The pentafunctional arom enzyme of Saccharomyces cerevisiae is a mosaic of monofunctional domains.
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ABSTRACT:
SUBMITTER: Duncan K
PROVIDER: S-EPMC1148286 | biostudies-other | 1987 Sep
REPOSITORIES: biostudies-other
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Project description:Several yeast strains have been engineered to express different cellulases to achieve simultaneous saccharification and fermentation of lignocellulosic materials. However, successes in these endeavors were modest, as demonstrated by the relatively low ethanol titers and the limited ability of the engineered yeast strains to grow using cellulosic materials as the sole carbon source. Recently, substantial enhancements to the breakdown of cellulosic substrates have been observed when lytic polysaccharide monooxygenases (LPMOs) were added to traditional cellulase cocktails. LPMOs are reported to cleave cellulose oxidatively in the presence of enzymatic electron donors such as cellobiose dehydrogenases. In this study, we coexpressed LPMOs and cellobiose dehydrogenases with cellobiohydrolases, endoglucanases, and ?-glucosidases in Saccharomyces cerevisiae. These enzymes were secreted and docked onto surface-displayed miniscaffoldins through cohesin-dockerin interaction to generate pentafunctional minicellulosomes. The enzymes on the miniscaffoldins acted synergistically to boost the degradation of phosphoric acid swollen cellulose and increased the ethanol titers from our previously achieved levels of 1.8 to 2.7 g/liter. In addition, the newly developed recombinant yeast strain was also able to grow using phosphoric acid swollen cellulose as the sole carbon source. The results demonstrate the promise of the pentafunctional minicellulosomes for consolidated bioprocessing by yeast.
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Project description:The AROM protein is a pentadomain protein catalysing steps two to six in the prechorismate section of the shikimate pathway in microbial eukaryotes. On the basis of amino acid sequence alignments and the properties of mutants unable to utilize quinic acid as a carbon source, the AROM protein has been proposed to be homologous throughout its length with the proteins regulating transcription of the genes necessary for quinate catabolism. The QUTR transcription repressor protein has been proposed to be homologous with the three C-terminal domains of the AROM protein and one-fifth of the penultimate N-terminal domain. We report here the results of experiments designed to overproduce the QUTR and AROM proteins and their constituent domains in Escherichia coli, the purpose being to facilitate domain purification and (in the case of AROM), complementation of E. coli aro- mutations in order to probe the degree to which individual domains are stable and functional. The 3-dehydroquinate dehydratase domain of the AROM protein and the 3-dehydroquinate dehydratase-like domain of the QUTR spectroscopy and fluorescence emission spectroscopy. The CD spectra were found to be virtually superimposable. The fluorescence emission spectra of both domains had the signal from the tryptophan residues almost completely quenched, giving a tyrosine-dominated spectrum for both the AROM- and QUTR-derived domains. This unexpected observation was demonstrated to be due to a highly unusual environment provided by the tertiary structure, as addition of the denaturant guanidine hydrochloride gave a typical tryptophan-dominated spectrum for both domains. The spectroscopy experiments had the potential to refute the biologically-based proposal for a common origin for the AROM and QUTR proteins; however, the combined biophysical data are consistent with the hypothesis. We have previously reported that the AROM dehydroquinate synthase and 3-dehydroquinate dehydratase are stable and functional as individual domains, but that the 5-enol-pyruvylshikimate-3-phosphate synthase is only active as part of the complete AROM protein or as a bi-domain fragment with dehydroquinate synthase. Here we report that the aromA gene (encoding the AROM protein) of Aspergillus nidulans contains a 53 nt intron in the extreme C-terminus of the shikimate dehydrogenase domain. This finding accounts for the previously reported observation that the AROM protein was unable to complement aroE- (lacking shikimate dehydrogenase) mutations in E. coli. When the intron is removed the correctly translated AROM protein is able to complement the E. coli aroE- mutation. An AROM-derived shikimate dehydrogenase domain is, however, non-functional, but function is restored in a bi-domain protein with e-dehydroquinate dehydratase. This interaction is not entirely specific, as substitution of the 3-dehydroquinate dehydratase domain with the glutathione S-transferase protein partially restores enzyme activity. Similarly an AROM-derived shikimate kinase domain is non-functional, but is functional as part of the complete AROM protein, or as a bi-domain protein with 3-dehydroquinate dehydratase.
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Project description:Limited proteolysis of the arom enzyme complex of Neurospora crassa by trypsin or subtilisin yielded a stable fragment of Mr 68000. This fragment, which was purified by two-dimensional polyacrylamide-gel electrophoresis, was shown by activity staining to contain the shikimate dehydrogenase active site, and by substrate labelling with 3-dehydroquinate and NaB3H4 to contain the 3-dehydroquinase active site. The fragment thus constitutes a bifunctional domain containing the two enzymic activities that are known, from genetic evidence, to be located adjacently at the C-terminal end of the pentafunctional arom polypeptide.
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Project description:The dehydroquinate synthase (DHQ synthase) functional domain from the pentafunctional AROM protein of Aspergillus nidulans has previously been overproduced in Escherichia coli [van den Hombergh, Moore, Charles and Hawkins (1992) Biochem J. 284, 861-867]. We now report the purification of this domain to homogeneity and subsequent characterization. The monofunctional DHQ synthase was found to retain efficient catalytic activity when compared with the intact pentafunctional AROM protein of Neurospora crassa [Lambert, Boocock and Coggins (1985) Biochem J. 226, 817-829]. The apparent kcat. was estimated to be 8 s-1, and the apparent Km values for NAD+ and 3-deoxy-D-arabino-heptulosonate phosphate (DAHP) were 3 microM and 2.2 microM respectively. These values are similar to those reported for the intact N. crassa enzyme, except that the apparent Km for NAD+ reported here is 15-fold higher. The monofunctional DHQ synthase domain is inactivated by treatment with chelating agents in the absence of substrates and is re-activated by the addition of metal ions; among those tested, Zn2+ gave the highest kcat./Km value. The enzyme is inactivated by diethyl pyrocarbonate; both the substrate, DAHP, and the product phosphate protected against inactivation. Size-exclusion chromatography suggested an M(r) of 43,000 for the monofunctional domain, indicating that it is monomeric and compactly folded. The c.d. spectrum confirmed that the domain has a compact globular conformation; the near-u.v. c.d. of zinc- and cobalt-reactivated domains were superimposable.
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Project description:We have demonstrated the co-purification in constant ratio of all five activities of the pentafunctional arom enzyme complex from Neurospora crassa. Progressive inactivation of the 3-dehydroquinate synthase component of the purified enzyme complex by chelating agents was blocked by the substrate, 3-deoxy-D-arabino-heptulosonate 7-phosphate, but not by the cofactor NAD+. Full activity was restored at Zn2+ concentrations as low as 0.05 nM. Atomic absorption data indicated that the intact enzyme complex contained 1 atom per subunit of tightly bound zinc. The arom 3-dehydroquinate synthase had a calculated turnover number of 19s-1, this being within the narrow range of values obtained for the other four activities of the intact multifunctional enzyme. The Km for 3-deoxy-D-arabino-heptulosonate 7-phosphate was 1.4 microM in a phosphate-free buffer; inorganic phosphate was a competitive inhibitor with respect to 3-deoxy-D-arabino-heptulosonate 7-phosphate.
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