The three-dimensional structure of a ternary complex of the purine repressor, PurR, bound to both its corepressor, hypoxanthine, and the 16-base pair purF operator site has been solved at 2.7 A resolution by x-ray crystallography. The bipartite structure of PurR consists of an amino-terminal DNA-binding domain and a larger carboxyl-terminal corepressor binding and dimerization domain that is similar to that of the bacterial periplasmic binding proteins. The DNA-binding domain contains a helix-turn-helix motif that makes base-specific contacts in the major groove of the DNA. Base contacts are also made by residues of symmetry-related alpha helices, the "hinge" helices, which bind deeply in the minor groove. Critical to hinge helix-minor groove binding is the intercalation of the side chains of Leu54 and its symmetry-related mate, Leu54', into the central CpG-base pair step. These residues thereby act as "leucine levers" to pry open the minor groove and kink the purF operator by 45 degrees.

Activation of gluatmine phosphoribosylpyrophosphate (RPPP) amidotransferase (GPATase) by binding of a PRPP substrate analog results in the formation of a 20 A channel connecting the active site for glutamine hydrolysis in one domain with the PRPP site in a second domain. This solvent-inaccessible channel permits transfer of the NH3 intermediate between the two active sites. Tunneling of NH3 may be a common mechanism for glutamine amidotransferase-catalyzed nitrogen transfer and for coordination of catalysis at two distinct active sites in complex enzymes. The 2.4 A crystal structure of the active conformer of GPATase also provides the first description of an intact active site for the phosphoribosyltransferase (PRTase) family of nucleotide synthesis and salvage enzymes. Chemical assistance to catalysis is provided primarily by the substrate and secondarily by the enzyme in the proposed structure-based mechanism. Different catalytic and inhibitory modes of divalent cation binding to the PRTase active site are revealed in the active conformer of the enzyme and in a feedback-inhibited GMP complex.

Multi-wavelength anomalous diffraction (MAD) has been used to determine the structure of the regulatory enzyme of de novo synthesis of purine nucleotides, glutamine 5-phosphoribosyl-1-pyrophosphate (PRPP) amidotransferase, from Bacillus subtilis. This allosteric enzyme, a 200-kilodalton tetramer, is subject to end product regulation by purine nucleotides. The metalloenzyme from B. subtilis is a paradigm for the higher eukaryotic enzymes, which have been refractory to isolation in stable form. The two folding domains of the polypeptide are correlated with functional domains for glutamine binding and for transfer of ammonia to the substrate PRPP. Eight molecules of the feedback inhibitor adenosine monophosphate (AMP) are bound to the tetrameric enzyme in two types of binding sites: the PRPP catalytic site of each subunit and an unusual regulatory site that is immediately adjacent to each active site but is between subunits. An oxygen-sensitive [4Fe-4S] cluster in each subunit is proposed to regulate protein turnover in vivo and is distant from the catalytic site. Oxygen sensitivity of the cluster is diminished by AMP, which blocks a channel through the protein to the cluster. The structure is representative of both glutamine amidotransferases and phosphoribosyltransferases.

No abstract

The modulation of the affinity of DNA-binding proteins by small molecule effectors for cognate DNA sites is common to both prokaryotes and eukaryotes. However, the mechanisms by which effector binding to one domain affects DNA binding by a distal domain are poorly understood structurally. In initial studies to provide insight into the mechanism of effector-modulated DNA binding of the lactose repressor family, we determined the crystal structure of the purine repressor bound to a corepressor and purF operator. To extend our understanding, we have determined the structure of the corepressor-free corepressor-binding domain of the purine repressor at 2.2 A resolution. In the unliganded state, structural changes in the corepressor-binding pocket cause each subunit to rotate open by as much as 23 degrees, the consequences of which are the disengagement of the minor groove-binding hinge helices and repressor-DNA dissociation.

Transcription of the Bacillus subtilis pur operon is repressed in response to a signal of excess adenine. We have purified the repressor protein and have identified, cloned, and overexpressed the purR regulatory gene that controls transcription initiation of the operon. B. subtilis purR encodes a 62-kDa homodimer that binds to the pur operon control region. The PurR binding site which overlaps the promoter encompasses "110 bp. The protein-DNA interaction is inhibited by 5-phosphoribosyl 1-pyrophosphate. A mutation that deletes the repressor binding site or one that disruptspurR abolishes binding activity in vitro and repression of transcription in vivo in response to the excess adenine signal. These results lead to a model in which an excessadenine signal is transmitted to PurR via the 5-phosphoribosyl 1-pyrophosphate pool. In addition, purR is autoregulated. There is no structural or mechanistic similarity between the B. subtilis and Escherichia coli purine repressors.The Bacillus subtilis purEKBC(orf)QLFMNHD operon (where orf is an open reading frame of unknown function), herein called thepur operon, encodes the 10 enzymes required for de novo synthesis of IMP (1). Transcription of the 12-gene operon from a oA-dependent promoter is initiated 242 nt upstream of thepurE, the first structural gene. Transcription of the operon is subject to dual regulation. Addition of adenine to cells results in repression of transcription initiation, whereas guanine addition signals premature transcription termination in the mRNA leader region preceding the purE gene (1, 2). A putative repressor protein that binds to the pur operon 5' flanking region has been partially purified (3). Although binding of the protein was specific for pur operon 5' flanking DNA, it was independent of putative effector molecules such as adenine, adenosine, or adenine nucleotides. The DNA site to which the protein bound was mapped to nt -145 to -29 relative to the start of transcription. A deletion ofpur operon 5' flanking DNA from -193 to -64 abolished repression by adenine whereas guanine-mediated regulation was retained. This finding supported the idea that the extended protein binding site identified in vitro was in fact a control site for repression of transcription. We now report a definitive identification of the B. subtilispur operon repressor and a model for regulation. MATERIALS AND METHODSBacterial Strains. The B. subtilis stains that were used included DEl, the prototrophic wild type (1); DE64, a Purl strain withpur operon control-site deletion from -193 to -64 (relative to +1, the start of transcription) (3); DElCZ, DE1, purC-lacZ CmR (3); DE64 CZ, DE64 purC-lacZ CmR (3); MWR, DE1 purR NmR, MWRCZ, DElCZ purR CmR NmR; MWR64CZ, DE64CZpurR CmR NmR. The purC-lacZ fusion was integrated into the pur operon, resulting in a Purphenotype. Strain MWRZ1 is DEl purR-lacZ purR+ CmR. A purR-lacZ translational fusion was integrated by homologous recombination into purR. Strain MWRZ2 is MWRZ1 purE::neo CmR NmR. Bacteria were grown in minimal (1) ...

The Escherichia coli pur regulon repressor protein was overproduced in a phage T7 expression system. The overexpressed repressor constituted approximately 35% of the soluble cellular protein. Pur repressor was purified to near homogeneity by two chromatographic steps. Hypoxanthine or guanine was required for binding of purified repressor to purF operator DNA. Apparent dissociation constants of 3.4 nM were determined for binding of holorepressor to purF operator and of 1.7 and 7.1 ,uM were determined for aporepressor interaction with guanine and hypoxanthine, respectively. A requirement for hypoxanthine or guanine for conversion of aporepressor to holorepressor in vitro supports the earlier report (U. Houlberg and K. F. Jensen, J. Bacteriol. 153:837-845, 1983) that these purine bases are involved in regulation of pur gene expression in Salmonella typhimurium and confirms that hypoxanthine and guanine are corepressors.

Glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase from Escherichia coli exhibits a basal PRPP-independent glutaminase activity having a kcat/Km that is 0.3% of fully active enzyme. Binding of PRPP activates the enzyme by a structural change that lowers the Km for glutamine 100-fold and couples glutamine hydrolysis to synthesis of 5-phosphoribosylamine. By analysis of the x-ray structure of the glutamine site containing bound 6-diazo-5-oxonorleucine, a glutamine affinity analog, and by site-directed mutagenesis we have identified residues important for glutamine binding, catalysis, and coupling with PRPP. Tyr74 is a key residue in the coupling between the sites for glutamine in the NH2-terminal domain and PRPP in the COOH-terminal domain. Arg73 and Asp127 have roles in glutamine binding. The x-ray structure indicates that there are no amino acid side chains sufficiently close to Cys1 to participate as a proton acceptor in formation of the thiolate needed for nucleophilic attack on the carboxamide of glutamine, nor as a general acid for amide nitrogen transfer. Based on the x-ray model of the glutamine site and analysis of a mutant enzyme we propose that the free NH2 terminus of Cys1 functions as the proton acceptor and donor. The results indicate that the side chain of Asn101 and the backbone nitrogen of Gly102 function to stabilize a tetrahedral oxyanion resulting from attack of Cys1 on the glutamine carboxamide. Cys1, Arg73, Asn101, Gly102, and Asp127 are conserved in the NH2-terminal domain of a subfamily of amidotransferases that includes asparagine synthetase, glucosamine 6-phosphate synthase, and glutamate synthase, implying a common function in the four enzymes. Tyr74, on the other hand, is conserved only in glutamine PRPP amidotransferase sequences consistent with a specific role in interdomain coupling. The catalytic framework of key glutamine site residues supports the assignment of glutamine PRPP amidotransferase to a recently described Ntn (NH2-terminal nucleophile) hydrolase family of enzymes.