Plant riboflavin biosynthesis. Cloning, chloroplast localization, expression, purification, and partial characterization of spinach lumazine synthase.
Lumazine synthase, which catalyzes the penultimate step of riboflavin biosynthesis, has been cloned from three higher plants (spinach, tobacco, and arabidopsis) through functional complementation of an Escherichia coli auxotroph. Whereas the three plant proteins exhibit some structural similarities to known microbial homologs, they uniquely possess N-terminal polypeptide extensions that resemble typical chloroplast transit peptides. In vitro protein import assays with intact chloroplasts and immunolocalization experiments verify that higher plant lumazine synthase is synthesized in the cytosol as a larger molecular weight precursor protein, which is post-translationally imported into chloroplasts where it is proteolytically cleaved to its mature size. The authentic spinach enzyme is estimated to constitute <0.02% of the total chloroplast protein. Recombinant "mature" spinach lumazine synthase 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 procedure. Apparent V(max) and K(m) values obtained with the purified plant protein are similar to those reported for microbial lumazine synthases. Electron microscopy and hydrodynamic studies reveal that native plant lumazine synthase is a hollow capsid-like structure comprised of 60 identical 16.5-kDa subunits, resembling its icosahedral counterparts in E. coli and Bacillus subtilis.
E. I. DuPont de Nemours Agricultural Products, Stine-Haskell Research Center, Newark, Delaware 19714, USA.
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Jordan
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Bacot
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Carlson
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Kessel
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Viitanen
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D001419:Bacteria / Q000201:enzymology; D001483:Base Sequence; D002736:Chloroplasts / Q000201:enzymology*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010947:Plants, Toxic; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012256:Riboflavin / Q000096:biosynthesis*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D018724:Spinacia oleracea / Q000201:enzymology* / Q000235:genetics; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics
D009097:Multienzyme Complexes; D011994:Recombinant Proteins; C044822:6,7-dimethyl-8-ribityllumazine synthase; D012256:Riboflavin