Amino acid utilization by Chlamydomonas reinhardtii: specific study of histidine

Phytoplankton live in fluctuating environments where many factors such as grazing pressure, sinking, light availability, nutrient uptake and turnover influence the distribution of phytoplankton in time and space. The purpose of this study was to investigate if under conditions of depletion of inorganic nitrogen, as recorded in summer in naturals waters, phytoplanktonic species have the capability of using organic nitrogen sources, including free or combined amino acids, in addition to inorganic nitrogen. The study has focussed on histidine, the degradation of which yielding potentially three nitrogen atoms for each molecule of histidine. Chlamydomonas reinhardtii (CCAP 11/32A) was cultivated axenically with two different sources of nitrogen (histidine and/or ammonium). In the presence of histidine as sole source of nitrogen, cell growth was comparable to that observed with the same concentration of nitrogen in ammonium form. In the presence of both histidine and ammonium, histidine degradation was observed only when the concentration of ammonium was depleted. Under these conditions, the first two enzymes of histidine degradation pathway, histidase (EC 4.3.1.3) and urocanase (EC 4.2.1.49) were produced and were co-ordinately regulated. Histidase activity was also controlled by succinate and glutamate as carbon sources. Histidase was purified 1018-fold and partially characterized. The molecular weight of the native enzyme was estimated to 152.4 kDa corresponding to four subunits of 38.1 kDa. The enzyme did not exhibit classical Michaelis-Menten kinetics but showed a relationship between the rate of catalysis (V) and the concentration of substrate (S), characteristic of negative allosteric behavior. A Hill coefficient of 4 was measured for histidine concentrations higher than 20.5 mM.