Eva Johansson

Inhibitor binding in a class 2 dihydroorotate dehydrogenase causes variations in the membrane-associated N-terminal domain.

The flavin enzyme dihydroorotate dehydrogenase (DHOD; EC 1.3.99.11) catalyzes the oxidation of dihydroorotate to orotate, the fourth step in the de novo pyrimidine biosynthesis of UMP. The enzyme is a promising target for drug design in different biological and clinical applications for cancer and arthritis. The first crystal structure of the class 2 dihydroorotate dehydrogenase from rat has been determined in complex with its two inhibitors brequinar and atovaquone. These inhibitors have shown promising results as anti‐proliferative, immunosuppressive, and antiparasitic agents. A unique feature of the class 2 DHODs is their N‐terminal extension, which folds into a separate domain comprising two α‐helices. This domain serves as the binding site for the two inhibitors and the respiratory quinones acting as the second substrate for the class 2 DHODs. The orientation of the first N‐terminal helix is very different in the two complexes of rat DHOD (DHODR). Binding of atovaquone causes a 12 Å movement of the first residue in the first α‐helix. Based on the information from the two structures of DHODR, a model for binding of the quinone and the residues important for the interactions could be defined. His 56 and Arg 136, which are fully conserved in all class 2 DHODs, seem to play a key role in the interaction with the electron acceptor. The differences between the membrane‐bound rat DHOD and membrane‐associated class 2 DHODs exemplified by the Escherichia coli DHOD has been investigated by GRID computations of the hydrophobic probes predicted to interact with the membrane.

Preliminary crystallographic analysis of the NAC domain of ANAC, a member of the plant-specific NAC transcription factor family

The NAC domain (residues 1-168) of ANAC, encoded by the abscisic acid-responsive NAC gene from Arabidopsis thaliana, was recombinantly produced in Escherichia coli and crystallized in hanging drops. Three morphologically different crystal forms were obtained within a relatively narrow range of conditions: 10-15% PEG 4000 and 0.1 M imidazole/malic acid buffer pH 7.0 in the reservoir, 3.2-7.7 mg ml(-1) protein stock and a 1:1 ratio of reservoir to protein solution in the hanging drop. One of the crystal forms, designated crystal form III, was found to be suitable for further X-ray analysis. Form III crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 62.0, b = 75.2, c = 80.8 A at 100 K. The unit-cell volume is consistent with two molecules in the asymmetric unit and a peak in the native Patterson map suggests the presence of a non-crystallographic twofold axis parallel to a crystallographic axis. Size-exclusion chromatography of the NAC domain showed that the dimeric state is also the preferred state in solution and probably represents the biologically active form. Data sets were collected from four potential heavy-atom derivatives of the form III crystals. The derivatized crystals are reasonably isomorphous with the non-derivatized crystals and the four data sets are being evaluated for use in structure determination by multiple isomorphous replacement.

Regulation of dCTP deaminase from Escherichia coli by nonallosteric dTTP binding to an inactive form of the enzyme.

The trimeric dCTP deaminase produces dUTP that is hydrolysed to dUMP by the structurally closely related dUTPase. This pathway provides 70–80% of the total dUMP as a precursor for dTTP. Accordingly, dCTP deaminase is regulated by dTTP, which increases the substrate concentration for half‐maximal activity and the cooperativity of dCTP saturation. Likewise, increasing concentrations of dCTP increase the cooperativity of dTTP inhibition. Previous structural studies showed that the complexes of inactive mutant protein, E138A, with dUTP or dCTP bound, and wild‐type enzyme with dUTP bound were all highly similar and characterized by having an ordered C‐terminal. When comparing with a new structure in which dTTP is bound to the active site of E138A, the region between Val120 and His125 was found to be in a new conformation. This and the previous conformation were mutually exclusive within the trimer. Also, the dCTP complex of the inactive H121A was found to have residues 120–125 in this new conformation, indicating that it renders the enzyme inactive. The C‐terminal fold was found to be disordered for both new complexes. We suggest that the cooperative kinetics are imposed by a dTTP‐dependent lag of product formation observed in presteady‐state kinetics. This lag may be derived from a slow equilibration between an inactive and an active conformation of dCTP deaminase represented by the dTTP complex and the dUTP/dCTP complex, respectively. The dCTP deaminase then resembles a simple concerted system subjected to effector binding, but without the use of an allosteric site.

Lid L11 of the glutamine amidotransferase domain of CTP synthase mediates allosteric GTP activation of glutaminase activity.

GTP is an allosteric activator of CTP synthase and acts to increase the kcat for the glutamine‐dependent CTP synthesis reaction. GTP is suggested, in part, to optimally orient the oxy‐anion hole for hydrolysis of glutamine that takes place in the glutamine amidotransferase class I (GATase) domain of CTP synthase. In the GATase domain of the recently published structures of the Escherichia coli and Thermus thermophilus CTP synthases a loop region immediately proceeding amino acid residues forming the oxy‐anion hole and named lid L11 is shown for the latter enzyme to be flexible and change position depending on the presence or absence of glutamine in the glutamine binding site. Displacement or rearrangement of this loop may provide a means for the suggested role of allosteric activation by GTP to optimize the oxy‐anion hole for glutamine hydrolysis. Arg359, Gly360 and Glu362 of the Lactococcus lactis enzyme are highly conserved residues in lid L11 and we have analyzed their possible role in GTP activation. Characterization of the mutant enzymes R359M, R359P, G360A and G360P indicated that both Arg359 and Gly360 are involved in the allosteric response to GTP binding whereas the E362Q enzyme behaved like wild‐type enzyme. Apart from the G360A enzyme, the results from kinetic analysis of the enzymes altered at position 359 and 360 showed a 10‐ to 50‐fold decrease in GTP activation of glutamine dependent CTP synthesis and concomitant four‐ to 10‐fold increases in KA for GTP. The R359M, R359P and G360P also showed no GTP activation of the uncoupled glutaminase reaction whereas the G360A enzyme was about twofold more active than wild‐type enzyme. The elevated KA for GTP and reduced GTP activation of CTP synthesis of the mutant enzymes are in agreement with a predicted interaction of bound GTP with lid L11 and indicate that the GTP activation of glutamine dependent CTP synthesis may be explained by structural rearrangements around the oxy‐anion hole of the GATase domain.

Structure of the dimeric form of CTP synthase from Sulfolobus solfataricus

CTP synthase catalyzes the last committed step in de novo pyrimidine-nucleotide biosynthesis. Active CTP synthase is a tetrameric enzyme composed of a dimer of dimers. The tetramer is favoured in the presence of the substrate nucleotides ATP and UTP; when saturated with nucleotide, the tetramer completely dominates the oligomeric state of the enzyme. Furthermore, phosphorylation has been shown to regulate the oligomeric states of the enzymes from yeast and human. The crystal structure of a dimeric form of CTP synthase from Sulfolobus solfataricus has been determined at 2.5u2005Å resolution. A comparison of the dimeric interface with the intermolecular interfaces in the tetrameric structures of Thermus thermophilus CTP synthase and Escherichia coli CTP synthase shows that the dimeric interfaces are almost identical in the three systems. Residues that are involved in the tetramerization of S. solfataricus CTP synthase according to a structural alignment with the E. coli enzyme all have large thermal parameters in the dimeric form. Furthermore, they are seen to undergo substantial movement upon tetramerization.

Structural and kinetic studies of the allosteric transition in Sulfolobus solfataricus uracil phosphoribosyltransferase: Permanent activation by engineering of the C-terminus.

Uracil phosphoribosyltransferase catalyzes the conversion of 5-phosphoribosyl-alpha-1-diphosphate (PRPP) and uracil to uridine monophosphate (UMP) and diphosphate (PP(i)). The tetrameric enzyme from Sulfolobus solfataricus has a unique type of allosteric regulation by cytidine triphosphate (CTP) and guanosine triphosphate (GTP). Here we report two structures of the activated state in complex with GTP. One structure (refined at 2.8-A resolution) contains PRPP in all active sites, while the other structure (refined at 2.9-A resolution) has PRPP in two sites and the hydrolysis products, ribose-5-phosphate and PP(i), in the other sites. Combined with three existing structures of uracil phosphoribosyltransferase in complex with UMP and the allosteric inhibitor cytidine triphosphate (CTP), these structures provide valuable insight into the mechanism of allosteric transition from inhibited to active enzyme. The regulatory triphosphates bind at a site in the center of the tetramer in a different manner and change the quaternary arrangement. Both effectors contact Pro94 at the beginning of a long beta-strand in the dimer interface, which extends into a flexible loop over the active site. In the GTP-bound state, two flexible loop residues, Tyr123 and Lys125, bind the PP(i) moiety of PRPP in the neighboring subunit and contribute to catalysis, while in the inhibited state, they contribute to the configuration of the active site for UMP rather than PRPP binding. The C-terminal Gly216 participates in a hydrogen-bond network in the dimer interface that stabilizes the inhibited, but not the activated, state. Tagging the C-terminus with additional amino acids generates an endogenously activated enzyme that binds GTP without effects on activity.

Concerted bifunctionality of the dCTP deaminase-dUTPase from Methanocaldococcus jannaschii: a structural and pre-steady state kinetic analysis.

Two mutant dCTP deaminase-dUTPases from Methanocaldococcus jannaschii were crystallised and the crystal structures were solved: E145A in complex with the substrate analogue alpha,beta-imido-dUTP and E145Q in complex with diphosphate. Both mutant enzymes were defect in the deaminase reaction and had reduced dUTPase activity. In the structure of E145Q in complex with diphosphate, the diphosphate occupied the same position as the beta- and gamma-phosphoryls of the nucleotide analogue in the E145A complex. The C-terminal region that is unresolved in the apo-form of the enzyme was ordered in both complexes and closed over the active site by interacting with the phosphate backbone of the nucleotide or with the diphosphate. A magnesium ion was readily observed to complex with all three phosphoryls in the nucleotide complex or with the diphosphate. A water molecule that is likely to be involved in the nucleotidyl diphosphorylase reaction was observed in the E145A:alpha,beta-imido-dUTP complex and positioned similarly as in the monofunctional trimeric dUTPase. A comparison of the active sites of the bifunctional enzyme and the monofunctional family members, dCTP deaminase and dUTPase, suggests similar reaction mechanisms. The similar side chain conformations in the deaminase site between the nucleotide and diphosphate complexes indicated a concerted re-arrangement, or induced fit, of the whole active site promoted by enzyme and nucleotide phosphoryl interactions. A pre-steady state kinetic analysis of the bifunctional reaction and the dUTPase half-reaction supported a conformational change upon substrate binding in both reactions and a concerted catalytic step for the bifunctional reaction.