In addition to their essential catalytic role in protein biosynthesis, aminoacyl-tRNA synthetases participate in numerous other functions, including regulation of gene expression and amino acid biosynthesis via transamidation pathways. Herein, we describe a class of aminoacyl-tRNA synthetase-like (HisZ) proteins based on the catalytic core of the contemporary class II histidyl-tRNA synthetase whose members lack aminoacylation activity but are instead essential components of the first enzyme in histidine biosynthesis ATP phosphoribosyltransferase (HisG). Prediction of the function of HisZ in Lactococcus lactis was assisted by comparative genomics, a technique that revealed a link between the presence or the absence of HisZ and a systematic variation in the length of the HisG polypeptide. HisZ is required for histidine prototrophy, and three other lines of evidence support the direct involvement of HisZ in the transferase function. (i) Genetic experiments demonstrate that complementation of an in-frame deletion of HisG from Escherichia coli (which does not possess HisZ) requires both HisG and HisZ from L. lactis. (ii) Coelution of HisG and HisZ during affinity chromatography provides evidence of direct physical interaction. (iii) Both HisG and HisZ are required for catalysis of the ATP phosphoribosyltransferase reaction. This observation of a common protein domain linking amino acid biosynthesis and protein synthesis implies an early connection between the biosynthesis of amino acids and proteins.
In a recent study, Palomo et al. identified available binding sites using the geometry-based algorithm fpocket () and hpocket programs to map the surface of GSK-3. Palomo et al. identified seven conserved binding sites (Figure ). These binding sites could be targeted for selective and effective modulation of protein-catalytic functions. Out of the seven identified binding sites on the surface of GSK-3, three consisted of the previously reported (Figure , sites 1-3) ATP, substrate, and peptides axin/fratide-binding sites. The four additional binding sites were newly identified to be non-ATP sites.
Sources of Drugs: Biological, marine, mineral and plant tissue cultures as sources of drugs;
Classification of Drugs: Morphological, taxonomical, chemical and pharmacological classification of drugs; Study of medicinally important plants belonging to the families with special reference to: Apocynacae, Solanaceae, Rutacease, Umbelliferae, Leguminosae, Rubiaceae, Liliaceae, Graminae, Labiatae, Cruciferae, Papaveraceae; Cultivation, Collection, Processing and Storage of Crude Drugs: Factors influencing cultivation of medicinal plants, Types of soils and fertilizers of common use. Pest management and natural pest control agents, Plant hormones and their applications, Polyploidy, mutation and hybridization with reference to medicinal plants. Quality Control of Crude Drugs: Adulteration of crude drugs and their detection by organoleptic, microscopic, physical, chemical and biological methods and properties. Introduction to Active Constituents of Drugs: Their isolation, classification and properties.
Systematic pharmacognostic study of the followings:
CARBOHYDRATES and derived products: agar, guar gum acacia, Honey, Isabagol, pectin, Starch, sterculia and Tragacanth; Lipids: Bees wax, Castor oil, Cocoa butter, Codliver oil, Hydnocarpus oil, Kokum butter, Lard, Linseed oil, Rice, Bran oil, Shark liver oil and Wool fat; RESINS: Study of Drugs Containing Resins and Resin Combinations like Colophony, podophyllum, jalap, cannabis, capsicum, myrrh, asafoetida, balsam of Tolu, balsam of Peru, benzoin, turmeric, ginger;
TANNINS: Study of tannins and tannin containing drugs like Gambier, black catechu, gall and myrobalan;
VOLATILE OILS: General methods of obtaining volatile oils from plants, Study of volatile oils of Mentha, Coriander, Cinnamon, Cassia, Lemon peel, Orange peel, Lemon grass, Citronella, Caraway, Dill, Spearmint, Clove, Fennel, Nutmeg, Eucalyptus, Chenopodium, Cardamom, Valerian, Musk, Palmarosa, Gaultheria, Sandal wood; Phytochemical Screening: Preparation of extracts, Screening of alkaloids, saponins, cardenolides and bufadienolides, flavonoids and leucoanthocyanidins, tannins and polyphenols, anthraquinones, cynogenetic glycosides, amino acids in plant extracts; FIBERS: Study of fibers used in pharmacy such as cotton, silk, wool, nylon, glass-wool, polyester and asbestos.
Study of the biological sources, cultivation, collection, commercial varieties, chemical constituents, substitutes, adulterants, uses, diagnostic macroscopic and microscopic features and specific chemical tests of following groups of drugs:
GLYCOSIDE CONTAINING DRUGS: Saponins : Liquorice, ginseng, dioscorea, sarsaparilla, and senega. Cardioactive glycosides: Digitalis, squill, strophanthus and thevetia, Anthraquinone cathartics: Aloe, senna, rhubarb and cascara, Others: Psoralea, Ammi majus, Ammi visnaga, gentian, saffron, chirata, quassia.
ALKALOID CONTAINING DRUGS: Pyridine-piperidine: Tobacco, areca and lobelia. Tropane: Belladonna, hyoscyamus, datura, duboisia, coca and withania. Quinoline and Isoquinoline: Cinchona, ipecac, opium. Indole: Ergot, rauwolfia, catharanthus, nux-vomica and physostigma. Imidazole: Pilocarpus. Steroidal: Veratrum and kurchi. Alkaloidal Amine: Ephedra and colchicum. Glycoalkaloid: Solanum. Purines: Coffee, tea and cola. Biological sources, preparation, identification tests and uses of the following enzymes: Diastase, papain, pepsin, trypsin, pancreatin. Studies of Traditional Drugs: Common vernacular names, botanical sources, morphology, chemical nature of chief constituents, pharmacology, categories and common uses and marketed formulations of following indigenous drugs: Amla, Kantkari, Satavari, Tylophora, Bhilawa, Kalijiri, Bach, Rasna, Punamava, Chitrack, Apamarg, Gokhru, Shankhapushpi, Brahmi, Adusa, Atjuna, Ashoka, Methi, Lahsun, Palash, Guggal, Gymnema, Shilajit, Nagarmotha and Neem. The holistic concept of drug administration in traditional systems of medicine. Introduction to ayurvedic preparations like Arishtas, Asvas, Gutikas, Tailas, Chumas, Lehyas and Bhasmas.
General Techniques of Biosynthetic Studies and Basic Metabolic Pathways/Biogenesis: Brief introduction to biogenesis of secondary metabolites of pharmaceutical importance. Terpenes: monoterpenes, sesquiterpenes, diterpenes, and triterpenoids. Carotenoids: a-carotenoids, ß-carotenes, vitamin A, Xanthophylls of medicinal importance. Glycosides: Digitoxin, digoxin, hecogenin, sennosides, diosgenin and sarasapogenin. Alkaloids: Atropine and related compounds, Quinine, Reserpine, Morphine, Papaverine, Ephedrine, Ergot and Vinca alkaloids. Lignans, quassanoids and flavonoids. Role of plant-based drugs on National economy: A brief account of plant based industries and institutions involved in work on medicinal and aromatic plants in India. Utilization and production of phyto-constituents such as quinine, calcium sennosides, podophyllotoxin, diosgenin, solasodine, and tropane alkaloids. Utilization of aromatic plants and derived products with special reference to sandalwood oil, mentha oil, lemon grass oil, vetiver oil, geranium oil and eucalyptus oil. World-wide trade in medicinal plants and derived products with special reference to diosgenin (disocorea), taxol (Taxus sps) digitalis, tropane alkaloid containing plants, Papain, cinchona, Ipecac, Liquorice, Ginseng, Aloe, Valerian, Rauwolfia and plants containing laxatives. Plant bitters and sweeteners. Plant Tissue Culture: Historical development of plant tissue culture, types of cultures, nutritional requirements, growth and their maintenance. Applications of plant tissue culture in pharmacognosy. Marine pharmacognosy: Novel medicinal agents from marine sources. Natural allergens and photosensitizing agents and fungal toxins. Herbs as health foods. Herbal cosmetics. Standardization and quality control of herbal drugs, WHO guidelines for the standardization of herbal drugs.
The HisZ subfamily described herein plays an important role in histidine biosynthesis that was not anticipated previously on the basis of sequence analysis. However, both the functional HisRS and a class of HisRS-like proteins specific to eukaryotes (GCN2) are involved in regulation of histidine biosynthesis (, ). The GCN2 class possesses a Ser-Thr kinase domain and a HisRS-like domain (, ). Binding of uncharged tRNA by the HisRS-like domain activates the kinase, which through phosphorylation of eIF2α increases the translation of the general amino acid control factor GCN4. These three families (functional HisRS, HisZ, and GCN2) share limited but significant sequence identity and are likely to share a common protein fold, as suggested by the class II aaRS catalytic domain. A phylogenetic analysis of the HisRS family suggests that the HisZ subfamily is the result of an early gene duplication in the bacteria, whereas the GCN2 subfamily represents a separate duplication of a primordial HisRS gene in the Eukarya (J.B. and C.F., unpublished results). The independent nature of these early duplications is supported by the fact that the functional roles of HisZ and GCN2 are distinct. Notably, we have no evidence at present that the apparent nonspecific RNA binding properties of HisZ have any functional significance.
Having eliminated the regulatory model, we begun to focus on a direct role of HisZ in histidine biosynthesis. Early literature in the his operon field showed that the ATP phosphoribosyltransferase (HisG, first enzyme involved in the histidine biosynthesis pathway) from enteric bacteria binds tRNA with an affinity of about 100 nM (). The observed affinity for tRNA and the apparent involvement of HisZ in histidine biosynthesis prompted consideration of a model in which HisZ serves as a functional subunit of the HisG enzyme. In support of this model, revertants of a L. lactis HisZ− deletion strain were selected, and these mutations were mapped within HisG (data not shown). Furthermore, we noticed a striking covariation between the presence of HisZ in bacterial genomes and the length of the HisG polypeptides, as deduced from the translational sequence (Table ). In all cases, the lengths of the HisG ORFs in genomes from taxa containing HisZ were shorter by some 80–100 amino acids, whereas other his biosynthetic enzyme polypeptide lengths remained unchanged. Sequence alignments of HisG genes reveal that the reduced length is accounted for by the deletion of a contiguous region of the C terminus (data not shown).
The evolution of histidine biosynthetic pathways was likely to be important in the development of cellular physiology, as a result of the essential catalytic role of histidine in the active sites of enzymes and because of the direct connections between nitrogen metabolism in general and purine, pyrimidine, and tryptophan biosynthesis in particular. The high energetic costs associated with the biosynthesis of each molecule of histidine likely exerted selective pressure on primitive organisms to develop multilevel regulatory circuits. Although the transcriptional controls observed in enteric bacteria require several cell generations for the new steady-state level to be reached, feedback inhibition of the first enzyme of the pathway serves as a major control that provides rapid regulation of biosynthetic activity as a function of the availability of exogenous amino acid (). The precise role of the HisZ subunit(s) in the transferase remains to be elucidated, but a model suggested by conservation of histidine binding site residues in HisZ (Fig. ) is that it allows allosteric control of the transferase by histidine and AMP. Notably, mutations in the E. coli HisG that confer unresponsiveness to feedback inhibitors map to the C-terminal part of HisG (), possibly in the segment absent in the short versions of HisG found in Gram-positive bacteria. This C-terminal extension of HisG shares no obvious sequence homology with aaRS or with any other known RNA binding domain (data not shown).
We therefore sought to address this model by identifying and characterizing proteins in contemporary organisms that contain isolated aaRS functional domains. The studies reported herein were undertaken to determine the relationship between naturally occurring fragments of HisRSs and fragments produced biochemically (). Herein, we describe a protein family (the HisZ family) related to the catalytic core of the contemporary class II HisRS whose members lack aminoacylation activity, as predicted by sequence comparisons with functional synthetases. The first member of the HisZ family was originally identified in Lactococcus lactis as an ORF of unknown function with significant homology to class II aaRSs, as indicated by sequences resembling the three diagnostic sequence motifs (). We present herein genetic and biochemical evidence that support its direct involvement as an essential subunit of ATP phosphoribosyltransferase (HisG), the first enzyme in histidine biosynthesis. Other members of the HisZ family are identified, and the implications of these observations for aaRS evolution are discussed.
The further involvement of aaRS or aaRS-like proteins in amino acid biosynthesis is also suggested by the existence of proteins that are based on the catalytic domains of an aaRS yet do not catalyze the aminoacylation reaction. A striking illustration is the asparagine synthetase A (AsnA), whose recently solved structure contains a class II aaRS catalytic domain (closely related to AspRS and AsnRS). The role of AsnA is to convert aspartate to asparagine via an amidation reaction involving a transient aspartyl-adenylate (). The high degree of structural homology between AsnA and AspRS and the observation that truncated aaRS catalytic domains retain residual catalytic activity [e.g., MetRS (), AlaRS (), and HisRS ()] lend support to proposals that synthetases arose by fusion of specialized tRNA interaction and editing domains to primordial catalytic domains capable of amino acid activation (). Obtaining corroborating evidence for this theory is hindered by the difficulty in distinguishing between homologous proteins that might have been antecedents to the aaRS and proteins that might have started as functional aaRS but lost aminoacylation capacity over evolution.
The first member of the HisRS-like family (designated herein as HisZ) was identified as the second ORF (Orf3) in the histidine biosynthesis operon of L. lactis (). This HisZ protein possesses all three sequence motifs diagnostic for the class II aaRSs and, because of its truncation immediately after motif 3, is missing the mixed α/β anticodon binding domain () found in the class IIa subgroup. Notably, HisZ also lacks several essential catalytic residues that have been shown by mutagenesis to be important for the aminoacylation function by class II aaRS in general and by the E. coli HisRS in particular (–) (Fig. ). The genome of L. lactis also includes a full-length version of HisRS that presumably fulfills the typical role of a functional aaRS in protein synthesis (). Subsequent to the description of the L. lactis HisZ, we identified additional HisRS/HisZ pairs in Bacilllus subtilis, Pseudomonas aeruginosa, Neisseria gonorrhoeae, Synechocystis sp. PC6803, and Aquifex aeolicus (Fig. ). The absence of one or both of the catalytic arginines in the histidine 1 peptide and the GLER peptide of motif 3 () suggests that none of these HisZ proteins is a functional aaRS in vivo. This is in marked contrast to the paralogous LysRS (LysU) and ThrRS (thrZ) in E. coli and B. subtilis, respectively, which are functional aaRSs (, ).
Protein synthesis requires the association of amino acids with the nucleotide triplets of the genetic code, a reaction mediated by tRNA adapters and their specific aminoacyl-tRNA synthetases (aaRSs). As reflected in the absence of some of the canonical 20 aaRSs in contemporary organisms and the duplication and truncation of aaRS in others, some variation in components involved in proteins synthesis has persisted over evolution (–). For example, contemporary archaebacterial and bacterial species possess transamidation pathways that use glutamyl-tRNAGln and aspartyl-tRNAAsn (produced by GluRS and AspRS, respectively) as substrates (–). These transamidation pathways account for the absence of GlnRS and AsnRS in these species. The aaRSs also regulate the biosynthetic operons responsible for tryptophan, branched-chain amino acids, and histidine (–) by attenuation mechanisms that couple transcription of the operon to translation of leader peptides rich in codons specific for the amino acids in question. Notably, both the transamidation pathways and the regulation by attenuation are dependent on the same aminoacylation reactions that generate aminoacylated tRNA for protein synthesis.