Crystallization and preliminary crystallographic investigations of avian 5-aminoimidazole-4-carboxamide ribonucleotide transformylase-inosine monophosphate cyclohydrolase expressed in Escherichia coli.

ATIC [5-aminoimidazole-4-carboxamide ribonucleotide transformylase (AICAR Tfase)-inosine monophosphate cyclohydrolase (IMPCH)] is a bifunctional enzyme that catalyzes the penultimate and final steps in the de novo purine biosynthesis pathway and thus is an attractive anticancer target. Recombinant avian ATIC has been purified from an Escherichia coli expression system and crystallized in a binary complex with methotrexate (MTX). Crystals were obtained from PEG 4000 or MPEG 5000 buffered at pH 7.0-7.2 and data were collected from a single crystal at 96 K to 2.3 A resolution at the Stanford Synchrotron Radiation Laboratory (SSRL). The crystals are monoclinic and belong to space group P2(1), with unit-cell dimensions a = 65.17, b = 105.93, c = 103.47 A, beta = 108.27 degrees. Assuming two molecules per asymmetric unit, the Matthews coefficient V(m) is 2.63 A(3) Da(-1) and the solvent volume is 52.9%.

Improving telephone contact rates of patients discharged from the emergency department.

STUDY OBJECTIVE: This study investigated the rate of incorrect contact telephone numbers recorded during emergency department registration, and evaluated whether postdischarge contact rates can be improved by verifying the best contact number with the patient before discharge. METHODS: A prospective study was conducted with convenience sampling at a tertiary care hospital with an annual census of 60,000. Patients older than 18 years were enrolled, and the "unverified" telephone numbers recorded at registration were entered on the data sheet. Patients were then asked, "What number can we reach you at to discuss lab or x-ray results?" These "verified" numbers and additional demographic data were entered on the data sheet. Within 1 week, 3 calls were made to both the unverified and the verified numbers. Calls were considered successful if the patient, a friend, family member, or coworker was reached, or if the patient returned a message left on an answering machine. RESULTS: Four hundred eighteen patients (43% men) were enrolled; 72 (17%) patients provided a different best contact number than the one recorded on the chart. When unverified numbers were called, only 68.9% of patients were reachable, whereas when verified numbers were called, 81.8% of patients were contacted (P <.01). No statistical difference was found between patients who were successfully contacted and those who were not with regard to age, sex, race, or time of visit. Patients insured by health maintenance organizations were more likely to be reached (P =.02). CONCLUSION: Verification of a best contact telephone number significantly improves the ability to contact patients after ED discharge.

Crystal structures of Escherichia coli dihydrofolate reductase complexed with 5-formyltetrahydrofolate (folinic acid) in two space groups: evidence for enolization of pteridine O4.

The crystal structure of Escherichia coli dihydrofolate reductase (ecDHFR, EC 1.5.1.3) as a binary complex with folinic acid (5-formyl-5,6,7,8-tetrahydrofolate; also called leucovorin or citrovorum factor) has been solved in two space groups, P6(1) and P6(5), with, respectively, two molecules and one molecule per asymmetric unit. The crystal structures have been refined to an R-factor of 14.2% at resolutions of 2.0 and 1.9 A. The P6(1) structure is isomorphous with several previously reported ecDHFR binary complexes [Bolin, J.T., Filman, D.J., Matthews, D.A., Hamlin, R.C., & Kraut, J. (1982) J. Biol. Chem. 257, 13650-13662; Reyes, V.M., Sawaya, M.R., Brown, K.A., & Kraut, J. (1995) Biochemistry 34, 2710-2723]; enzyme and ligand conformations are very similar to the P6(1) 5,10-dideazatetrahydrofolate complex. While the two enzyme subdomains of the P6(1) structure are nearly in the closed conformation, exemplified by the methotrexate P6(1) binary complex, in the P6(5) structure they are in an intermediate conformation, halfway between the closed and the fully open conformation of the apoenzyme [Bystroff, C., Oatley, S.J., & Kraut, J. (1990) Biochemistry 29, 3263-3277]. Thus crystal packing strongly influences this aspect of the enzyme structure. In contrast to the P6(1) structure, in which the Met-20 loop (residues 9-23) is turned away from the substrate binding pocket, in the P6(5) structure the Met-20 loop blocks the pocket and protrudes into the cofactor binding site. In this respect, the P6(5) structure is unique. Additionally, positioning of a Ca2+ ion (a component of the crystallization medium) is different in the two crystal packings: in the P6(1) structure it lies at the boundary between the two molecules of the asymmetric unit, while in P6(5) it coordinates two water molecules, the hydroxyl group of an ethanol molecule, and the backbone carbonyl oxygens of Glu-17, Asn-18, and Met-20. The Ca2+ ion thus stabilizes a single turn of 3(10) helix (residues 16-18 in the Met-20 loop), a second unique feature of the P6(5) crystal structure. The disposition of the N5-formyl group in these structures indicates formation, at least half of the time, of an intramolecular hydrogen bond between the formyl oxygen and O4 of the tetrahydropterin ring. This observation is consistent with the existence of an enol-keto equilibrium in which the enolic tautomer is favored when a hydrogen-bond acceptor is present between O4 and N5. Such would be the case whenever a water molecule occupies that site as part of a hypothetical proton-relay mechanism. Two arginine side chains, Arg-52 in the P6(5) structure and Arg-44 in molecule A of the P6(1) structure, are turned away drastically from the ligand (p-aminobenzoyl)glutamic acid moiety as compared with previously reported DHFR binary complex structures. As in the ecDHFR dideazatetrahydrofolate complex, in both the P6(1) and P6(5) structures a water molecule bridges pteridine O4 and Trp-22(N epsilon 1) with ideal geometry for hydrogen bonding, perhaps contributing to the slow release of 5,6,7,8-tetrahydrofolate from the enzyme-product complex. When either the P6(1) or the P6(5) structures are superimposed with the NADPH holoenzyme [Sawaya, M. R. (1994) Ph.D. Dissertation, University of California, San Diego], we find that the distances between the nicotinamide C4 and pteridine C6 and C7 are very short, 2.1 and 1.7 A in the P6(1) case and 2.0 and 1.4 A in the P6(5) case, perhaps in part explaining the more rapid release of tetrahydrofolate from the enzyme-product complex when NADPH is bound.

Isomorphous crystal structures of Escherichia coli dihydrofolate reductase complexed with folate, 5-deazafolate, and 5,10-dideazatetrahydrofolate: mechanistic implications.

Crystal structures of Escherichia coli dihydrofolate reductase (ecDHFR, EC 1.5.1.3) in binary complexes with folate, 5-deazafolate (5dfol), and 5,10-dideazatetrahydrofolate (ddTHF) have been refined to R-factors of 13.7%, 14.9%, and 14.5%, respectively, all at 1.9 A. All three are isomorphous with a previously reported binary complex of ecDHFR with methotrexate (MTX), in space group P6(1), two molecules per asymmetric unit [Bolin, J. T., Filman, D. J., Matthews, D. A., Hamlin, R. C., & Kraut, J. (1982) J. Biol. Chem. 257, 13650-13662]. A hitherto unobserved water molecule is hydrogen bonded to the pteridine N5 and O4 in both molecules of the asymmetric unit of the folate complex (but not the 5dfol or ddTHF complexes), supporting the hypothesis that N5 protonation of bound substrate, an important step of the DHFR reaction, occurs by way of such a water molecule. There is no indication of a hydrogen bond between N8 of 5dfol and the backbone carbonyl of Ile-5, suggesting that the bacterial enzyme, unlike the human enzyme [Davies, J. F., II, Delcamp, T. J., Prendergast, N. J., Ashford, V. A., Freisheim, J. H., & Kraut, J. (1990) Biochemistry 29, 9467-9479], does not favor protonation at N8. Perhaps this explains why bacterial DHFR is much less effective than vertebrate DHFR in folate reduction. When the ecDHFR.NADPH complex (space group P3221; M. R. Sawaya, in preparation) is superimposed on the folate and 5dfol complexes, the distances from pteridine C6 to nicotinamide C4 were found to be 2.9 and 2.8 A, respectively, in close agreement with the theoretically calculated optimal distance in the transition state for hydride transfer [Wu, Y. D., & Houk, K. N. (1987) J. Am. Chem. Soc. 109, 906-908, 2226-2227]. In contrast to the planar ring system of folate or 5dfol, the reduced pteridine ring of ddTHF is severely puckered and bent toward the nicotinamide pocket, with the reduced pyridine ring assuming a half-chair type of conformation. This change in shape causes the pteridine ring to bind with O4 closer to Trp-22(N epsilon 1) by over 0.5 A, so that an invariant water molecule now bridges these two atoms with ideal hydrogen bonds. Furthermore, while the pABA rings of folate and 5dfol are nearly coincident and closer to the alpha C helix than to the alpha B helix, those of MTX and ddTHF are displaced along the binding crevice by approximately 1.1 and 0.6 A, respectively, and are equidistant from alpha B and alpha C.(ABSTRACT TRUNCATED AT 400 WORDS)

Substrate recognition and splice site determination in yeast tRNA splicing.

S. cerevisae tRNA introns interrupt the gene at a constant position in the anticodon loop. Pre-tRNAs are matured by an endonuclease and a ligase. The endonuclease alone can accurately release the intron from the pre-tRNA. Here, we investigate the mechanism of splice site selection by the endonuclease. We propose that it initially recognizes features in the mature domain common to all tRNAs. Once positioned on the enzyme, the splice sites are recognizable because they are a fixed distance from the mature domain. To test this hypothesis, we developed a system for synthesizing pre-tRNA by bacteriophage T7 RNA polymerase. To search for recognition sites, we made several mutations. Mutations of C56 and U8 strongly affect endonuclease recognition of pre-tRNA. With insertion and deletion mutations, we show that the anticodon stem determines splicing specificity. The sequence and structure of the intron are not strong determinants of splice site selection.

A synthetic substrate for tRNA splicing.

A novel method for the synthesis of precursor tRNA as substrate for in vitro splicing is reported. A construct consisting of the Saccharomyces cerevisiae pre-tRNAPhe gene under the control of a bacteriophage T7 promoter was assembled from a set of synthetic oligonucleotides and cloned into an M13 vector. By the use of T7 RNA polymerase, BstNI-runoff transcripts were produced. The resulting pre-tRNA was shown to possess mature termini and was accurately spliced by highly purified yeast tRNA-splicing endonuclease and ligase. Using this synthetic pre-tRNA, the kinetic parameters of the tRNA-splicing endonuclease were also determined. Use of this system provides several advantages for the study of tRNA-splicing mechanisms. Mutant tRNA precursors can be readily synthesized. It is also possible to synthesize large quantities of pre-tRNA for structural studies.

Mutational analysis of the coordinate expression of the yeast tRNAArg-tRNAAsp gene tandem.

tRNA genes occur in the yeast genome as highly dispersed and independent transcriptional units. The 5'-tRNAArg-tRNAAsp-3' gene tandem, separated by a 10-base-pair spacer sequence, thus represents a rare case of tight clustering. Previous in vitro studies did not reveal any primary transcript from the tRNAAsp gene, but rather a dimeric precursor containing both gene sequences plus spacer, which undergoes a series of maturation steps. This seems anomalous since the tRNAAsp gene contains the sequences necessary for its own transcription. We found that site-directed mutation of the highly conserved C at position 56 to a G in the tRNAArg gene suppresses all transcription and does not activate the tRNAAsp gene. Precise deletion of the entire tRNAArg gene gives a similar result. Rescue of tRNAAsp gene transcription is effected either by the precise deletion of both the tRNAArg gene and spacer or by the precise deletion of this gene with concomitant introduction of an artificial RNA polymerase III start site in the spacer. This artificial start site is ineffective if the tRNAArg gene is present upstream.