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

The phylogeny of the mammalian heme peroxidases and the evolution of their diverse functions

The mammalian heme peroxidases (MHPs) are a medically important group of enzymes. Included in this group are myeloperoxidase, eosinophil peroxidase, lactoperoxidase, and thyroid peroxidase. These enzymes are associated with such diverse diseases as asthma, Alzheimer’s disease and inflammatory vascular disease. Despite much effort to elucidate a clearer understanding of the function of the 4 major groups of this multigene family, we still do not have a clear understanding of their relationships to each other

Stability properties of an ancient plant peroxidase

Plant (Class III) peroxidases have numerous applications throughout biotechnology but their thermal and oxidative stabilities may limit their usefulness. Horseradish peroxidase isoenzyme C (HRPC) has good catalytic turnover and is moderately resistant to heat and to excess (oxidizing) concentrations of hydrogen peroxide. In contrast, HRP isoenzyme A2 (HRPA2) has better oxidative but poorer thermal stability, while soybean peroxidase (SBP) displays enhanced thermal stability. Intrigued by these variations amongst closely related enzymes, we previously used maximum likelihood methods (with application of Bayesian statistics) to infer an amino acid sequence consistent with their most recent common ancestor, the ‘Grandparent’ (GP). Here, we report the cloning and expression of active recombinant GP protein in Escherichia coli. GP’s half-inactivation temperature was 45 oC, notably less than HRPC’s, but its resistance to excess H2O2 was 2-fold greater. This resurrected GP protein enables a greater understanding of plant peroxidase evolution and serves as a test-bed to explore their ancestral properties

Positive selection neighboring functionally essential sites and disease-implicated regions of mammalian reproductive proteins

<p>Abstract</p> <p>Background</p> <p>Reproductive proteins are central to the continuation of all mammalian species. The evolution of these proteins has been greatly influenced by environmental pressures induced by pathogens, rival sperm, sexual selection and sexual conflict. Positive selection has been demonstrated in many of these proteins with particular focus on primate lineages. However, the <it>mammalia </it>are a diverse group in terms of mating habits, population sizes and germ line generation times. We have examined the selective pressures at work on a number of novel reproductive proteins across a wide variety of <it>mammalia</it>.</p> <p>Results</p> <p>We show that selective pressures on reproductive proteins are highly varied. Of the 10 genes analyzed in detail, all contain signatures of positive selection either across specific sites or in specific lineages or a combination of both. Our analysis of SP56 and Col1a1 are entirely novel and the results show positively selected sites present in each gene. Our findings for the Col1a1 gene are suggestive of a link between positive selection and severe disease type. We find evidence in our dataset to suggest that interacting proteins are evolving in symphony: most likely to maintain interacting functionality.</p> <p>Conclusion</p> <p>Our <it>in silico </it>analyses show positively selected sites are occurring near catalytically important regions suggesting selective pressure to maximize efficient fertilization. In those cases where a mechanism of protein function is not fully understood, the sites presented here represent ideal candidates for mutational study. This work has highlighted the widespread rate heterogeneity in mutational rates across the <it>mammalia </it>and specifically has shown that the evolution of reproductive proteins is highly varied depending on the species and interacting partners. We have shown that positive selection and disease are closely linked in the Col1a1 gene.</p

A Case-by-Case Evolutionary Analysis of Four Imprinted Retrogenes

Retroposition is a widespread phenomenon resulting in the generation of new genes that are initially related to a parent gene via very high coding sequence similarity. We examine the evolutionary fate of four retrogenes generated by such an event; mouse Inpp5f_v2, Mcts2, Nap1l5, and U2af1-rs1. These genes are all subject to the epigenetic phenomenon of parental imprinting. We first provide new data on the age of these retrogene insertions. Using codon-based models of sequence evolution, we show these retrogenes have diverse evolutionary trajectories, including divergence from the parent coding sequence under positive selection pressure, purifying selection pressure maintaining parent-retrogene similarity, and neutral evolution. Examination of the expression pattern of retrogenes shows an atypical, broad pattern across multiple tissues. Protein 3D structure modeling reveals that a positively selected residue in U2af1-rs1, not shared by its parent, may influence protein conformation. Our case-by-case analysis of the evolution of four imprinted retrogenes reveals that this interesting class of imprinted genes, while similar in regulation and sequence characteristics, follow very varied evolutionary paths

The phylogeny of the mammalian heme peroxidases and the evolution of their diverse functions

Abstract Background The mammalian heme peroxidases (MHPs) are a medically important group of enzymes. Included in this group are myeloperoxidase, eosinophil peroxidase, lactoperoxidase, and thyroid peroxidase. These enzymes are associated with such diverse diseases as asthma, Alzheimer's disease and inflammatory vascular disease. Despite much effort to elucidate a clearer understanding of the function of the 4 major groups of this multigene family, we still do not have a clear understanding of their relationships to each other. Results Sufficient signal exists for the resolution of the evolutionary relationships of this family of enzymes. We demonstrate, using a root mean squared deviation statistic, how the removal of the fastest evolving sites aids in the minimisation of the effect of long branch attraction and the generation of a highly supported phylogeny. Based on this phylogeny we have pinpointed the amino acid positions that have most likely contributed to the diverse functions of these enzymes. Many of these residues are in close proximity to sites implicated in protein misfolding, loss of function or disease. Conclusion Our analysis of all available genomic sequence data for the MHPs from all available completed mammalian genomes, involved sophisticated methods of phylogeny reconstruction and data treatment. Our study has (i) fully resolved the phylogeny of the MHPs and the subsequent pattern of gene duplication, and (ii), we have detected amino acids under positive selection that have most likely contributed to the observed functional shifts in each type of MHP.</p

The phylogeny of the mammalian heme peroxidases and the evolution of their diverse functions-6

Cates are shown on all nodes. The TPO primate clade appears here as a polytomy as the branch lengths are extremely short, however, this is in fact resolved with a low Bootstrap of 56%. The star symbol denotes those branches that were treated as foreground in the selection analysis. The analysis of the resolved phylogeny using gene tree species tree reconciliation method implemented in GeneTree. The large filled circles represent gene duplication events, and the red branches indicate gene losses.<p><b>Copyright information:</b></p><p>Taken from "The phylogeny of the mammalian heme peroxidases and the evolution of their diverse functions"</p><p>http://www.biomedcentral.com/1471-2148/8/101</p><p>BMC Evolutionary Biology 2008;8():101-101.</p><p>Published online 27 Mar 2008</p><p>PMCID:PMC2315650.</p><p></p

The phylogeny of the mammalian heme peroxidases and the evolution of their diverse functions-3

Ely selected in MPO, in blue is the heme binding site. Example of the affect on hydrogen bonding of one such mutation at positively selected position 496 in human MPO from Asparagine to Phenylalanine.<p><b>Copyright information:</b></p><p>Taken from "The phylogeny of the mammalian heme peroxidases and the evolution of their diverse functions"</p><p>http://www.biomedcentral.com/1471-2148/8/101</p><p>BMC Evolutionary Biology 2008;8():101-101.</p><p>Published online 27 Mar 2008</p><p>PMCID:PMC2315650.</p><p></p

The phylogeny of the mammalian heme peroxidases and the evolution of their diverse functions-5

He entire dataset. The bootstrap support values from 1000 replicates are shown on all nodes. (b) Resolved phylogeny following site stripping, the cow sequence for LPO can be seen to take an unusual place on the phylogeny.<p><b>Copyright information:</b></p><p>Taken from "The phylogeny of the mammalian heme peroxidases and the evolution of their diverse functions"</p><p>http://www.biomedcentral.com/1471-2148/8/101</p><p>BMC Evolutionary Biology 2008;8():101-101.</p><p>Published online 27 Mar 2008</p><p>PMCID:PMC2315650.</p><p></p