residues in the reactions catalyzed by crotonases and the eukaryotic MGCHs.     

Structures of the core oligosaccharide and O-units in the R- and SR-type lipopolysaccharides of reference strains of Pseudomonas aeruginosa O-serogroups

Highly phosphorylated core oligosaccharides and those substituted with one O-antigen repeating unit were obtained by mild acid degradation or strong alkaline hydrolysis of lipopolysaccharide samples from 23 reference strains representing all Pseudomonas aeruginosa O-serogroups. Studies by high-resolution electrospray ionization mass spectrometry and two-dimensional NMR spectroscopy revealed both conserved and variable structural features of the lipopolysaccharides of various O-serogroups. The upstream terminal saccharide of the O-antigen, which contributes most to the immunospecificity of the bacteria, was defined in 11 from a total of 13 O-serogroups. The data obtained link together the known biosynthesis pathways, genetics and serology of the P. aeruginosa lipopolysaccharide.     

The RGK family: a regulatory tail of small GTP-binding proteins

RGK proteins are small Ras-related GTP-binding proteins that function as potent inhibitors of voltage-dependent calcium channels, and two members of the family, Gem and Rad, modulate Rho-dependent remodeling of the cytoskeleton. Within the Ras superfamily, RGK proteins have distinct structural and regulatory characteristics. It is an open question as to whether RGK proteins catalyze GTP hydrolysis in vivo. Binding of calmodulin and the 14-3-3 protein to RGK proteins controls downstream pathways. Here, we discuss the structural and functional properties of RGK proteins and highlight recent work by Beguin and colleagues addressing the mechanism of Gem regulation by calmodulin and 14-3-3.     

Evolutionary trace analysis of the alpha-D-phosphohexomutase superfamily

The alpha-D-phosphohexomutase superfamily is composed of four related enzymes that catalyze a reversible, intramolecular phosphoryl transfer on their sugar substrates. The enzymes in this superfamily play important and diverse roles in carbohydrate metabolism in organisms from bacteria to humans. Recent structural and mechanistic studies of one member of this superfamily, phosphomannomutase/phosphoglucomutase (PMM/PGM) from Pseudomonas aeruginosa, have provided new insights into enzyme mechanism and substrate recognition. Here we use sequence-sequence and sequence-structure comparisons via evolutionary trace analysis to examine 71 members of the alpha-D-phosphohexomutase superfamily. These analyses show that key residues in the active site, including many of those involved in substrate contacts in the P. aeruginosa PMM/PGM complexes, are conserved throughout the enzyme family. Several important regions show class-specific differences in sequence that appear to be correlated with differences in substrate specificity exhibited by subgroups of the family. In addition, we describe the translocation of a 20-residue segment containing the catalytic phosphoserine of phosphoacetylglucosamine mutase, which uniquely identifies members of this subgroup.