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).
According to current models, the aaRS evolved from simpler enzymes based on the canonical class I and class II catalytic domains that possessed amino acid activation function but were limited in their ability to catalyze sequence specific aminoacylation of tRNA (). This follows from the modular organization of the aaRS, which is evident in their three-dimensional structures and in the retention of limited catalytic activity by the isolated class conserved core domains produced biochemically (–, ). Two noteworthy contemporary proteins that are based on isolated aaRS catalytic or tRNA binding domains are AsnA and the biotin repressor (BirA), which show homology to the isolated catalytic domains of AspRS and SerRS, respectively (, ). Both AsnA and BirA catalyze reactions that include adenylated intermediates, which is in marked contrast to the HisZG enzyme. The absence of adenylation function in HisZ and its putative role as the functional binding site for regulation by histidine lends credence to the model that the first aaRS progenitors may in fact have been simple amino acid binding proteins. Later evolutionary improvements might have included the ability to activate amino acids by condensation with ATP and the ability to bind tRNA in a sequence specific fashion. This latter step required the accrual of additional RNA binding domains capable of folding independently. Along these lines, the recent report that ribosomal protein L25, which plays a critical role in 5S RNA binding, is structurally related to the anticodon binding domains of GlnRS is highly significant ().
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.
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.
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.
Gebhardt et al. showed application of emodin (82) and its ethylenediamine analog 83 as non-ATP competitive inhibitors of GSK-3 (Table ). Addition of the ethylenediamine group on the emodin nucleus increased potency of inhibition (IC50 0.56±0.02 µM, 83), reduced cytotoxicity and generated an insulin sensitizing effect mediated by increasing hepatocellular glycogen and fatty acid biosynthesis. Selectivity's of compounds 82 and 83 were evaluated against twelve protein kinases including eleven of human protein kinases. Compound 83 showed high selectivity towards GSK-3β but 82 failed to do so.
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 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 (, ).
Additional experiments using a strain carrying an in-frame deletion of HisZ demonstrated that the deletion conferred a failure to grow on minimal medium in the absence of exogenously supplied histidine (Fig. ). This deficiency could be relieved by trans complementation with HisZ or by inclusion of histidinol, the substrate for histidinol dehydrogenase (HisD) and the last intermediate in the histidine biosynthetic pathway. These results confirm that HisZ is unlikely to be involved in regulation of the his operon. However, failure to grow in the absence of added histidine or histidinol indicated a role in histidine biosynthesis at a step before the step catalyzed by HisD.