Isolation and Characterization of a Histidine Biosynthetic Gene in Arabidopsis Encoding a Polypeptide with Two Separate Domains for Phosphoribosyl-ATP Pyrophosphohydrolase and Phosphoribosyl-AMP Cyclohydrolase

Phosphoribosyl-ATP pyrophosphohydrolase (PRA-PH) and phosphoribosyl-AMP cyclohydrolase (PRA-CH) are encoded by HIS4 in yeast and by hisIE in bacteria and catalyze the second and the third step, respectively, in the histidine biosynthetic pathway. By complementing a hisI mutation of Escherichia coli with an Arabidopsis cDNA library, we isolated an Arabidopsis cDNA (At-IE) that possesses these two enzyme activities. The At-IE cDNA encodes a bifunctional protein of 281 amino acids with a calculated molecular mass of 31,666 D. Genomic DNA-blot analysis with the At-IE cDNA as a probe revealed a single-copy gene in Arabidopsis, and RNA-blot analysis showed that the At-IE gene was expressed ubiquitously throughout development. Sequence comparison suggested that the At-IE protein has an N-terminal extension of about 50 amino acids with the properties of a chloroplast transit peptide. We demonstrated through heterologous expression studies in E. coli that the functional domains for the PRA-CH (hisI) and PRA-PH (hisE) resided in the N-terminal and the C-terminal halves, respectively, of the At-IE protein.

Complex regulation of human inducible nitric oxide synthase gene transcription by Stat 1 and NF-κB

The human inducible nitric oxide synthase (hiNOS) gene is expressed in several disease states and is also important in the normal immune response. Previously, we described a cytokine-responsive enhancer between −5.2 and −6.1 kb in the 5′-flanking hiNOS promoter DNA, which contains multiple nuclear factor κβ (NF-κB) elements. Here, we describe the role of the IFN-Jak kinase-Stat (signal transducer and activator of transcription) 1 pathway for regulation of hiNOS gene transcription. In A549 human lung epithelial cells, a combination of cytokines tumor necrosis factor-α, interleukin-1β, and IFN-γ (TNF-α, IL-1β, and IFN-γ) function synergistically for induction of hiNOS transcription. Pharmacological inhibitors of Jak2 kinase inhibit cytokine-induced Stat 1 DNA-binding and hiNOS gene expression. Expression of a dominant-negative mutant Stat 1 inhibits cytokine-induced hiNOS reporter expression. Site-directed mutagenesis of a cis-acting DNA element at −5.8 kb in the hiNOS promoter identifies a bifunctional NF-κB/Stat 1 motif. In contrast, gel shift assays indicate that only Stat 1 binds to the DNA element at −5.2 kb in the hiNOS promoter. Interestingly, Stat 1 is repressive to basal and stimulated iNOS mRNA expression in 2fTGH human fibroblasts, which are refractory to iNOS induction. Overexpression of NF-κB activates hiNOS promoter–reporter expression in Stat 1 mutant fibroblasts, but not in the wild type, suggesting that Stat 1 inhibits NF-κB function in these cells. These results indicate that both Stat 1 and NF-κB are important in the regulation of hiNOS transcription by cytokines in a complex and cell type-specific manner.

Site-directed spin labeling and chemical crosslinking demonstrate that helix V is close to helices VII and VIII in the lactose permease of Escherichia coli.

Site-directed chemical cleavage of lactose permease indicates that helix V is in close proximity to helices VII and VIII. To test this conclusion further, permease containing a biotin-acceptor domain and paired Cys residues at positions 148 (helix V) and 228 (helix VII), 148 and 226 (helix VII), or 148 and 275 (helix VIII) was affinity purified and labeled with a sulfhydryl-specific nitroxide spin label. Spin-spin interactions are observed with the 148/228 and 148/275 pairs, indicating close proximity between appropriate faces of helix V and helices VII and VIII. Little or no interaction is evident with the 148/226 pair, in all likelihood because position 226 is on the opposite face of helix VII from position 228. Broadening of the electron paramagnetic resonance spectra in the frozen state was used to estimate distance between the 148/228 and the 148/275 pairs. The nitroxides at positions 148 and 228 or 148 and 275 are within approximately 13-15 A. Finally, Cys residues at positions 148 and 228 are crosslinked by dibromobimane, a bifunctional crosslinker that is approximately 5 A. long, while no crosslinking is detected between Cys residues at positions 148 and 275 or 148 and 226. The results provide strong support for a structure in which helix V is in close proximity to both helices VII and VIII and is oriented in such a fashion that Cys-148 is closer to helix VII.

Interaction between human tRNA synthetases involves repeated sequence elements.

Aminoacyl-tRNA synthetases (tRNA synthetases) of higher eukaryotes form a multiprotein complex. Sequence elements that are responsible for the protein assembly were searched by using a yeast two-hybrid system. Human cytoplasmic isoleucyl-tRNA synthetase is a component of the multi-tRNA synthetase complex and it contains a unique C-terminal appendix. This part of the protein was used as bait to identify an interacting protein from a HeLa cDNA library. The selected sequence represented the internal 317 amino acids of human bifunctional (glutamyl- and prolyl-) tRNA synthetase, which is also known to be a component of the complex. Both the C-terminal appendix of the isoleucyl-tRNA synthetase and the internal region of bifunctional tRNA synthetase comprise repeating sequence units, two repeats of about 90 amino acids, and three repeats of 57 amino acids, respectively. Each repeated motif of the two proteins was responsible for the interaction, but the stronger interaction was shown by the native structures containing multiple motifs. Interestingly, the N-terminal extension of human glycyl-tRNA synthetase containing a single motif homologous to those in the bifunctional tRNA synthetase also interacted with the C-terminal motif of the isoleucyl-tRNA synthetase although the enzyme is not a component of the complex. The data indicate that the multiplicity of the binding motif in the tRNA synthetases is necessary for enhancing the interaction strength and may be one of the determining factors for the tRNA synthetases to be involved in the formation of the multi-tRNA synthetase complex.

Conformational dependence of electron transfer across de novo designed metalloproteins.

Flash photolysis and pulse radiolysis measurements demonstrate a conformational dependence of electron transfer rates across a 16-mer helical bundle (three-helix metalloprotein) modified with a capping CoIII(bipyridine)3 electron acceptor at the N terminus and a 1-ethyl-1'-ethyl-4,4'- bipyridinium donor at the C terminus. For the CoIII(peptide)3-1-ethyl-1'-ethyl-4,4'-bipyridinium maquettes, the observed transfer is a first order, intramolecular process, independent of peptide concentration or laser pulse energy. In the presence of 6 M urea, the random coil bundle (approximately 0% helicity) has an observed electron transfer rate constant of kobs = 900 +/- 100 s-1. In the presence of 25% trifluoroethanol (TFE), the helicity of the peptide is 80% and the kobs increases to 2000 +/- 200 s-1. Moreover, the increase in the rate constant in TFE is consistent with the observed decrease in donor-acceptor distance in this solvent. Such bifunctional systems provide a class of molecules for testing the effects of conformation on electron transfer in proteins and peptides.

The redox/DNA repair protein, Ref-1, is essential for early embryonic development in mice.

The DNA-binding activity of AP-1 proteins is modulated, in vitro, by a posttranslational mechanism involving reduction oxidation. This mode of regulation has been proposed to control both the transcriptional activity and the oncogenic potential of Fos and Jun. Previous studies revealed that reduction of oxidized Fos and Jun by a cellular protein, Ref-1, stimulates sequence-specific AP-1 DNA-binding activity. Ref-1, a bifunctional protein, is also capable of initiating the repair of apurinic/apyrymidinic sites in damaged DNA. The relationship between the redox and DNA repair activities of Ref-1 is intriguing; both activities have been suggested to play an important role in the cellular response to oxidative stress. To investigate the physiological function of Ref-1, we used a gene targeting strategy to generate mice lacking a functional ref-1 gene. We report here that heterozygous mutant mice develop into adulthood without any apparent abnormalities. In contrast, homozygous mutant mice, lacking a functional ref-1 gene, die during embryonic development. Detailed analysis indicates that death occurs following blastocyst formation, shortly after the time of implantation. Degeneration of the mutant embryos is clearly evident at embryonic day 5.5. These findings demonstrate that Ref-1 is essential for early embryonic development.

A three-hybrid system to detect RNA-protein interactions in vivo.

RNA-protein interactions are pivotal in fundamental cellular processes such as translation, mRNA processing, early development, and infection by RNA viruses. However, in spite of the central importance of these interactions, few approaches are available to analyze them rapidly in vivo. We describe a yeast genetic method to detect and analyze RNA-protein interactions in which the binding of a bifunctional RNA to each of two hybrid proteins activates transcription of a reporter gene in vivo. We demonstrate that this three-hybrid system enables the rapid, phenotypic detection of specific RNA-protein interactions. As examples, we use the binding of the iron regulatory protein 1 (IRP1) to the iron response element (IRE), and of HIV trans-activator protein (Tat) to the HIV trans-activation response element (TAR) RNA sequence. The three-hybrid assay we describe relies only on the physical properties of the RNA and protein, and not on their natural biological activities; as a result, it may have broad application in the identification of RNA-binding proteins and RNAs, as well as in the detailed analysis of their interactions.

Complementation of an Escherichia coli adhE mutant by the Entamoeba histolytica EhADH2 gene provides a method for the identification of new antiamebic drugs.

The pathogenic protozoan parasite Entamoeba histolytica, the cause of amebic dysentery and amebic liver abscess, is an obligate anaerobe, and derives energy from the fermentation of glucose to ethanol with pyruvate and acetyl coenzyme A as intermediates. We have isolated EhADH2, a key enzyme in this pathway, that is a NAD+- and Fe2+-dependent bifunctional enzyme with acetaldehyde dehydrogenase and alcohol dehydrogenase activities. EhADH2 is the only known eukaryotic member of a newly defined family of prokaryotic multifunctional enzymes, which includes the Escherichia coli AdhE enzyme, an enzyme required for anaerobic growth of E. coli. Because of the critical role of EhADH2 in the amebic fermentation pathway and the lack of known eukaryotic homologues of the EhADH2 enzyme, EhADH2 represents a potential target for antiamebic chemotherapy. However, screening of compounds for antiamebic activity is hampered by the cost of large scale growth of Ent. histolytica, and difficulties in quantitating drug efficacy in vitro. To approach this problem, we expressed the EhADH2 gene in a mutant strain of E. coli carrying a deletion of the adhE gene. Expression of EhADH2 restored the ability of the mutant E. coli strain to grow under anaerobic conditions. By screening compounds for the ability to inhibit the anaerobic growth of the E. coli/EhADH2 strain, we have developed a rapid assay for identifying compounds with anti-EhADH2 activity. Using bacteria to bypass the need for parasite culture in the initial screening process for anti-parasitic agents could greatly simplify and reduce the cost of identifying new therapeutic agents effective against parasitic diseases.

Deciphering the mechanism for the assembly of aromatic polyketides by a bacterial polyketide synthase.

Aromatic polyketides are assembled by a type 11 (iterative) polyketide synthase (PKS) in bacteria. Understanding the enzymology of such enzymes should provide the information needed for the synthesis of novel polyketides through the genetic engineering of PKSs. Using a previously described cell-free system [B.S. & C.R.H. (1993) Science 262, 1535-1540], we studied a PKS enzyme whose substrate is not directly available and purified the TcmN polyketide cyclase from Streptomyces glaucescens. TcmN is a bifunctional protein that catalyzes the regiospecific cyclization of the Tcm PKS-bound linear decaketide to Tcm F2 and the 0-methylation of Tcm D3 to Tcm B3. In the absence of TcmN, the decaketide formed by the minimal PKS consisting of the TcmJKLM proteins undergoes spontaneous cyclization to form some Tcm F2 as well as SEK15 and many other aberrant shunt products. Addition of purified TcmN to a mixture of the other Tcm PKS components both restores and enhances Tcm F2 production. Interestingly, Tcm F2 but none of the aberrant products was bound tightly to the PKS. The results described support the notion that the polyketide cyclase, not the minimal PKS, dictates the regiospecificity for the cyclization of the linear polyketide intermediate. Furthermore, because the addition of TcmN to the TcmJKLM proteins results in a significant increase of the total yield of decaketide, interactions among the individual components of the Tcm PKS complex must give rise to the optimal PKS activity.

Leukotriene A4 hydrolase: protection from mechanism-based inactivation by mutation of tyrosine-378.

Leukotriene A4 (LTA4) hydrolase [(7E,9E,11Z,14Z)-(5S,6S)-5,6-epoxyicosa-7, 9,11,14-tetraenoate hydrolase; EC 3.3.2.6] is a bifunctional zinc metalloenzyme that catalyzes the final step in the biosynthesis of the potent chemotactic agent leukotriene B4 (LTB4). LTA4 hydrolase/aminopeptidase is suicide inactivated during catalysis via an apparently mechanism-based irreversible binding of LTA4 to the protein in a 1:1 stoichiometry. Previously, we have identified a henicosapeptide, encompassing residues Leu-365 to Lys-385 in human LTA4 hydrolase, which contains a site involved in the covalent binding of LTA4 to the native enzyme. To investigate the role of Tyr-378, a potential candidate for this binding site, we exchanged Tyr for Phe or Gln in two separate mutants. In addition, each of two adjacent and potentially reactive residues, Ser-379 and Ser-380, were exchanged for Ala. The mutated enzymes were expressed as (His)6-tagged fusion proteins in Escherichia coli, purified to apparent homogeneity, and characterized. Enzyme activity determinations and differential peptide mapping, before and after repeated exposure to LTA4, revealed that wild-type enzyme and the mutants [S379A] and [S380A]LTA4hydrolase were equally susceptible to suicide inactivation whereas the mutants in position 378 were no longer inactivated or covalently modified by LTA4. Furthermore, in [Y378F]LTA4 hydrolase, the value of kcat for epoxide hydrolysis was increased 2.5-fold over that of the wild-type enzyme. Thus, by a single-point mutation in LTA4 hydrolase, catalysis and covalent modification/inactivation have been dissociated, yielding an enzyme with increased turnover and resistance to mechanism-based inactivation.

Seed and vascular expression of a high-affinity transporter for cationic amino acids in Arabidopsis.

In most plants amino acids represent the major transport form for organic nitrogen. A sensitive selection system in yeast mutants has allowed identification of a previously unidentified amino acid transporter in Arabidopsis. AAT1 encodes a hydrophobic membrane protein with 14 membrane-spanning regions and shares homologies with the ecotropic murine leukemia virus receptor, a bifunctional protein serving also as a cationic amino acid transporter in mammals. When expressed in yeast, AAT1 mediates high-affinity transport of basic amino acids, but to a lower extent also recognizes acidic and neutral amino acids. AAT1-mediated histidine transport is sensitive to protonophores and occurs against a concentration gradient, indicating that AAT1 may function as a proton symporter. AAT1 is specifically expressed in major veins of leaves and roots and in various floral tissues--i.e., and developing seeds.

Leukotriene A4 hydrolase: mapping of a henicosapeptide involved in mechanism-based inactivation.

Leukotriene A4 (LTA4) hydrolase [7E,9E,11Z,14Z)-(5S,6S)-5,6-epoxyicosa-7,9 ,11,14-tetraenoate hydrolase; EC 3.3.2.6] is a bifunctional zinc metalloenzyme which converts LTA4 into the chemotactic agent leukotriene B4 (LTB4). Suicide inactivation, a typical feature of LTA4 hydrolase/aminopeptidase, occurs via an irreversible, apparently mechanism-based, covalent binding of LTA4 to the protein in a 1:1 stoichiometry. Differential lysine-specific peptide mapping of unmodified and suicide-inactivated LTA4 hydrolase has been used to identify a henicosapeptide, encompassing the amino acid residues 365-385 of human LTA4 hydrolase, which is involved in the binding of LTA4, LTA4 methyl ester, and LTA4 ethyl ester to the native enzyme. A modified form of this peptide, generated by lysine-specific digestion of LTA4 hydrolase inactivated by LTA4 ethyl ester, could be isolated for complete Edman degradation. The sequence analysis revealed a gap at position 14, which shows that binding of the leukotriene epoxide had occurred via Tyr-378 in LTA4 hydrolase. Inactivation of the epoxide hydrolase and the aminopeptidase activity was accompanied by a proportionate modification of the peptide. Furthermore, both enzyme inactivation and peptide modification could be prevented by preincubation of LTA4 hydrolase with the competitive inhibitor bestatin, which demonstrates that the henicosapeptide contains functional elements of the active site(s). It may now be possible to clarify the molecular mechanisms underlying suicide inactivation and epoxide hydrolysis by site-directed mutagenesis combined with structural analysis of the lipid molecule, covalently bound to the peptide.