Plant riboflavin biosynthesis - Cloning, chloroplast localization expression, purification, and partial characterization of spinach lumazine synthase

Lumazine synthase, which catalyzes the penultimate step of riboflavin biosy nthesis, has been cloned from three higher plants (spinach, tobacco, and ar abidopsis) through functional complementation of an Escherichia coli auxotr oph. Whereas the three plant proteins exhibit some structural similarities to known microbial homologs, they uniquely possess N-terminal polypeptide e xtensions that resemble typical chloroplast transit peptides. In vitro prot ein import assays with intact chloroplasts and immunolocalization experimen ts verify that higher plant lumazine synthase is synthesized in the cytosol as a larger molecular weight precursor protein, which is post-translationa lly imported into chloroplasts where it is proteolytically cleaved to its m ature size. The authentic spinach enzyme is estimated to constitute <0.02% of the total chloroplast protein. Recombinant "mature" spinach lumazine syn thase is expressed in E. coli at levels exceeding 30% of the total soluble protein and is readily purified to homogeneity using a simple two-step proc edure. Apparent V-max and K-m values obtained with the purified plant prote in are similar to those reported for microbial lumazine synthases. Electron microscopy and hydrodynamic studies reveal that native plant lumazine synt hase is a hollow capsid-like structure comprised of 60 identical 16.5-kDa s ubunits, resembling its icosahedral counterparts in E. coli and Bacillus su btilis.