The work reported in this thesis was undertaken to fulfil three principal objectives :
il The investigation of a fungal ALAD.
The HEM2 gene encoding ALAD in Saccharomyces cerevisiae (hereafter called yeast) has been isolated and sequenced, the encoded protein expressed in low quantities (~ 2 0 0 pg/1) and shown to be zinc dependent [Myers et a l, 1987; Borralho et a l, 1990]. However, sequence data for other fungi is not available and the only other fungal ALADs to have been even broadly characterised are those from Neurospora crassa [Muthukrishnan et a l,
1968; 1972] and Ustilago sphaerogena [Komai and Neilands, 1968 and 1969]. Ustilago
sphaerogena ALAD was reported to be zinc dependent but the results are difficult to
interpret fully as only partial purification of the enzyme was achieved [Komai and Neilands, 1969]. The metal dependency of the enzyme from Neurospora has not been reported.
Studies on an ALAD from a fungus are of interest for two main reasons. Firstly, it is important to determine whether the metal dependency of all fungal ALADs resembles that from yeast and Ustilago and secondly, a fungal ALAD may be a very good target for fungicides as evidence exists supporting a controlling role in the tetrapyrrole pathway. For example, studies on the pathway of haem biosynthesis in Neurospora crassa indicated that ALAD is present in small amounts, has a lower than other enzymes in the pathway and is subject to regulation by the endogenous levels of protoporphyrin in a feedback mechanism [Muthukrishnan et a l, 1968; Chandrika et a l, 1980]. ALAD also forms a rate limiting step for tetrapyrrole biosynthesis in yeast [Labbe-Bois and Volland, 1977].
Yeast ALAD was chosen as a model fungal ALAD in this study. In order to obtain enzyme in sufficient quantity for characterisation and inhibition studies, an efficient over expression and purification system was developed and this is described in chapter 3. In addition, the isolation of a gene encoding ALAD in other fungi was investigated and this is also discussed in chapter 3.
Chapter 1 : Introduction to tetrapyrroles and 5-aminolaevulinic acid dehydratase
ii") Comparative studies on the ALADs from E. coli. veast and pea.
As discussed earlier, on the basis of metal binding, enzyme physical properties and sequence analysis, there appear to be differences between ALADs from different sources. However, investigators have used a variety of experimental approaches and the variation in results could reflect this rather than underlying biochem ical differences between ALADs. A comparative study of ALADs was therefore undertaken using three ALADs. The enzyme from yeast was taken as a representative of fungal ALADs as discussed above. The enzyme from E. coli was taken as a representative of the mammalian type of ALAD since it shares their requirement for zinc ions, their octameric structure and their sulphydryl group sensitivity to oxidation [Spencer and Jordan, 1993; Mitchell and Jaffe, 1993]. In addition, an efficient overexpression system for the hemB gene encoding ALAD
in E. coli had been developed [Roessner et al., 1995]. The ALAD from Pisum sativum
(hereafter called pea) was taken to represent the plant type of ALAD since it is known to bind magnesium [M.P. Timko, pers. commun.].
The enzymes were purified to homogeneity as described in chapter 3 and characterised in terms of physical properties such as subunit structure, metal binding and pH dependent kinetics as discussed in chapter 4. The inhibition of the enzymes was also investigated with the hypothesis that if the classes of enzyme are truly different, it should be possible to identify inhibitors which selectively inhibit the activity of one class of enzyme. The inhibitors identified provide a tool to investigate the mechanism of action and arrangement o f residues at the active site of ALADs and also form the basis for rational design of specific herbicides or insecticides. This work is discussed in chapter 5.
iiil Structural studies on veast ALAD.
A clearer framework for the discussion of the differences between ALADs would be provided by a three dimensional structure for the enzyme. However, at the start of this work, structural information was limited. Electron microscopy of bovine ALAD had been reported, revealing the arrangement of the eight subunits at the corners o f a cube-like structure with dihedral symmetry [Wu et a l, 1974] and small-angle X-ray scattering had provided more detail, describing the protein as a quadratic arrangement o f four stacks, composed o f two subunits each [Pilz et a l, 1988]. A general model for ALAD has been proposed in which four pairs o f subunits are arranged around a fourfold symmetry axis [Jaffe et a l, 1995]. In addition, the crystallisation and preliminary X-ray diffraction data for the bovine ALAD have been reported although the resolution of this work is poor [Carrell e t a l , 1996].
In light of this dearth of structural information, structural studies were undertaken with the three enzymes in this study (from E. coli, yeast and pea). In chapter 6 o f this work, the
Chapter 1 ; Introduction to tetrapyrroles and 5-aminolaevulinic acid dehydratase
crystallisation of yeast ALAD is described with the substantial progress made towards the determination of the structure of the enzyme by X-ray diffraction methods. In addition, the secondary structure of the three ALADs has been extensively investigated using circular dichroism and these results are discussed in chapters 4 and 6.