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PhzD from Pseudomonas aeruginosa is an isochorismatase involved in phenazine biosynthesis. Phenazines are antimicrobial compounds that provide Pseudomonas with a competitive advantage in certain environments and may be partly responsible for the persistence of Pseudomonas infections. In vivo, PhzD catalyzes the hydrolysis of the vinyl ether functional group of 2-amino-2-deoxyisochorismate, yielding pyruvate and trans-2,3-dihydro-3-hydroxyanthranilic acid, which is then utilized in the phenazine biosynthetic pathway. PhzD also catalyzes hydrolysis of the related vinyl ethers isochorismate, chorismate, and 4-amino-4-deoxychorismate. Here we report the 1.5 A crystal structure of native PhzD, and the 1.6 A structure of the inactive D38A variant in complex with isochorismate. The structures reveal that isochorismate binds to the PhzD active site in a trans-diaxial conformation, and superposition of the structures indicates that the methylene pyruvyl carbon of isochorismate is adjacent to the side chain carboxylate of aspartate 38. The proximity of aspartate 38 to isochorismate and the complete loss of activity resulting from the conversion of aspartate 38 to alanine suggest a mechanism in which the carboxylate acts as a general acid to protonate the substrate, yielding a carbocation/oxocarbonium ion that is then rapidly hydrated to form a hemiketal intermediate, which then decomposes spontaneously to products. The structure of PhzD is remarkably similar to other structures from a subfamily of alpha/beta-hydrolase enzymes that includes pyrazinamidase and N-carbamoylsarcosine amidohydrolase. However, PhzD catalyzes unrelated chemistry and lacks a nucleophilic cysteine found in its close structural relatives. The vinyl ether hydrolysis catalyzed by PhzD represents yet another example of the catalytic diversity seen in the alpha/beta-hydrolase family, whose members are also known to hydrolyze amides, phosphates, phosphonates, epoxides, and C-X bonds.

The class kappa glutathione (GSH) transferase is an enzyme that resides in the mitochondrial matrix. Its relationship to members of the canonical GSH transferase superfamily has remained an enigma. The three-dimensional structure of the class kappa enzyme from rat (rGSTK1-1) in complex with GSH has been solved by single isomorphous replacement with anomalous scattering at a resolution of 2.5 A. The structure reveals that the enzyme is more closely related to the protein disulfide bond isomerase, dsbA, from Escherichia coli than it is to members of the canonical superfamily. The structures of rGSTK1-1 and the canonical superfamily members indicate that the proteins folds have diverged from a common thioredoxin/glutaredoxin progenitor but did so by different mechanisms. The mitochondrial enzyme, therefore, represents a fourth protein superfamily that supports GSH transferase activity. The thioredoxin domain functions in a manner that is similar to that seen in the canonical enzymes by providing key structural elements for the recognition of GSH. The hydroxyl group of S16 is within hydrogen-bonding distance of the sulfur of bound GSH and is, in part, responsible for the ionization of the thiol in the E*GSH complex (pKa = 6.4 +/- 0.1). Preequilibrium kinetic experiments indicate that the k(on) for GSH is 1 x 10(5) M(-1) s(-1) and k(off) for GS- is approximately 8 s(-1) and relatively slow with respect to turnover with 1-chloro-2, 4-dinitrobenzene (CDNB). As a result, the KM(GSH) (11 mM) is much larger than the apparent Kd(GSH) (90 microM). The active site has a relatively open access channel that is flanked by disordered loops that may explain the relatively high turnover number (280 s(-1) at pH 7.0) toward CDNB. The disordered loops form an extensive contiguous patch on one face of the dimeric enzyme, a fact that suggests that the protein surface may interact with a membrane or other protein partner.

The x-ray analysis of the monoclinic form of yeast tRNA~h. has been taken to a resolution of 2.5 A by the method of isomorphous replacement. The model ropsed at 3 A has been confirmed and extended to revea a ditional features of the tertiary structure and of the stereochemistry. An extensive hydrogen bonding network is described involving specific interactions between bases and the ribose-phosphate backbone. The structure of a -U base pair has ben solved.In this paper we present the second stage of the x-ray analysis of the monoclinic form of yeast phenylalanine tRNA. A year ago we proposed a molecular model based on the interpretation of an electron density map at 3 A resolution calculated with phases obtained from isomorphous replacement (1). A similar model was also proposed by Kim et al. (2) for the closely related orthorhombic form. Our map was of sufficient quality to trace the ribose-phosphate chain and to assign all except five nucleotides to peaks in the electron density. These nucleotides lay in a corner of the molecule where the density was strong, but could not be interpreted with as much confidence as the rest of the structure because of tight intra-and intermolecular packing, and two alternative chain tracings were given. In the rest of the molecule we were able to define unambiguously a number of base-base interactions involved in maintaining the tertiary structure, and to describe the extensive stacking interactions present. Many of these interactions could be related to invariances or semiinvariances in the generalized nucleotide sequence of tRNA ( Fig. 1). We showed how other species of tRNA could be accommodated in the same tertiary structure by coordinated changes of sequence (1, 3). The results of a companion study of the chemical reactivity of yeast tRNAPhe were found to be in good accord with the model (4, 5).The x-ray analysis has now been extended to 2.5 A by the method of isomorphous replacement using six heavy atom derivatives to produce an improved electron density map. The structure reported previously (1) has been confirmed and the tracing of the chain in the uncertain region is now unambiguous. The crystallographic discrepancy index, R, has been reduced from 46 to 39%, a value as good as that for protein structure determinations at a comparable stage. The atomic coordinates are unlikely to suffer any large changes in subsequent refinement, and are therefore being published (8).The 2.5 A model allows us to complete the detailed description of base-base interactions in the molecule (Fig. 2). It has also revealed a large number of base-backbone interactions and a new semi-invariant base pair. A hydrogen-bonding network runs through most of the molecule, apart from the exposed amino-acid and anticodon stems. A number of the ribose groups in the nonhelical regions are of the 2'-endo type rather than the 3'-endo found in RNA helices. We now have a picture of the ordered complexity of a folded RNA molecule, a complexity as great as that of a protein. An interesting by-product is...

Aminodeoxychorismate synthase is part of a heterodimeric complex that catalyzes the two-step biosynthesis of 4-amino-4-deoxychorismate, a precursor of p-aminobenzoate and folate in microorganisms. In the first step, a glutamine amidotransferase encoded by the pabA gene generates ammonia as a substrate that, along with chorismate, is used in the second step, catalyzed by aminodeoxychorismate synthase, the product of the pabB gene. Here we report the X-ray crystal structure of Escherichia coli PabB determined in two different crystal forms, each at 2.0 A resolution. The 453-residue monomeric PabB has a complex alpha/beta fold which is similar to that seen in the structures of homologous, oligomeric TrpE subunits of several anthranilate synthases of microbial origin. A comparison of the structures of these two classes of chorismate-utilizing enzymes provides a rationale for the differences in quaternary structures seen for these enzymes, and indicates that the weak or transient association of PabB with PabA during catalysis stems at least partly from a limited interface for protein interactions. Additional analyses of the structures enabled the tentative identification of the active site of PabB, which contains a number of residues implicated from previous biochemical and genetic studies to be essential for activity. Differences in the structures determined from phosphate- and formate-grown crystals, and the location of an adventitious formate ion, suggest that conformational changes in loop regions adjacent to the active site may be needed for catalysis. A surprising finding in the structure of PabB was the presence of a tryptophan molecule deeply embedded in a binding pocket that is analogous to the regulatory site in the TrpE subunits of the anthranilate synthases. The strongly bound ligand, which cannot be dissociated without denaturation of PabB, may play a structural role in the enzyme since there is no effect of tryptophan on the enzymic synthesis of aminodeoxychorismate. Extensive sequence similarity in the tryptophan-binding pocket among several other chorismate-utilizing enzymes, including isochorismate synthase, suggests that they too may bind tryptophan for structural integrity, and corroborates early ideas on the evolution of this interesting enzyme family.