BCH/PPA 503 -- Plant Biochemistry
Lecture Fourteen
Isoprenoids

 

LECTURE 17:  INTRODUCTION:

OVERVIEW OF SECONDARY METABOLIC PATHWAYS;  GENERAL ASPECTS OF PRIMARY/SECONDARY PATHWAYS AND METABOLIC REGULATION;

                                    ISOPRENOIDS: source and nomenclature

 

LECTURE 18:  ISOPRENOIDS:  synthetic mechanisms; examples of regulation.

 

LECTURE 19:  ISOPRENOIDS:  biosynthesis of select isoprenoids;  more examples of metabolic regulation 

LECTURE 20:  PHENYLPROPANOIDS:  secondary metabolites derived from phenylalanine

 

LECTURE 21:  PHENYLPROPANOIDS: lignin and lignin precursors.

 

 


LECTURE 17:   INTRODUCTION:

 OVERVIEW OF SECONDARY METABOLIC PATHWAYS;  GENERAL ASPECTS OF PRIMARY/SECONDARY PATHWAYS AND METABOLIC REGULATION

 

Themes:

 

1.      “Primary” (1˚) and “Secondary” (2˚) Pathways

 

The metabolic pathways to be discussed over the next several lectures can be all considered to generate both 1˚ and 2˚ metabolites and therefore have aspects of both 1˚ and 2˚ pathways.

“Updated” definition of secondary products: “Plant Natural Products” 

 

Plant Natural Products:  Those products (chemical compounds) of metabolism that are not essential for normal growth, development or reproduction of the plant or organism.  In this sense they are “secondary”.  

 

Definition of primary products:  substances present and required in all organisms [necessary for the growth, development and completion of the life cycle of an organism].

 

Ecology…

 

 

 

 

Phytoanticipants

 

 

L(I)R

 

SAR

 

 

 

2.      “Breadth” of diversity of secondary metabolism

There are several tens of thousands of secondary compounds identified in higher plants.  All the more remarkable considering only 20-30% of them have been investigated thus far.

Functions.

[Also keep in mind information derived from other organisms:

e.g., not just plants:  algae, fungi, bacteria]

 

 

3.      . Regulation -- Concept: the additional impact of secondary product formation in regard to primary metabolic pathways and cellular homeostasis

 

Keeping in mind the basic definition of pathways that lead to the synthesis of secondary compounds, consider:

 

a.       Types of regulatory mechanisms likely to be possible

 

b.       [e. g., when, where, how, expressed]

 

 

Implications for the types of regulation we might expect:

 

a.      inducibility (i.e., not constitutive;  elicited)

 

b.      temporal

 

c.      developmental;

 

d.      tissue specificity


Because of this diversity (in function and in pathway of the natural product and its regulation), the biochemistry of most secondary compounds produced in plants is not well studied.  Therefore, in the areas that are covered in this section, namely isoprenoids and phenylpropanoids, examples will be taken from the species and specific pathways that are most well studied, to illustrate major principles.

 

 

So, the above general themes should be kept in mind when considering all of the specific pathways we will cover. 

 

For example:

If a “true” secondary plant pathway is not normally expressed under most circumstances, how does the induction of the upregulation of such a pathway impact supply or “primary” pathways?

 

With such a pronounced chemical diversity of natural products, what is (are) the functional basis (bases) for this diversity?

 

 

With the numerous types of regulation normally associated with secondary pathways, (e.g., inducible, temporal, developmental, and tissue [and organelle] specificity) all of these levels of regulation must be considered as an integrated whole in order to fully understand how a pathway is regulated.  How is this achieved?

 

 

ISOPRENOIDS    (outline)

 

 

Fall into both primary and secondary metabolites/pathways.

 

Present in all living organisms – but unusually diverse:  (> 25,000 distinct plant isoprenoids identified!!),  possibly the largest class of natural products.

This is all the more remarkable when considering that only 20-30% of all plants have been investigated so far.

 

Historical:

 

Isoprene (2-methyl-1,3-butadiene) recognized as the basic constituent of “terpenes” by O. Wallach (Nobel prize in chemistry, 1910).

 Ruzicka (N.P. in chemistry, 1939) formulated “biogenic isoprene rule” which hypothesized that there was such a universal isoprene precursor.

Bloch and Lynen (N.P. in medicine or physiology, 1964) established the basic elucidation of the biosynthetic pathway.  Their discoveries concerned the mechanism and regulation of the metabolism of cholesterol and fatty acids, which relate to the understanding of steroid and fat metabolism.  Bloch showed that acetyl CoA is the precursor for steroid biosynthesis;  and Lynen identified the “active isoprene” (isopentenyl pyrophosphate), as the precursor to all isoprenoids, which confirmed Ruzicka’s speculation 25 years earlier.

 

 

Some metabolites/nomencature

Precursor

Classes

Examples

C#

Functions

 Location

 

 

 

 

IPP

Prenyl group

Cytokinin

5

Growth regulator

 

 

 

 

Menthol

10

Aroma, fragrance, plant-insect interactions,

 

 

 

 

 

Phytoalexins

15

Plant-pathogen interactions, Membrane structure/function

 

 

Phytol

20

 

 

 

 
Brassinosteroids 30
Carotenoids 40
Ubiquinone >40
Latex >>40

 

 

[]

 

Biosynthesis

Acetyl CoA is the precursor leading to isopentenyl pyrophosphate (IPP)

Isopentenyl pyrophosphate (IPP) is the universal precursor for all isoprenoid synthesis

 

How IPP is made in the "Mevalonate pathway":

[]

Initial steps of the pathway:  three molecules of acetyl-CoA are fused to produce the six-carbon (C6) 3-hydroxy-3-methylglutaryl-CoA (HMG)-CoA.

            The first two reactions are catalyzed by acetyl-CoA acetyltransferase and HMG-CoA synthase.  From here, HMG-CoA, is reduced in two steps, each requiring NADPH.  This step, catalyzed by HMG-CoA reductase (HMGR), is extremely important in animal systems, in that it is the rate-limiting reaction in cholesterol biosynthesis.  In plants, this step is much more complex (and controversial), not necessarily being a rate-limiting step, but still a “committed” or irreversible step.  [See Newman & Chappell. 1999. Isoprenoid Biosynthesis in Plants: Carbon Partitioning Within the Cytoplasmic Pathway (Critical Rev. in Biochem. and Mol. Biol. 34: 95-106)].

  

Research on HMGR had been hampered because it is a membrane-bound enzyme that made it difficult to purify and characterize.  This situation has greatly improved because of the isolation of genes from several species.  Claims that HMGR is present in the endoplasmic reticulum have by demonstration of co-translational insertion into the ER [Denbow, et al., J. Biol. Chem. 271: 9710-15. 1996].  In addition, the two transmembrane regions of the protein, which mediate insertion into the ER membrane, are conserved in all genes encoding HMGR to date.

Note that several “HMG” genes encoding HMGR can be present within the same plant species and species and display different levels of expression in different tissues, and differences in inducibility characteristics (e.g., elicitation by pathogen challenge; induction dependent on developmental stage). 

Isozymes

 

Isoforms

 

Allozmyes

 

Perhaps because of this variable results have been found when HMGR is overexpressed in transgenic lines.  In some cases, HMGR overexpression results in an increase in the levels of sterols, such as in tobacco, but no changes in other isoprenoid end products such as sesquiterpenes [Chappell et al., Ann. Rev. Plant Physiol Plant Mol. Biol. 46: 521-47 1995].  However, in other cases, such as HMGR overexpression in Arabidopsis, no effect was seen in sterol or other isoprenoid accumulation [Re et al., Plant Journal 7:771-84  1995].

 

 

Mevalonate formed from the reduction of HMG-CoA is sequentially phosphorylated by two distinct soluble enzymes, mevalonate kinase and 5-phosphomevalonate kinase.  The 5-pyrophosphomevalonate formed is then converted to IPP by a concerted decarboxylative elimination by pyrophosphomevalonate decarboxylase, requiring ATP and a divalent metal ion (see mevalonate pathway figure).

 

 

Isopentenyl pyrophosphate is the basic C5 building block for isoprenoid chain formation.  

 

 

 

THE "OTHER" PATHWAY FOR THE SYNTHESIS OF THE "UNVERSAL PRECURSOR", IPP :

 

It had been repeatedly observed that in tracer studies that labeled substrates such as mevalonate, make very poor precursors for many isoprenoids in plants.  There was, however, no reasonable alternative to the well defined “mevalonate pathway” as described above for the synthesis of IPP.  Studies in bacteria, on the other hand, indicated that radiolabeled precursors, such as 13C glucose, acetate, and pyruvate, were incorporated into bacterial isoprenoids.  If labeled intermediates of the mevalonate pathway were used in incorporation studies with E. coli cells, no labeled isoprenoid products were detected.  It was then determined that a C3 glycolysis product and pyruvate, were somehow formed to produce the C5 isoprenoid [Rohmer, et al., Biochem. J. 295: 517-24. 1993]. In 1994, a similar route for mevalonate-independent synthesis was first observed in plants (labeling pattern was identical to E. coli).   Since that time a large number studies have basically confirmed that there are two distinct pathways that operate in different plant cell compartments.  [Arigoni et al., PNAS 94: 10600-05. 1997;  Lange & Croteau PNAS 95: 2100-104 1998;  Lange & Croteau PNAS 96: 13714-19  1999]. 

 

Outline of the DXP pathway for the biosynthesis of IPP, and the proposed role of IPK. The circled P denotes the phosphate moiety. The large open arrow indicates several as-yet-unidentified steps.

 

The DXP pathway for the biosynthesis of  IPP

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It had been repeatedly observed that in tracer studies that labeled substrates such as mevalonate, made very poor precursors for many isoprenoids in plants. There was, however, no reasonable alternative to the well defined "mevalonate pathway" 

 

Two distinct pathways are utilized by plants for the biosynthesis of isopentenyl diphosphate, the universal precursor of isoprenoids. The classical acetate/mevalonate pathway operates in the cytosol, whereas plastidial isoprenoids originate via a novel mevalonate-independent route that involves a transketolase-catalyzed condensation of pyruvate and D-glyceraldehyde-3-phosphate to yield 1-deoxy-D-xylulose-5- phosphate as the first intermediate. Based on in vivo feeding experiments, rearrangement and reduction of deoxyxylulose phosphate have been proposed to give rise to 2-C-methyl-D-erythritol-4-phosphate as the second intermediate of this pyruvate/glyceraldehyde-3-phosphate pathway (1-3). The cloning of an Escherichia coli gene encoding an enzyme capable of converting 1-deoxy-D-xylulose-5-phosphate to 2-C- erythritol-4-phosphate was recently reported. A cloning strategy was developed for isolating the gene encoding a plant homolog of this enzyme from peppermint (Mentha  piperita), and the identity of the resulting cDNA was confirmed by heterologous expression in E. coli. Unlike the microbial reductoisomerase, the plant ortholog encodes a preprotein bearing an N-terminal plastidial transit peptide that directs the enzyme to plastids where the mevalonate-independent pathway operates in plants. The peppermint gene comprises an open reading frame of 1425 nucleotides which, when the plastidial targeting sequence is excluded, encodes a deduced enzyme of approximately 400 amino acid residues with a mature size of about 43.5 kDa. 

 

 

BIOSYNTHESIS OF HIGHER MOL. WT. TERPENOIDS

See "Prenyl transferases catalyze head to tail addition of active isoprene units"

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  • Reading Assignment for the 2nd isoprenoid lecture (LECTURE 18: ISOPRENOIDS: synthetic mechanisms; examples of regulation):

    • a)      REQUIRED:

    1 -   Newman and Chappell (1999) Isoprenoid Biosynthesis in Plants: Carbon Partitioning Within the Cytoplasmic Pathway. Crit. Rev. in Biochem. and Mol. Biol. 34: 95-106. 

    2 -    Chapter 24, sections 24.4 -24.5 of the Biochemistry & Molecular Biology of Plants class text.

     

    b)      OPTIONAL:

    1 -   McConkey, et al. (2000) Developmental Regulation of Monoterpene Biosynthesis in the Glandular Trichomes of Peppermint. Plant Physiol. 122: 215-223.  

    2 -    Bohlmann, et al., (2000) Terpenoid Secondary Metabolism in Arabidopsis thaliana: cDNA cloning, Characterization, and Functional Expression of a Myrcene/(E)-ß-Ocimene Synthase. Arch. of Biochem. and Biophys. 375: 261-269.

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