Publications

• Phosphoribosyl anthranilate isomerase from Thermotoga maritima is an extremely stable and active homodimer.

  Sterner R., Kleemann G.R., Szadkowski H., Lustig A., Hennig M., Kirschner K.

  The metabolism of hyperthermophilic microorganisms can function properly at temperatures close to 100 degrees C. It follows that they are equipped with both thermostable enzymes and mechanisms that handle labile metabolites. We wanted to understand how stable and active phosphoribosyl anthranilate ... >> More

  The metabolism of hyperthermophilic microorganisms can function properly at temperatures close to 100 degrees C. It follows that they are equipped with both thermostable enzymes and mechanisms that handle labile metabolites. We wanted to understand how stable and active phosphoribosyl anthranilate isomerase (tPRAI) from the hyperthermophile Thermotoga maritima is at its optimum growth temperature of 80 degrees C, and how its thermolabile substrate, N-(5'-phosphoribosyl)-anthranilate (PRA), is protected from rapid decomposition. To this end, the trpF gene of T. maritima was expressed heterologously in Escherichia coli and tPRAI was purified. In contrast to most PRAIs from mesophiles, which are monomers with the eightfold beta alpha (or TIM) barrel fold, tPRAI is a homodimer. It is strongly resistant toward inactivation by temperatures up to 95 degrees C, by acidification to pH 3.2, and by proteases in the presence and absence of detergents. tPRAI is about 35-fold more active at its physiologic temperature than is the enzyme from E. coli (ePRAI) at 37 degrees C. This high catalytic efficiency of tPRAI is likely to complete successfully with the rapid spontaneous hydrolysis of PRA at 80 degrees C. Thus, with respect to both stability and function, tPRAI appears well adapted to the extreme habitat of T. maritima. Single crystals of tPRAI have been obtained that are suitable for X-ray analysis at high resolution. << Less

  Protein Sci 5:2000-2008(1996) [PubMed] [EuropePMC]

• Two-fold repeated (beta alpha)4 half-barrels may provide a molecular tool for dual substrate specificity.

  Kuper J., Doenges C., Wilmanns M.

  Some bacterial genomes contain an incomplete set of genes encoding phosphoribosyl isomerases, raising the question of whether there exists broadened substrate specificity for the missing gene products. To investigate the underlying molecular principles of this hypothesis, we have determined the cr ... >> More

  Some bacterial genomes contain an incomplete set of genes encoding phosphoribosyl isomerases, raising the question of whether there exists broadened substrate specificity for the missing gene products. To investigate the underlying molecular principles of this hypothesis, we have determined the crystal structure of the bifunctional enzyme PriA from Streptomyces coelicolor at 1.8 A resolution. It consists of a (betaalpha)(8)-barrel fold that is assembled by two symmetric (betaalpha)(4) half-barrels. The structure shows how its active site may catalyse the isomerization reactions of two different substrates, and we provide a plausible model of how the smaller of the two substrates could be bound in two different orientations. Our findings expand the half-barrel ancestor concept by demonstrating that symmetry-related half-barrels could provide a smart solution to cope with dual substrate specificity. The data may help to unravel molecular rationales regarding how organisms with miniature genomes can keep central biological pathways functional. << Less

  EMBO Rep. 6:134-139(2005) [PubMed] [EuropePMC]

• The role of the TRP1 gene in yeast tryptophan biosynthesis.

  Braus G.H., Luger K., Paravicini G., Schmidheini T., Kirschner K., Hutter R.

  Transcription of the gene for phosphoribosyl-anthranilate isomerase (TRP1) from the TRP1 promoter is initiated only approximately half as frequently as, for example, from the TRP3 promoter, but TRP1 mRNA is approximately twice as stable as TRP3 mRNA. Therefore, the steady state amount of TRP1 mRNA ... >> More

  Transcription of the gene for phosphoribosyl-anthranilate isomerase (TRP1) from the TRP1 promoter is initiated only approximately half as frequently as, for example, from the TRP3 promoter, but TRP1 mRNA is approximately twice as stable as TRP3 mRNA. Therefore, the steady state amount of TRP1 mRNA in yeast cells, grown without amino acid limitation, is similar to the steady-state amount of TRP3 mRNA. The protein concentration of both enzymes in yeast cells is about the same, but the basal specific enzyme activity in permeabilized cells of the TRP1 gene product N-(5'-phosphoribosyl-1)-anthranilate isomerase is about 2-3 times higher than that of any of the other TRP enzymes. According to the kinetic parameters of the purified isomerase protein, the enzyme is more active than, for example, the purified TRP3 enzyme indoleglycerol-phosphate synthase. It is suggested that the TRP1 gene of Saccharomyces cerevisiae might be the result of a rearrangement event, separating the N-(5'-phosphoribosyl-1)-anthranilate isomerase domain from the indoleglycerol-phosphate synthase domain and putting the catalytically more active isomerase domain behind a weak and nonregulated constitutive promoter. << Less

  J Biol Chem 263:7868-7875(1988) [PubMed] [EuropePMC]

• Tryptophan biosynthetic genes in eukaryotic microorganisms.

  Hutter R., Niederberger P., DeMoss J.A.

  In recent years more information about tryptophan biosynthesis in eukaryotic microorganisms has become available. The emphasis has been on genetics and biochemistry of the pathway. Eukaryotes manifest a trend toward fewer genes and toward multifunctional proteins, while prokaryotes have a greater ... >> More

  In recent years more information about tryptophan biosynthesis in eukaryotic microorganisms has become available. The emphasis has been on genetics and biochemistry of the pathway. Eukaryotes manifest a trend toward fewer genes and toward multifunctional proteins, while prokaryotes have a greater tendency toward separate activity domains but the genes tend to be clustered genetically. Cloning of various structural tryptophan biosynthetic genes and studies on their expression in homologous and heterologous hosts have made it possible to analyze promoter structures in detail and to define structural elements involved in regulated gene expression. Comparisons of homologous genes from different organisms have highlighted the conservation of the activity domains or parts therefrom involved in the catalysis of single steps. These studies also point to a stringent maintenance of domains responsible for protein-protein aggregation. Physiological studies will be facilitated by the availability of single cloned genes and especially the artificial gene cluster containing all five TRP genes from yeast. The range of physiological manipulation has thus been enormously broadened. With chromosomal mutations it has been possible to study primarily downward modulation of a pathway. We can now initiate studies on upward modulation, since enzyme levels appear to increase in proportion to gene dose. The new range of downward and upward modulation in the levels of single enzymes and combinations of enzymes may contribute to a better understanding of flux regulation and its influence on the overall physiology of an organism. << Less

  Annu Rev Microbiol 40:55-77(1986) [PubMed] [EuropePMC]

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