Evolutionary and Paleobiochemical Analyses of Heme Peroxidases

The focus of this thesis is to study the evolution of enzyme specificity, and the application of evolutionary theory to the design of enzymes with desirable characteristics for industry. The approach presented here, applied to the heme peroxidases, marries bioinformatic methods with evolutionary theory and biochemical validations. Heme peroxidases catalyse the oxidation of a variety of electron donors by hydrogen peroxide. These enzymes can be classified into two major families that arose from independent evolutionary events; the plant and the animal peroxidases. The first results chapter, Chapter 2, deals with the animal (mammalian) heme peroxidases known collectively as the MHP. Four main superfamilies of MHP have been classified; myeloperoxidase (MPO), eosinophil peroxidase (EPO), lactoperoxidase (LPO) and thyroid peroxidase (TPO).These comprise a functionally diverse multigene family of enzymes associated with such diseases as asthma, Alzheimer’s disease and inflammatory vascular disease. This study has determined how the enzymes in the multigene family of MHP are related. The order of gene duplication events has been traced, with an MPO-EPO-LPO most recent common ancestor (MRCA) arising from a gene duplication with extant TPO. A further duplication event gave rise to (i) the MPO-EPO MRCA, and (ii) the lineage leading to extant LPO. The final and most recent duplication of the MPO-EPO MRCA resulted in the extant MPO and EPO clades. This phylogeny was subsequently used to predict the amino acids that have most likely contributed to each of the diverse functions of MHP. Positively selected sites have been identified, through the use of Bayesian estimation, unique to all four MHP. Using MPO as a case study, in vitro analyses on the impact of mutating these positions, specifically mutants Y500F and L504T, indicates a disruption to the biosynthesis and loss of enzymatic activity in our mutants supporting our in silico predictions. This work is described in results Chapter 3. Finally, Chapter 4 details the analysis of ancestral protein reconstruction within the plant peroxidase gene family. The phylogeny of plant peroxidases had previously been resolved; this allowed for the generation of the ancestral enzyme, estimated age approx. 113 million years old. This enzyme was cloned, expressed and found to be active. Catalytic and stability properties of this unique enzyme have been ascertained. Together, these analyses provide a valuable insight into enzyme function through molecular evoultionary analyses of sequence data and serve to bridge the gap between protein sequence, structure, and function.

Name dc:type.qualificationname

phd

Level dc:type.qualificationlevel

doctoral

Grantor dc:publisher.institution

Dublin City University

Year dc:date.issued

2010

Author dc:creator

• Loughran, Noeleen B.

• Biotechnology
• Bioinformatics
• Biochemistry
• Enzymology

Language dc:language

en