Investigating the biosynthesis of heam d1 in pseudomonas aeruginosa: a cofactor for dissimilatory nitrite reductase.

Abstract

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 future.