Enzymatic Synthesis of the Crithidia Factors in Cell-free Extracts of Serratia indica

Abstract

The concomitant production of formic acid and pterin compounds from guanosine-5'-triphosphate (GTP) has been found in cell-free extracts of Serratia indica. Among the pterin compounds, L-threo-neopterin-the major Crithidia factor in S. indica-, a cyclic phosphate of neopterin (cNP), D-erythro-neopterin and 6-hydroxymethyl pterin were detected and isolated. Formate-¹⁴C elimination from GTP-8-¹⁴C was quantitatively distributed in the ethyl acetate layer in the ehyl acetate-hydrochloric acid partition system. Carbon 8 of GTP was released as formic acid. Enzymatic production of formate and cNP was linear for 2 hr at 37°C. Formate production was proportional to the enzyme concentration. The optimum pH for formate elimination was observed around pH 8.6. Optimum temperature for the production of formate and cNP was 50°C. The apparent Km value of GTP for formate production was 6.2×10^-b_M. Formate eliminating activity was activated by disodium phosphate but was inhibited by Mg²⁺ or AMP. Incorporation of GTP-U-¹⁴C into pterin compounds was also regulated with disodium phosphate. Effective incorporation into cNP and D-erythro-neopterin occurred in the presence of phosphate. When phosphate was omitted from the system, however, effective incorporation into 6-hydroxymethyl pterin was observed. The biosynthetic process of the Crithidia factors, i.e. L-threo-neopterin and cNP, from GTP in S. indica is also discussed.