Studies on the AROM Pentafunctional Enzyme in Saccharomyces cerevisiae and Aspergillus nidulans

Gillies, Fiona McGregor (1994) Studies on the AROM Pentafunctional Enzyme in Saccharomyces cerevisiae and Aspergillus nidulans. PhD thesis, University of Glasgow.

Abstract

Aromatic amino acids are synthesised via the shikimate pathway. AROM is a pentafunctional enzyme which catalyses the five central steps of the shikimate pathway and is found in fungi and yeast. This thesis describes the purification and characterisation of AROM from overexpressing strains of Saccharomyces cerevisiae and Aspergillus nidulans. S. cerevisiae AROM was purified 30-fold with a 10% yield from a yeast overexpression strain which exploited the ubiquitin-fusion system of yeast. The purified protein was found to possess all five enzyme activities in a similar ratio to that observed in crude extract and had a subunit molecular weight of 175kDa. The main S. cerevisiae protein was shown to have several minor, lower molecular weight contaminants following SDS PAGE and three of these were found to cross-react with anti-AROM antibodies raised against Neurospora crassa AROM. The peak AROM fraction eluted from gel filtration chromatography was found to be composed of two proteins which were separable by native PAGE and both of which were shown to have shikimate DH activity. The poor recovery and multiple protein bands suggested that during the AROM preparation limited proteolysis was occurring despite a number of anti-proteinase measures. No means of eliminating limited proteolysis during S. cerevisiae AROM isolation were found and purification studies were carried out on the AROM of A. nidulans in the hope that proteolysis might not be as problematic in this species. A rapid procedure for the purification of A. nidulans AROM from the overexpresion strain A. nidulans 1314 has been developed which results in 13-fold purification and a 9% yield. The subunit molecular weight was estimated at 175kDa and the native molecular weight suggests that the protein is a dimer. The N-terminal DHQ synthase activity in the AROM protein was found to be severly deficient in both crude extract and the purified protein from A. nidulans 1314. This was independently attributed to the introduction of a missense muta tion in this region of the polypeptide. A preliminary limited proteolysis study was carried out on AROM purified from A. nidulans 1314 which suggested that the proteolysis pattern is complicated and which show that the DHQase and shikimate DH enzyme activities are least susceptible to limited proteolysis.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: J R Coggins
• Keywords: Biochemistry
• Date of Award: 1994
• Depositing User: Enlighten Team
• Unique ID: glathesis:1994-76043
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 19 Nov 2019 17:04
• Last Modified: 19 Nov 2019 17:04
• URI: https://theses.gla.ac.uk/id/eprint/76043