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).