PURIFICATION AND PROPERTIES OF 3-HEXULOSEPHOSPHATE SYNTHASE FROM METHYLOMONAS M15

3-Hexulosephosphate synthase [EC 5.3.1.6], the 1st enzyme of the ribulose monophosphate cycle, was purified 15-fold from methanol-grown Methylomonas M 15. The purification procedure involved chromatography on DEAE-cellulose, Sephadex G-75 and DEAE-Sephadex A-50. The purified enzyme was > 95% pure as judged by analytical polyacrylamide gel electrophoresis. The MW was calculated to be 43,000 from sedimentation equilibrium experiments. Electrophoresis in sodium dodecylsulfate gels gave a single band corresponding to a MW of 22,000. The enzyme catalyzes specifically the condensation of formaldehyde with ribulose 5-phosphate to yield D-arabino-3-hexulose 6-phosphate. The Km values were 1.1 mM for formaldehyde and 1.6 mM for ribulose 5-phosphate. A bivalent cation is essential for activity and stability of the enzyme; Mg2+ and Mn2+ serve best for this purpose. The optimum of pH for enzyme activity is 7.5-8.0.