Limited proteolysis of aspartokinase I-homoserine dehydrogenase I from Escherichia coli by type VI protease from Streptomyces griseus yields five proteolytic fragments, three of which are dimeric, the other two being monomeric. One of the monomeric fragments (27 kilodaltons) exhibits residual aspartokinase activity, while the second one (33 kilodaltons) possesses residual homoserine dehydrogenase activity. The smallest of the dimeric species (2 X 25 kilodaltons) is inactive; the two other dimers exhibit either only homoserine dehydrogenase activity (2 X 59 kilodaltons) or both activities (hybrid fragment, 89 + 59 kilodaltons). This characterization of the proteolytic species in terms of molecular weight, subunit structure, and activity leads to the proposal of a triglobular model for the native enzyme. In addition, the time course of the formation of the various fragments was followed by measuring enzymatic activity and performing gel electrophoretic analysis of the protein mixture at defined time intervals during proteolysis. On the basis of the results of these studies, a reaction scheme describing the succession of events during proteolysis is given.
Species of coryneform bacteria (Corynebacterium glutamicum, Brevibacterium flavum, and B. ammoniagenes) utilize pretyrosine [fl-(1-carboxy-4-hydroxy-2,5cyclohexadien-1-yl) alanine] as an intermediate in L-tyrosine biosynthesis. Pretyrosine is fonned from prephenate via the activity of at least one species of aromatic aminotransferase which is significantly greater with prephenate as substrate than with either phenylpyruvate or 4-hydroxyphenylpyruvate. Pretyrosine dehydrogenase, capable of converting pretyrosine to L-tyrosine, has been partially purified from all three species. Each of the three pretyrosine dehydrogenases is catalytically active with either nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate as cofactors. The Km values for nicotinamide adenine dinucleotide phosphate in C. glutamicum and B. flavum are 55 AM and 14.2 ,uM, respectively, and corresponding Km values for nicotinamide adenine dinucleotide are 350,M and 625,uM, respectively. The molecular weights of pretyrosine dehydrogenase in C. glutamicum and in B. flavum are both about 158, 000, compared with 68,000 molecular weight in B. ammoniagenes. In all three species the enzyme is not feedback inhibited by L-tyrosine. Results obtained with various auxotrophic mutants, which were used to manipulate intemal concentrations of L-tyrosine, suggest that pretyrosine dehydrogenase is expressed constitutively. Pretyrosine dehydrogenase is quite sensitive to p-hydroxymercuribenzoic acid, complete inhibition being achieved at 10 to 25 uM concentrations. This inhibition is readily reversed by thiol reagents such as 2mercaptoethanol. Coryneform organisms, like species of blue-green bacteria,
Wild-type Brevibacterium flavum has shown to possess arogenate dehydrogenase activity and I prephenate dehydrogenase, thereby providing presur evidence that arogenate (previously named "pretyrosine' obligatory intermediate of L-tyrosine biosynthesis. A s enzymological pattern has been discerned in extracts mad wild-type cultures of various species of cyanobacteria. cation of rigorous molecular genetic criteria in confirm of the exclusive role of arogenate in L-tyrosine synthes made possible by the isolation of an auxotrophic muti hibiting a nutritional requirement for L-tyrosine. The n was found to lack activity for arogenate dehydrogenase accumulate substantial amounts of arogenate behind the I block during starvation for L-tyrosine. arogenate branchlet for L-tyrosine biosynthesis. Wild-type cells of Corynebacterium glutamicum, Brevibacterium flavum, and B. ammoniagenes lack prephenate dehydrogenase activity and were shown to contain prephenate aminotransferase and arogenate dehydrogenase, the two enzyme activities illustrated above.More rigorous establishment of conclusions based upon data derived from wild-type cells would be provided by the demonstration that tyrosine auxotrophy corresponds to the mutational loss of arogenate dehydrogenase and that arogenate accumulates behind the mutant block. The latter approach has been fulfilled in B. flavum, and this system now offers the most rigorous documentation to date of an exclusive role in vivo of the arogenate branchlet for L-tyrosine biosynthesis.
MATERIALS AND METHODS
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