|Haem d1 is a modified tetrapyrrole unique to the periplasmic enzyme nitrite reductase
where it acts in catalysing the reduction of nitrite (NO2
-) to nitric oxide (NO), as part
of denitrification. As with all modified tetrapyrroles, haem d1 shares a common
biosynthetic pathway starting from 5-aminolaevulinic acid (ALA), up to the formation
of uroporphyrinogen III (UIII). UIII is the branch point from which the pathway
diverges to form the various metallo-prosthetic groups including vitamin B12.
The precise mechanism of transformation from UIII to haem d1 is unknown.
Examination of both structures shows a requirement of methylation at C2 and C7;
decarboxylation of acetate side chains at C12 and C18; loss of propionic side chains at
C3 and C8 with subsequent oxidation at C3 and C8; dehydrogenation of C17
propionate side chain gives the acrylate substituent and ferrochelation. Of particular
interest is the addition of oxygen to the macrocycle under anaerobic conditions. Only
one other intermediate, compound 800, has been isolated thus far but it is unknown
how it is part of the pathway. Genetic studies have implicated seven nir genes, called
nirF, nirD, nirL, nirG, nirH, nirJ and nirE, are required for haem d1 biogenesis.
Here, experiments and data show for the first time that it proceeds from UIII to
precorrin-2 using the enzyme NirE. This study is the first to experimentally show the
production of precorrin-2 as part of the pathway using anaerobic enzyme assays.
This thesis illustrates the intense work that has focused on cloning the genes
individually and as multigene constructs in an attempt to characterise the proteins
overproduced. Heterologous expression in Escherichia coli has been successful as
well as the development of a homologous expression system in Pseudomonas
aeruginosa. The data represented shows the various aspects entailed in the
optimisation of overproduction and the stabilisation of the Nir proteins. It also
documents the first concerted attempt to take the operon and engineer strains to make
haem d1 both in vivo and in vitro, using the Link and Lock method to clone the nir
genes consecutively into a plasmid. This thesis therefore provides a foundation for
understanding the molecular biology and biochemistry of haem d1 synthesis for the