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Title: X-Ray structure of porphobilinogen deaminase from A. Thaliana at 1.5A resolution
Author: Roberts, Andrea
ISNI:       0000 0004 2672 631X
Awarding Body: University of Southampton
Current Institution: University of Southampton
Date of Award: 2008
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The enzyme, porphobilinogen deaminase (PBGD), catalyses the stepwise polymerization of the monopyrrole, porphobilinogen (PBG), to give the linear hydroxymethylbilane, preuroporphyrinogen. Preuroporphyrinogen is then cyclised, with rearrangement, to give uroporphyrinogen III, the ubiquitous precursor of haems, chlorophylls and corrins. In Arabidopsis thaliana, PBGD is the fifth enzyme of the chlorophyll biosynthetic pathway. The mature A. thaliana PBGD protein of 320 amino acids was expressed from two synthetic genes using the pT7-7 vector in Escherichia coli strain BL21 (DE3). One construct was identical to the nucleotide sequence of the A. thaliana HEMC (AT5G08280) coding region and the other was similar, but with an E. coli codon bias. Neither recombinant enzyme contained the chloroplast import sequence and possessed N-termini of NH2-MVAV… rather than NH2- CVAV… A high proportion (over 90%) of the expressed protein was found to be insoluble and much time was spent increasing the yield of soluble enzyme and obtaining sufficient material for crystal preparation. No significant differences in expression were noted for the two constructs and both purified enzymes had Mr values of 34,928 ± 4 as measured by mass spectrometry. Crystals were obtained from screens using 30% PEG 4000, 0.1M NaAc and 0.1M MgCl2, pH 4.6, containing 2mM dithothreitol. Data from suitable crystals were collected at the ESRF at Grenoble and were in space group C2 with unit cell dimensions of 142.1Å x 37.36Å x 55.37Å; α=90.00º, β=104.83º and γ=90.00º. A structure for the A. thaliana PBGD was obtained at 1.5Å resolution by molecular replacement using the E. coli PBDG enzyme as search model. Programmes used for the refinement included the CCP4 suite, MOSFLM, SORTMTZ, SCALA, TRUNCATE and HKL VIEW. The structure of the A. thaliana PBGD enzyme shows the presence of three domains, each of approximately 100 residues. A deep cavity between domains I and II constitutes the active site and harbours the dipyrromethane cofactor. Domain III provides the attachment site for the cofactor which is covalently bound to Cys 254. The structure shows, for the first time, a flap or “lid” over the active site, not previously observed in the E. coli and human PBGD structures. The differences and similarities between the A. thaliana PBGD structure and deaminase structures from E. coli and human sources are discussed. As security, a selenomethionine derivative of the enzyme was also prepared and crystals were obtained for possible multiwavelength anomalous dispersion (MAD) experiments. Two mutants of A. thaliana PBGD, D95N and R161K, were prepared and the proteins were isolated. The D95N mutation led to an inactive enzyme, whereas the R161K mutation yielded an enzyme with 10% activity, and a lowered pH optimum, since the mutation substituted one of the six conserved active site arginine residues. The thesis presents, for the first time, the X-ray structure of a PBGD from a higher plant, Arabidopsis thaliana, and is the first time that the “lid” over the active site has been resolved. The importance of the active site “lid” in the functioning of the enzyme is discussed.
Supervisor: Shoolingin-Jordan, Peter Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QH301 Biology ; QC Physics ; QA76 Computer software