An aminoacyl-tRNA synthetase paralog with a catalytic role in histidine biosynthesis

Abstract

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.

Figure 1

Structure-based sequence alignment of HisRS-like proteins (HisZ) and representative functional histidyl-tRNA synthetases (HisRSs). Species names are abbreviated as follows: Ll, L. lactis; Bs, B. subtilis; Pa, P. aeriginosa; Ng, N. gonorrhoeae; Ss, Synechocystis sp.; Aa, A. aeolicus; Ec, E. coli; Sc, Saccharomyces cerevisae; Mj, Methanococcus jannaschii. Numbers at the left of the sequences indicate positions of first aligned residues in the block, whereas the bracketed numbers indicate spacing between the elements of the blocks. Swiss-Prot, Protein Identification Resource, and National Center for Biotechnology Information (NCBI)/GenBank accession numbers are indicated to the right. UFG indicates sequences retrieved from the unfinished genomes of the NCBI server. The three diagnostic class II motifs and HisRS-specific peptide motifs are indicated and are overlaid by a schematic representation of HisRS secondary structure (25, 28). Highly conserved neutral, acidic, and basic residues are outlined in yellow, red, and blue, respectively; and key residues (E. coli HisRS numbering) that denote significant differences between the HisRS functional and HisRS-like proteins are labeled.

Figure 2

RNA binding by L. lactis HisZ determined by filter binding experiments. Assays were performed as described (19). (a) ³²P-labeled L. lactis tRNA^His (●) and hairpin ribozyme (□) were incubated at 25°C with HisZ protein over a concentration range of 5 nM to 1 mM. The binding constant for tRNA reported in the text represents the global fit of five experiments. (b) Competition experiments between labeled L. lactis tRNA^His transcripts (<5 nM) and unlabeled nonspecific RNA competitors over the range of 0 to 100 nM (●, E. coli 16S/23S RNA; □, hairpin ribozyme) and in the presence of 500 nM HisZ.

Figure 3

Genetic evidence for the role of HisZ in histidine biosynthesis. L. lactis strains differing by the presence (WT) or the absence (HisZ⁻) of HisZ were grown at 30°C on chemically defined medium (CDM) without histidine (−his) or supplemented with 0.01% histidine (+his) or histidinol (+hol). plcZ stands for a high-copy-number plasmid derivative of pJIM2279 (22) Em^r (erythromycin resistance) and containing HisZ gene under control of a constitutive promoter.

Figure 4

HisZ and HisG are both required for PRPP-ATP transferase activity. (a) Complementation of an E. coli HisG mutant strain by L. lactis HisG and HisZ. E. coli strains differing by the presence (E. coli) or the absence (HisG⁻) of HisG were grown at 37°C on minimal medium supplemented with 0.0025% amino acids (arginine, leucine, and methionine) without (−his) or with (+his) histidine. Growth on plates was assayed by spotting 5-μl drops of washed rich-medium overnight culture on plates containing minimal medium, and growth kinetics were measured. The HisG⁻ strain was complemented with a high copy number plasmid vector (pBS, Stratagene) containing various L. lactis his DNA fragments. Plasmid pZG [formally pIL704 (15)] has a 2.2-kb fragment containing the HisZ and HisG genes in tandem. Plasmids pZ (expressing the HisZ gene) and pG (expressing the HisG gene) are derivatives of pZG with a 538-bp deletion in the HisG gene or a 600-bp deletion in the HisZ gene, respectively. (b) Physical association of HisZ and HisG. Typical SDS/polyacrylamide gel showing L. lactis HisG (lane 1), HisZ (lane 2), and HisZ/HisG (lane3) purified by affinity chromatography (17) from overexpression E. coli strains. For homogenous HisG and HisZ (lanes 1 and 2), expression vectors were as described above. L stands for a protein ladder (BenchMark, GIBCO/BRL). (c) Effect of L. lactis HisZ and/or L. lactis HisG proteins on the PRPP conversion, detected by A₂₉₀. The purified proteins were provided at a final concentration of 100 nM, and the reaction was initiated by the addition of PRPP (0.5 mM). The HisZ-HisG trace refers to a preparation from the tandem expression construct. The accumulation of PR-ATP product was monitored by A₂₉₀ (24).