Thesis

Research and thesis

Steven Zachary Hanley

Funding body

BBSRC

Research Institutions

University Of Sheffield (main); IACR-Rothamsted

Supervisors

Dr Steven L. Kelly (Sheffield); Dr David Hallahan (Rothamsted)

PhD Examiners

Dr Kelvin Smith (QMC); Dr Graham Warren (Sheffield)

1. Summary/Abstract
2. Thesis outline and description of aims and objectives
3. Postscript

Summary/Abstract

The hydroxylation of geraniol is an important step in the formation of medicinally-important alkaloids in Catharanthus roseus. This reaction is catalysed by a CYP enzyme (haem-thiolate monooxygenase) and there is evidence to suggest that a member of the CYP71 family is specifically involved [Hallahan et al. (1992) Plant Physiology 98: 1290-7].

Two CYP71 enzymes (CYP71A5 from Nepeta racemosa and CYP71B1 from Thlaspi arvensae) were heterologously expressed in both Nicotiana tabacum and C. roseus. CYP71B1 was also heterologously expressed in Saccharomyces cerevisiae. Microsomes from S.cerevisiae and N.tabacum were assayed spectrophotometrically with a number of potential substrates in order to ascertain probable in vivo activities of the two enzymes. No interaction was observed with geraniol or structurally-related compounds, strongly suggesting that neither enzyme was involved in alkaloid metabolism. Strong interactions were observed with both enzymes and the herbicide chlortoluron, and transgenic plants grown on chlortoluron-containing medium were studied. The CYP71B1 enzyme also interacted with the polyaromatic compound benzo-(a)-pyrene but no metabolism was observed. A weak interaction was also observed for this enzyme with the herbicide glyphosate.

C. roseus plants expressing the two CYP71 enzymes were analysed for alterations in the production of the monomeric alkaloids ajmalicine and serpentine. Despite the evidence above suggesting that neither enzyme performed the hydroxylation of geraniol levels of these alkaloids were altered in transgenic plants.

A 'PCR prospecting' approach was undertaken in order to identify further candidate geraniol 10-hydroxylases from C. roseus. A cDNA encoding a novel CYP enzyme was also isolated from C.roseus using partial sequence data from other workers [Meijer (1993) Ph.D thesis, University Of Leiden]. The enzyme was determined to be a member of the CYP71A subfamily and therefore a potential candidate as the geraniol 10-hydroxylase of C. roseus.

Thesis Outline and Description of Aims and Objectives

The aim of this research project was to study several plant haem-thiolate enzymes of the CYP71 family. Experimental results were reported in chapters 3-6 of the thesis. These experiments were designed and carried out in order to answer the following questions:

1. Does the CYP71A5 enzyme of Nepeta racemosa perform the specific hydroxylation of the monoterpenoid geraniol at the tenth carbon position?
2. What is the in planta metabolic role of the CYP71B1 enzyme of Thlaspi arvensae?
3. Can the heterologous expression of haem-thiolate monooxygenase enzymes in Catharanthus roseus alter alkaloid biosynthesis?
4. What is the value of the 'PCR prospecting' method of identifying novel haem-thiolate enzymes?
5. What is the superfamilial affiliation and potential function of the novel haem-thiolate enzyme from Catharanthus roseus for which partial cDNA sequence is presented?

The aims and objectives of each chapter can be summarized as follows:

CHAPTER 1 - Introduction

The function of this chapter was to present an overview of the state of knowledge of plant haem-thiolate enzyme systems contemporaneous with the experiments reported in this thesis

(With 12 figures and 5 tables)

CHAPTER 2 - Materials & Methods

This chapter aimed to outline the materials and methods employed in the experiments described in subsequent chapters. Unpublished techniques, variations on published methods or methods not detailed in the literature cited were to be described in detail in order to facilitate any subsequent replication of experiments.

(With 4 figures and 3 tables)

CHAPTER 3 - The Production Of Transgenic Plants Harbouring Xenologous Haem-Tholate Enzymes

The experiments reported in chapter three were undertaken in order to clone both the CYP71A5 and CYP71B1 cDNAs into separate expression vectors and to introduce these novel recombinant plasmids into the genomes of tobacco and Madagascan periwinkle plants. Methods based on the use of two different Agrobacterium species would be devised, performed, validated and optimized for each plant species.

(With 20 figures and 5 tables)

CHAPTER 4 - Biochemical Analyses Of Plant CYP Enzymes In Heterologous Hosts

The aim of the experiments reported in chapter four was to analyse spectroscopically the interactions between each of the heterologously-expressed enzymes (CYP71A5 and CYP71B1) and various potential substrate molecules. Transformation of Saccharomyces cerevisiae with an expression cassette containing the CYP71B1 cDNA would also be undertaken to allow a wider survey of potential substrates for this protein.

(With 34 figures and 5 tables)

CHAPTER 5 - Attempts To Identify Novel Haem-Thiolate Enzyme Systems From Plants Via The Polymerase Chain Reaction

The experiments reported in chapter five were performed to isolate novel haem-thiolate cDNAs from the higher plant Catharanthus roseus using a 'PCR prospecting' approach. These experiments would incorporate the use of a variety of primers designed with reference to publicly-available plant haem-thiolate cDNA sequences and varying reaction conditions calculated using several different algorithms.

(With 17 figures and 3 tables)

CHAPTER 6 - Isolation And Analysis Of A Novel Haem-Thiolate Enzyme From A lambda-ZAP2 Library Of Catharanthus roseus

The aims of the experiments reported in chapter six were to produce sequence data encoding a novel Catharanthus roseus haem-thiolate enzyme and to analyse this partial data to allow predictions of the superfamilial position and possible metabolic role of this protein. Other data sets of partial sequences would be shown to be amenable to this approach.

(With 16 figures and 6 tables)

CHAPTER 7 - Discussion And Perspectives

The results presented in chapters 3-6 were used to answer the questions posed above in this chapter, which presents a discussion of the results in the context of the plant haem-thiolate field of research. An extract is presented below:

Is N.racemosa CYP71A5 a geraniol 10-hydroxylase?

The work in this thesis, coupled with other reported experiments by Hallahan and co-workers, would suggest that CYP71A5 is not a geraniol 10-hydroxylase enzyme. The expression pattern of CYP71A5 does not correlate with hydroxylation activity (Hallahan, personal communication). Heterologous expression experiments in tobacco showed that the active site of the enzyme does not have a biochemically-significant affinity for the structurally-similar monoterpenoids geraniol, nerol or citronellol.

What possible metabolic role does T.arvensae CYP71B1 play?

Substrate-binding experiments with T.arvensae CYP71B1 expressed in both Saccharomyces cerevisiae and N. tabacum failed to indicate an endogenous metabolic role for this enzyme.

Can heterologous CYP enzyme expression alter alkaloid metabolism in C.roseus?

The preliminary observation of an effect of either heterologously-expressed CYP71A5 or CYP71B1 on alkaloid anabolism in C.roseus suggested that these enzymes may interact with the biosynthetic pathway of the alkaloids at some point.

What is the value of the 'PCR prospecting' approach?

While the technique of 'PCR prospecting' has proved successful in several cases, it may nevertheless be eclipsed by new developments such as the computer methods in chap 6. The current emphasis on genome sequencing of many organisms of interest may mean that PCR prospecting becomes outmoded (particularly if advances in genome sequencing continue to reduce the costs of such major undertakings, rendering economical the comprehensive analysis of large C-value organisms such as higher plants).

To what CYP family does the CR10 sequence belong?

The methods used here strongly suggest that the CR10 sequence represents a member of the CYP71 family and probably a member of the CYP71A subfamily.

Appendix 1

References And Sources

Appendix 2

List Of Suppliers

Appendix 3

List Of Known Plant Haem-Thiolate Enzymes At Time Of Submission

Postscript

The thesis ended with the following:

Since the experiments reported and discussed in this thesis were designed and carried out there have been several developments in the study of plant haem-thiolate monooxygenases germane to the subjects of this work.

There have been no further publications on the endogenous roles of the N.racemosa CYP71A5 or T.arvensae CYP71B1 proteins, nor any further work on haem-thiolate monooxygenases in these plants. However, the enormous multiplicity of plant haem-thiolates is becoming apparent, suggesting that both plants may possess many more CYP isoforms. Since the allocation of CYP99 designation the CYP nomenclature system is to use CYP701A1, CYP702A1, etc. to include new families (Nelson, personal communication).

Several important new sequences have been released into the public domain, including some cyp51a1 cDNAs from sorghum (U74319) and wheat (two partial sequences, designated Y09291and Y09292, previously listed as unavailable). Kahn et al. (1996) h ave reported that the CYP51A1 isoform from sorghum does not bind lanosterol, campesterol, sitosterol or stigmasterol (analogues of its in vivo substrate obtusifoliol found in other organisms). This is in contrast to the work of Grausem et al. (1995) in which the yeast CYP51A1 orthologue (in vivo substrate lanosterol) could overcome a deficiency in obtusifoliol 14alpha-demethylation in transgenic tobacco calli. Van den brink et al. (1996) have reported complementation of Aspergillus niger eburicol 14alpha-demethylase by the Penicillium italicum CYP51A1 orthologue (which also metabolizes eburicol in vivo) in demethylase inhibition experiments. These studies suggest that the CYP51A family members share complex relationships and in some cases are not interchangeable. A detailed phylogenetic study of the CYP51A1 orthologues has yet to be published.

There have been other developments in the field of plant CYP enzyme studies. Chu and Cho (1996) report salicylic acid-induction of several haem-thiolate isoforms in oilseed rape (Brassica napus) implying that these enzymes are involved in defence mechanisms. The CYP90 enzyme of Arabidopsis thaliana has been implicated in steroid hormone metabolism and cell elongation (Szekeres et al., 1996). Sequences of several maize haem-thiolate monooxygenases of unknown function but presumed commercial importance remain outside the public domain (see Nelson, WWW). Beyond the plant kingdom, the first archaean CYP enzyme sequence (the extremely atypical CYP119A1 from Sulfolobus sulfotaricus, accession number U51337) has opened up the third kingdom of life to researchers in this field.

Other scientific work continues to impinge on the CYP enzyme world. The genomes of several organisms have been sequenced in their entirety and released into the public domain (e.g. S. cerevisiae and E. coli), inciting a large amount of computer searching using techniques akin to those described in chapter 6.Other, larger genome sequencing programs (e.g. Arabidopsis, Triticum, Homo) continue to release new sequences as ESTs, some of which have homology to CYP enzymes and show the promise of future research e.g. sixty-eight distinct haem-thiolate monooxygenase cDNA sequences have been identified in the first 63% of the Ceanorhabdidtis elegans (nematode) genome. This emphatically demonstrates the underappreciated diversity of the CYP superfamily. The two major WWW sites devoted to the dissemination of haem-thiolate monooxygenase information (Nelson, WWW; Degtyarenko, WWW) continue to expand with updates becoming more regular. The study of the extremely diverse haem-thiolate monooxygenase superfamily, which began as the biochemistry of unusual pigments in mammalian livers, continues to grow and expand with our understanding of the breadth and diversity of life.