X-Ray structure of porphobilinogen deaminase from A. Thaliana at 1.5A resolution

Roberts, Andrea (2008) X-Ray structure of porphobilinogen deaminase from A. Thaliana at 1.5A resolution. University of Southampton, School of Biological Sciences, Doctoral Thesis, 253pp.

Abstract

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;
?=90.00º, ?=104.83º and ?=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.

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