Biosynthesis of Natural Products Containing …article.aascit.org/file/pdf/9000726.pdfInternational Journal of Biological Sciences and Applications 2015; 2(5): 48-66 52 Fig. 5. The

International Journal of Biological Sciences and Applications

2015; 2(5): 48-66

Published online October 20, 2015 (http://www.aascit.org/journal/ijbsa)

ISSN: 2375-3811

Keywords Natural products,

Phosphonic Esters,

Phosphinic Acids,

2-AEP, Bialaphos,

Fosfomycin,

Phosphinothricin,

FR900098,

Phosphatidylethanolamine

Received: September 28, 2015

Revised: October 15, 2015

Accepted: October 17, 2015

Biosynthesis of Natural Products Containing Phosphonic Esters and Acids: Review

Azza A. Kamel

Chemical Industries Division, National Research Centre (NRC), Elbohouth Street, Dokki, Cairo,

Egypt

Email address [email protected]

Citation Azza A. Kamel. Biosynthesis of Natural Products Containing Phosphonic Esters and Acids:

Review. International Journal of Biological Sciences and Applications.

Vol. 2, No. 5, 2015, pp. 48-66.

Abstract Natural products containing carbon-phosphorus bond (phosphonic esters and phosphinic

acids) have found widespread use in medicine and agriculture. As such, natural products

are the active components not only of most traditional medicines but also many modern

medicines. Furthermore, because the structural diversity of natural products exceeds that

readily achievable by chemical synthesis, and synthetic analogs can be prepared with

improved potency and safety, natural products are often used as starting points for drug

discovery. In fact, natural products are the inspiration for approximately one half of U.S.

Food and Drug Administration (FDA)-approved drugs. Recent years have seen a renewed

interest in the biochemistry and biology of these compounds with the cloning of the

biosynthetic gene clusters for several family members. This article aims to assay the

classification and biosynthesis of some natural products containing C-P bond. The current

knowledge regarding the metabolic pathways and enzymes involved in the production of a

number of natural products containing C-P bond (6-representative examples) was briefly

scrutinized. These compounds including: 2-AEP, bialaphos, fosfomycin, phosphinothricin,

FR900098, and phosphatidylethanolamine. The emphasis is on the recent information

added to this topic. Although the author has attempted the review to be encyclopedic with

respect to the topic, the article is not exhaustive.

1. Introduction

Since very ancient ages, man used plants and then crude extracts in the treatment of

diseases, as poisons, toxins, hormones, and stimulants. Recently, scientists have been

interested in both structure elucidation and synthesis of natural products, isolated from

plants, animals, insects, or lower organisms. The ability to extend nature's chemistry

through combinatorial biosynthesis—altering functional groups, regiochemistry and

scaffold backbones through the manipulation of biosynthetic enzymes—offers unique

opportunities to create natural product analogs [1].

A natural product is a chemical compound or substance produced by a living organism

that is found in nature. In the broadest sense, natural products include any substance

produced by life. Natural products can also be prepared by chemical synthesis (both

semi-synthesis and total synthesis) and have played a central role in the development of

the field of organic chemistry by providing challenging synthetic targets. The term natural

product has also been extended for commercial purposes to refer to cosmetics, dietary

supplements, and foods produced from natural sources without added artificial ingredients

[2-4].

49 Azza A. Kamel: Biosynthesis of Natural Products Containing Phosphonic Esters and Acids: Review

Chemistry of natural products is the branch of organic

chemistry that deals with the study of organic compounds

isolated from natural sources, such as plants and animals. This

science comprises extraction, isolation, structure elucidation,

synthesis and biogenesis. The work in this field is divided into

two major branches: a) the isolation and structure elucidation

of natural products; b) the synthesis of natural products [2].

Natural products containing carbon-phosphorus bond

(phosphonic and phosphinic acids) have found widespread use

in medicine and agriculture as phosphorus plays a

fundamental role in the microbial cell from the physiological

and biochemical points of view [2,3]. It is suggested [4] that

phosphonates, as well as phosphites, emerged at an early stage

of the Earth's evolution and could be prebiotic phosphorus

carriers. They are usually small molecules characterized by

the presence of a stable covalent C–P bond and have been

isolated from a wide variety of external life forms.

Furthermore, a significant percentage of biogenic phosphorus

is believed to occur in the C–P linkage. However, more and

more data show the ability of bacteria to utilize the more

reduced organophosphorus compounds as phosphorus sources,

in particular, phosphonates characterized by a single

carbon-to-phosphorus (C-P) bond. In contrast to the more

labile O-P, N-P, and S-P linkages, the C-P bond is extremely

resistant to chemical hydrolysis, thermal decomposition, and

photolysis. Only prokaryotic microorganisms are able to

cleave this bond [5].

To date, only a few number of the complete phosphonate

biosynthetic pathways has been unrevealed. In this article six

representative examples of natural products containing C-P

bond are shed light including 2-AEP, bialaphos, fosfomycin,

phosphinothricin, FR900098, and phosphatidylethanolamine.

2. Classification of Natural Products

Fig. 1. Biosynthesis of primary and secondary metabolites (From Wikipedia, the free encyclopedia).

International Journal of Biological Sciences and Applications 2015; 2(5): 48-66 50

Following Albrecht Kossel's original proposal in 1891 [6],

natural products are often divided into two major classes, the

primary and secondary metabolites [7,8] .

In contrast, secondary metabolites have an extrinsic

function that mainly affects other organisms.

Secondary metabolites are not essential to survival but do

increase the competitiveness of the organism within its

environment. Because of their ability to modulate biochemical

and signal transduction pathways, some secondary

metabolites have useful medicinal properties.

Natural products especially within the field of organic

chemistry are often defined as primary and secondary

metabolites (Fig. 1). A more restrictive definition limiting

natural products to secondary metabolites is commonly used

within the fields of medicinal chemistry and pharmacognosy

[8].

2.1. Primary Metabolites

Primary metabolites [7-13] have an intrinsic function that is

essential to the survival of the organism that produces them.

They are associated with essential cellular functions such as

nutrient assimilation, energy production, and

growth/development. They have a wide species distribution

that spans many phyla and frequently more than one kingdom

(Fig. 2). Primary metabolites include carbohydrates, lipids,

amino acids, and nucleic acids [7,8] which are the basic

building blocks of life [9]. Primary metabolites that are

involved with energy production include respiratory and

photosynthetic enzymes. Enzymes in turn are composed of

amino acids and often non-peptidic cofactors that are essential

for enzyme function [10]. The basic structures of cells and of

organisms are also composed of primary metabolites. These

include cell membranes (e.g. phospholipids), cell walls (e.g.

peptidoglycan, chitin), and cytoskeletons (proteins) [11].

DNA and RNA which store and transmit genetic information

are composed of nucleic acid primary metabolites [10].

Primary metabolite enzymatic cofactors include members

of the vitamin B family. Vitamin B1 as thiamine diphosphate

is a coenzyme for pyruvate dehydrogenase, 2-oxoglutarate

dehydrogenase, and transketolase which are all involved in

carbohydrate metabolism. Vitamin B2 (riboflavin) is a

constituent of FMN and FAD which are necessary for many

redox reactions. Vitamin B3 (nicotinic acid or niacin),

synthesized from tryptophan is a component of the coenzymes

NAD+ and NADP

+ which in turn are required for electron

transport in the Krebs cycle, oxidative phosphorylation, as

well as many other redox reactions. Vitamin B5 (pantothenic

acid) is a constituent of coenzyme A, a basic component of

carbohydrate and amino acid metabolism as well as the

biosynthesis of fatty acids and polyketides.

Fig. 2. Molecular building blocks of life (From Wikipedia, the free encyclopedia).

Vitamin B6 (pyridoxol, pyridoxal, and pyridoxamine) as

pyridoxal 5′-phosphate is a cofactor for many enzymes

especially transaminases involve in amino acid metabolism.

Vitamin B12 (cobalamins) contains a corrin ring similar in

structure to porphyrin and is an essential coenzyme for the

catabolism of fatty acids as well for the biosynthesis of


methionine [12].

First messengers are signaling molecules that control

metabolism or cellular differentiation. These signaling

molecules include hormones and growth factors in turn are

composed of peptides, biogenic amines, steroid hormones,

auxins, gibberellins, etc. These first messengers interact with

cellular receptors which are composed of proteins. Cellular

receptors in turn activate second messengers are used to relay

the extracellular message to intracellular targets. These

signaling molecules include the primary metabolites cyclic

nucleotides, diacyl glyceroletc [13].

2.2. Secondary Metabolites

In contrast to primary metabolites, secondary metabolites

[14,15]

are dispensable and not absolutely required for

survival. Furthermore, they typically have a narrow species

distribution (Fig. 3). Some secondary metabolites have a

broad range of functions. These include pheromones that act

as social signaling molecules with other individuals of the

same species, communication molecules that attract and

activate symbiotic organisms, agents that solubilize and

transport nutrients (siderophores etc.), and competitive

weapons (repellants, venoms, toxins etc.) that are used against

competitors, prey, and predators [14]. For many other

secondary metabolites, the function is unknown. One

hypothesis is that they confer a competitive advantage to the

organism that produces them. An alternative view is that, in

analogy to the immune system, these secondary metabolites

have no specific function, but having the machinery in place to

produce these diverse chemical structures is important and a

few secondary metabolites are therefore produced and

selected for [15].

Fig. 3. Representative examples of each of the major classes of secondary metabolites (From Wikipedia, the free encyclopedia).

3. Classification of Natural Products

Containing C-P Bond

3.1. Phospholipids

Phospholipids [16] are fat derivatives in which one fatty

acid has been replaced by either a phosphonate group or a

phosphate group and one of several nitrogen-containing

molecules. 2-Aminoethylphosphonic acid (2- AEP) is an

example for phospholipids containing a phosphonate group.

Whereas, examples for phospholipids containing a phosphate

group include phosphatidyl ethanolamine (also known as

Cephalin, represented in Fig. 4); phosphatidylcholine;

Lecithin and Phosphocholine.

Fig. 4. Structure of phosphatidyl ethanolamine (From Wikipedia, the free

encyclopedia).

A phospholipid, (as shown in Fig. 5) looks like a bubble

with two long chains attached. There are three major kinds of

lipids in living organisms, one of them are phospholipids as

demonstrated in Fig. 5.


Fig. 5. The parts of a phospholipid molecule (From Wikipedia, the free encyclopedia).

This example is phosphatidylcholine, represented (A) schematically, (B) by a formula, (C) as a space-filling model, and (D) as a symbol. The kink resulting from

the cis-double bond is exaggerated for emphasis. Phospholipids, present in biomembranes -which are the walls of the cells and organelles (inside the cells)- are

made of three types of molecules: [a] (two fatty acyl groups); [b] (1 glycerol); and [c] (a phosphate group which provides the hydrophilic characteristic).

3.2. Phosphorus Derivatives Containing

Nucleotides

Including compounds in which a methylene group (-CH2),

an imido group, or a sulfur atom replaces an oxygen atom

bridging two phosphorus atoms, e.g. nucleoside triphosphate

(Fig. 6) [17].

Fig. 6. Nucleoside triphosphate (From Wikipedia, the free encyclopedia).



Nucleic Acids

Such as, oligophosphates; phosphoguanidine;

phosphocreatine (represented in Fig. 7); bisnucleoside

phosphates and cyclic phosphates; phosphoramidic acids or

amidophosphoricacids [18].

Fig. 7. Phosphocreatine also known as creatine phosphate or PCr, is a phosphorylated creatine molecule that serves as a rapidly mobilizable reserve of

high-energy phosphates in skeletal muscle and the brain (From Wikipedia, the free encyclopedia).


Polysaccharides

An example is: Glycerol-3-phosphate which is a phosphoric

ester of glycerol (a component of glycerophospholipids) (Fig.

8).

Fig. 8. Structure of glycerol-3-phosphate (From Wikipedia, the free

encyclopedia).

4. Biosynthesis of Some Natural

Products Containing C-P Bond

Phosphonates are widespread among naturally occurring

compounds in all kingdoms of wildlife. Their industrial

synthesis became possible since 1905 after the discovery of

the Arbuzov reaction [9].

Moreover, a growing number of synthetic

organophosphonates has found applications in industrial,

agricultural, and domestic products and are ultimately

disposed of to soils or natural waters. The matter is that

organophosphorus compounds with the C-P bond are

widespread among man-made chemical substances,

xenobiotics, which uncontrollably enter the environment and

become a toxic factor. The search for the ways of phosphonate

biodegradation to understand molecular mechanisms of this

process is an urgent basic problem of physicochemical

biology that would also contribute to the solution of practical

problems of environmental protection biotechnology.


The ability to degrade phosphonates is relatively

widespread, occurring in Gram-positive and Gram-negative

bacteria as well as in fungi. Three classes of enzyme capable

of breaking the C-P bond of phosphonates are known:

PA-hydrolase, an enzyme specific for PA breakdown;

phosphonatase, which specifically degrades 2-AEP; and

C-P-lyase, which cleaves the C-P bond in a broad spectrum of

phosphonates. The C-P-lyase activity can be detected in the

whole organisms; nevertheless, it has never been convincingly

assayed in cell extracts, and this has limited attempts to

understand the mechanism of the enzyme, which has been

suggested to involve a redox-dependent free radical

mechanism. The C–P-lyase complex has a broad substrate

specificity [19] and can act upon unsubstituted alkyl and aryl

organophosphonates (general formula R–PO3H2). The two C–

P hydrolases that act on phosphonoacetaldehyde (OHC–CH2–

PO3H2) and phosphonoacetate (HOOC–CH2–PO3H2) are

essentially specific to their respective substrates (Fig. 9) [20].

Fig. 9. The hydrolytic cleavage of the C–P bond of phosphonopyruvate (McGrath, 2013 [20]).

A fourth C–P bond-metabolizing enzyme,

phosphoenolpyruvate phosphomutase (PEP mutase),

catalyzes the intramolecular rearrangement and

interconversion of 3-phosphonopyruvate (COOH–CO–CH2–

PO3H2) and phosphoenolpyruvate (COOH–C(=CH2)–O–

PO3H2). Phosphonopyruvate formation by this route is a key

step in the biosynthesis of all known natural products that

contain the C–P bond [21].

One of the most widely distributed of such biogenic C–P

compounds is 2-amino-3-phosphonopropionic acid,

commonly called phosphonoalanine (HOOC–CH-(NH2)–

CH2–PO3H2); it is formed through the transamination of

phosphonopyruvate by many lower organisms, such as the sea

anemone Zoanthussociatus and the protozoan Tetrahymena

pyriformis. The earlier studies on its biodegradation showed

that 47 of 100 randomly chosen environmental bacterial

isolates had the ability to utilize phosphonoalanine as a sole

phosphorus source for growth [22]. Subsequently, Ternan et al.

[23] reported the isolation of an environmental

Burkholderiacepacia strain capable of growth on

phosphonoalanine as sole source of carbon, nitrogen, and

phosphorus. Cell extracts of the isolate contained a previously

unknown Pi-independent activity designated

phosphonopyruvate hydrolase (PPH) that catalyzed the

hydrolytic cleavage of the C–P bond of phosphonopyruvate to

yield pyruvate and Pi (Fig. 9).

Other representatives of biogenic phosphonates are

antibiotics synthesized by Streptomyces. They include

phosphonomycin (1,2-cis-epoxyprolylphosphonic acid)

(known also as fosfomycin), an inhibitor of biosynthesis of

UDP-N-acetylmuramic acid essential for microbial cell wall

formation and bialaphos (L-alanyl-L-alanyl-phosphinothricin),

an inhibitor of glutamine synthetase in Escherichia coli and

plants. Biogenic phosphonates also include

phosphonopyruvateand phosphonoacetate (PA) [24].

The structures of some of the simplest biogenic


phosphonates are given in Fig. 10. Other representatives of

biogenic phosphonates include phosphonic acids widely used

in scientific research [25,26] and as drugs [26].

Fig. 10. Structures of orthophosphate and some biogenic phosphonates (From Wikipedia, the free encyclopedia).

The biosynthesis of phosphonolipids and other C-P

containing macromolecules has been extensively reviewed

elsewhere [27]. Almost, phosphonate/phosphonite

biosynthetic pathways involve the same initial steps, which

were first solved by analysis of AEP biosynthesis in

Tetrahymena. Therefore, the biosynthesis of this common

component of C-P containing macromolecules and small

molecules will be discussed first.

4.1. Biosynthesis of 2-AEP

2-Aminoethylphosphonic acid (2-AEP) is a constituent of

lipids, named, as phosphonolipids. Besides in lipids, 2-AEP

was found to be a constituent of proteins and polysaccharides.

2-AEP found in flagellates from rumen protozoa [28] was the

first known natural compound with the C-P bond. Further

investigations established that these compounds have been

found in protozoa, flagellates, coelenterates, mollusks, the

lower fungi, and even in man [29-33]. This compound and its

N-methylated derivative is the most abundant and ubiquitous

of the biogenic phosphonates. The biosynthesis of AEP from

phosphoenolpyruvate (PEP) (Fig. 11) is the shortest known

pathway for the construction of a natural phosphonate. It is

formed [34] by the decarboxylation of phosphonopyruvate to

phosphonoacetaldehyde, which is subsequently transaminated.

Degradation of the molecule involves a reverse

transamination to phosphonoacetaldehyde, followed by

cleavage of the C–P bond to yield acetaldehyde and Pi, as

demonstrated in the following equation.

Fig. 11. The biosynthesisof AEP from phosphoenolpyruvate (PEP). (From Wikipedia, the free encyclopedia).

The enzymes responsible for the completion of the biosynthetic reaction are:

pyruvate aminotransferase [EC 2.6.1.37; (1)] encoded by the phnW gene, and

phosphonoacetaldehyde hydrolase [phosphonatase; EC 3.11.1.1;(2)] encoded

by the phnX gene.

On the other hand, another biosynthetic reaction pathway

leading to the natural product, 2-AEP in Tetrahymena

pyriformis has been elucidated by Barry et.al [35]. Incubation

of [32

P] PEP and [14

C] PEP with T. pyriformis cellular

homogenate fortified with Mg2+

and alanine/pyridoxal

phosphate, yielded 2-aminoethylphosphonate as the minor

reaction product (2–5% yield) and phosphoglycerate and

pyruvate plus orthophosphate as the major products. Inclusion

of thiamine pyrophosphate in the reaction mixture increased

the yield of 2-aminoethylphosphonate by a factor of 10.

Incubation of phosphonoacetaldehyde or phosphonopyruvate

in the cellular homogenate also provided

2-aminoethylphosphonate. The cellular homogenate catalyzed

the transformation of phosphonoacetaldehyde to

2-aminoethylphosphonate in ca. 80% yield. However, the

maximum yield of 2-aminoethylphosphonic acid obtained by

use of phosphonopyruvate was only 15%. The major reaction

pathways induced by treatment of phosphonopyruvate with

the cellular extract involved its competitive conversion to PEP

and pyruvate plus orthophosphate.

Biosynthesis of phosphonates has been studied by the

example of 2-AEP to elucidate the mechanism of the C-P bond

formation (Fig. 12). It was shown that the first reaction in the

pathway of the C-P bond formation is the intramolecular

[2-aminoethylphosphonate] [phosphonoacetaldehyde]

H2O

(2)H2N [CH2] PO3H2 OHC CH2 PO3H2

pyruvate alanine

(1)+

(acetaldehyde) (Pi)

OHC CH3 H2PO4

-


restructuring of phosphoenolpyruvate to phosphonopyruvate

(Fig. 12, III A), catalyzed by

phosphoenolpyruvate-phosphomutase. Phosphonopyruvate

under the action of enzyme

phosphonopyruvate-decarboxylase is then converted to

phosphonoacetaldehyde which is the key intermediate in the

biosynthesis formation of many other phosphonates.

Phosphonoacetaldehyde can be converted either to

hydroxymethylphosphonic acid during the bialophos

biosynthesis (described in Section 4.2.) or to

2-hydroxypropylphosphonic acid during the phosphonomycin

biosynthesis (described in Section 4.3.). The finding of

enzymes responsible for the C-P bond biosynthesis suggested

that under appropriate conditions in a cell they could also

realize back reactions, i.e., to participate in decomposition of

the C-P bond. Indeed, as it was shown later,

phosphoenolpyruvate-phosphomutase is involved in

biodegradation of phosphonoalanine. It is suggested that the

ability to synthesize phosphonates could give

microorganisms- due to the presence of the chemically stable

and phosphatase-resistant C-P bond in these compounds-

certain biological advantages for survival in a potentially

phosphate-limited marine environment [5].

Fig. 12. Enzymes cleaving the C-P bond (From Wikipedia, the free encyclopedia).

H2N

PO3H2

H CH3

+ P1

O

H

PO3H2

O2AEP-pyruvateaminotransferase Phosphonatase

Phosphonatase( i )

HO

PO3H2

O

HO CH3P1

O

+

( ii ) Phosphonoacetate hydrolase

2-aminoethylphosphonate 2-phophonoacetaldehyde acetaldehyde

phosphonacetate acetate

HOOC C

PO3H2

H2

CH2

HOOC C

PO3H2

H2

O

phsphoenolpyruvate phosphonpyruvate

( iii) Phosphonopyruvate hydrolase

Aphsphoenolpyruvate

mutase

B

HOOC C

PO3H2

H2

O

H3C COOH

O

+ P1

phosphonpyruvate

phosphonpyruvatehydrolase

pyruvate

R P

PO3H2

OH

OH

O

H3C R P1+

P1+C-P lyase

( IV ) C-P lyase

R = H, CH3, H2N CH2-

C-P lyase


4.2. Biosynthesis of Bialaphos

Bialaphos (Fig.13):

(2S)-2-amino-4-[hydroxy(methyl)phosphinoyl]butyryl-L-ala

nyl-L-alanine; or (L-alanyl-L-alanyl-phosphinothricin), is a

natural herbicide produced by the bacteria Streptomyces

hygroscopicus [36] and Streptomyces viridochromogenes.

Bialaphos is a protoxin, non-toxic inhibitor of glutamine

synthetase in Escherichia coli and plants. When it is

metabolized by the plant, the glutamic acid analog

L-phosphinothricin is released which inhibits glutamine

synthetase. This results in the accumulation of ammonium and

disruption of primary metabolism [37]. Bialaphos is made up

of two alanine residues and phosphinothricin and is commonly

used as a gene sector in plants.

Fig. 13. Structure of bialaphos (From Wikipedia, the free encyclopedia).

The genes involved in the alanylation step in the

biosynthesis of a herbicide, bialaphos have been isolated and

studied by Nakashita et al [24]. It is produced by Streptomyces

hygroscopicus. Three bialaphos-non-producing mutants,

NP60, NP61 and NP62, isolated from S. hygroscopicus by

treatment with N-methyl-N'-nitro-N-nitrosoguanidine were

defective for the alanylation step and were not restored to

productivity by any locus of the gene cluster previously

cloned. Three plasmids were isolated using NP60, NP61 and

NP62 as recipients. The genes which restored productivity to

NP61 and NP62 hybridized to the contiguous region of the

bialaphos biosynthetic gene cluster. The gene cluster involved

in the bialaphos production was about 35 Kb long. The gene

which restored productivity to NP60 did not hybridize to the

bialaphos biosynthetic gene cluster. VM3 and VM4, putative

alanylation blocked mutants, were derived from a bialaphos

producer by gene replacement of an unidentified region of the

biosynthetic gene cluster with an in vitro altered DNA

sequence. The genes which restored productivity to VM3 and

VM4 were located between the genes which code for

phosphinomethylmalic acid synthase and

demethylphosphinothricin acetyltransferase in the cluster.

These results suggest that multiple genes are involved in the

alanylation step [38].

4.3. Biosynthesis of Fosfomycin

The research activities of the last years concentrated on

fosfomycin “referred also as phosphonomycin” (Fig. 14).

(1R,2S)-Epoxypropylphosphonic acid known as fosfomycin,

is a member of a class of compounds called phosphonic acids,

which is clinically utilized, highly effective antibiotic.

Fosfomycin is a low molecular weight natural product that

was originally isolated by Merck researchers from

Streptomyces fradiae, S. wedmorensis, and S.

viridochromogenes and was later shown to also be produced

by Pseudomonas syringae, and Pseudomonas viridiflava. In

the United States, fosfomycintromethamine

(tris(hydroxymethyl)aminomethane) is used under the name

Monurol® and is an FDA-approved drug that has become the

first choice for treatment of certain types of infections [39-44].

Attractively, fosfomycin has been proven effective for the

treatment of cephalosporin- and penicillin-resistant

Streptococcus pneumonia and is effective against

methicillin-resistant [41] and vancomycin-resistant [42]

strains of Staphylococcus aureus. In other countries like Japan,

Germany, Spain and France the compound is also used for

gastrointestinal infections [43].

Fig. 14. Structure of fosfomycin (From Wikipedia, the free encyclopedia).

Fosfomycin inactivates

UDP-N-acetyl-glucosamine-3-O-enolpyruvyltransferase

(MurA), an essential enzyme that catalyzes the first

committed step in cell wall biosynthesis [45-47], by covalent

alkylation of an active site cysteine [48,49].

Pioneering studies from the Seto laboratory on the

biosynthesis of fosfomycin, using genetic techniques [50]

complemented with in vivo experiments using isotopically

labeled precursors provided evidence for the biosynthetic

pathway shown in Fig. 15.

Antibiotics containing C-P bond as a class are virtually

uncharacterized on the genetic and biochemical level.

Although the biosynthetic pathway for fosfomycin production

in S. wedmorensis has been proposed, heterologous

production of fosfomycin has never been achieved, suggesting

the possibility that additional required genes remain to be

identified [51]. The enzymes encoded by fom 1-4 are


generally thought to catalyze consecutive steps as displayed in

Fig. 15, where PEP is converted to phosphonopyruvate (PnPy)

by PEP mutase, followed by a decarboxylation to form

phosphonoacetaldehyde (PnAA).

Fig. 15. The biosynthesis of fosfomycin (From Wikipedia, the free encyclopedia).

These first two steps are a common theme in the

biosynthesis of most phosphonates, where the

decarboxylation reaction derives the unfavorable PEP mutase

reaction forward. PnAA is known to be a biosynthetic

intermediate for several phosphonates such as bialaphos,

fosfomycin, 2-AEP, and 2-hydroxyethylphosphonate (HEP).

Both PEP mutase (Fom1) and PnPy decarboxylase (Fom2)

activities have been mapped to the corresponding genes in the

fosfomycin biosynthetic pathway. In the subsequent step of

the pathway, PnAA is proposed to be attacked by a methyl

anion derived from methylcobalamin (MeCbl) to form

2-hydroxypropylphosphonate (HPP). This step is unusual, as

it would involve an unprecedented use of methylcobalamin.

Since the methyltransferase encoded by fom3 has not been

characterized in vitro due to difficulties with functional

expression and purification, this proposed conversion is still

purely speculative. In the final step, HPP is oxidized to form

the epoxide, completing the biosynthesis of fosfomycin. This

unusual epoxidation step catalyzed by Fom4 has been

characterized in detail with several different crystal structures

reported for the enzyme. While the mechanistic details are still

under debate, there is a consensus that this enzyme catalyzes

the conversion of HPP to fosfomycin in a stereospecific

manner using a mononuclear metal. Thus, several important

features of fosfomycin biosynthesis have been discerned on

the biochemical level [51-55].

Finally, the enzyme C-P lyase is thought to catalyze the

direct scission of inactivated C-P bond to yield inorganic

phosphate and the corresponding hydrocarbons. It is

responsible for cleavage of alkylphoshponates, which are

utilized as sole phosphorus sources by many bacteria. C-P

lyase manifests its activity only in cells and has never been

reliably found in cell-free extracts (testing of such activity in

vitro turned out to be incorrect). This considerably limits the

possibility of understanding the mechanism of action of this

enzyme. However, several hypothetical models for the

mechanism of C-P lyase-catalyzed degradation of the C-P

bond have been proposed. The first working hypothesis

proposed that E. coli cells, growing in aerobic conditions,

oxidize alkylphosphonic acids at the first carbon atom bound

directly to phosphorus via incorporation of atomic or

molecular oxygen in this position [56-60].

The resulting α-hydroperoxy, α-hydroxy, α-keto, or

phophomonoesters could be easily utilized by cells. However,

the study of degradation of alkylphosphonates by cells of this

bacterium using isotopic-labeled material revealed no

intermediates corresponding to the mechanism proposed. In

fact, the main phenomenon of degradation of

methylphosphonic acid by E. coli cells is the predominant

formation of methane as the final reaction product in a ratio of

1:1 to intracellular phosphorus formed from

alkylphosphonates. Ethane, propane, butane, pentane, and

hexane are formed by these cells using appropriate derivatives

of phosphonic acid. A thorough study of degradation products

of the above alkylphosphonates showed also the presence of

ethene, propene, butene, etc., respectively. Results of the

analysis of degradation products of alkylphosphonates do not

thus fit the initially proposed mechanism and assume the

possibility of another mechanism.

A redox-dependent radical- based dephosphorylation

mechanism; probably including also participation of transition

metals in the reaction is depicted in Fig. 16 [61-65].


Fig. 16. Presumed mechanism of the C-P-lyase action (McGrath, 2013 [63]).

According to this mechanism, the process is as follows: a)

C–P lyase catalyses the reaction that converts a phosphonate

to a hydrocarbon and inorganic phosphate (Pi). b)

Specifically, using methylphosphonic acid as an example,

PhnI, a nucleotide phosphorylase, catalyses the displacement

of adenine from ATP by a phosphonate moiety in the

presence of PhnGHL to form a triphosphate ester. This is

hydrolysed by a phosphohydrolase, PhnM, to yield inorganic

pyrophosphate (PPi) and 5-phosphoribosyl-1- phosphonate.

The latter serves as a substrate for PhnJ-mediated cleavage of

the C–P bond in an S-adenosylmethionine (SAM)-dependent

radical-based reaction, resulting in release of the

phosphonate alkyl group and the production of

5-phosphoribosyl-1,2-cyclic phosphate. Part b image is

reproduced [63-65]. The formation of alkanes and alkenes as

a result of bacterial degradation of alkylphosphonates and

chemical degradation of alkylphosphonates with lead

tetraacetate is an important link between biodegradation and

the chemical model system, indicating the radical-based

mechanism of phosphonate degradation via the C-P-lyase

pathway.

4.4. Biosynthesis of Phosphinothricin

Phosphinothricin (PT), a non-proteinogenic amino acid

found in a number of peptide antibiotics, is the only known

phosphinic acid natural product (Fig. 17). In the early 1970s

independent groups in Germany and Japan discovered the

compound as a component of a tripeptide antibiotic

(PT-Ala-Ala) produced by Streptomyces viridochromogenes

(designated phosphinothricin-tripeptide, PTT)

[66]

or

Streptomyces hygroscopicus (designated bialaphos) [67]. PT

was later found as a component of phosalacine, a PT-Ala-Leu

tripeptide produced by Kitasatospora phosalacina [68], and

trialaphos (PT-Ala-Ala-Ala), a tetrapeptide produced by

Streptomyces hygroscopicus KSB-1285 [69].

Fig. 17. Structure of phosphinothricin (PT) (From Wikipedia, the free

encyclopedia).

PT is a structural analog of glutamate and a potent

inhibitor of glutamine synthetase. As a free amino acid, PT

has relatively poor antibiotic activity, probably due to

inefficient transport. Many organisms, however, readily take

up the peptide versions that are hydrolyzed by cytoplasmic

peptidases releasing the active component. Although PTT

has excellent antibacterial activity in vitro when minimal

media are used, it is a relatively poor therapeutic antibiotic

because its activity can easily be counteracted by the

presence of glutamine in host tissues. However, because

glutamine synthetase plays an essential role in pH


homeostasis in plants, PT is an outstanding herbicide and

both the tripeptide and synthetic versions of the monomer are

widely used in agriculture [70].

Interest in the unique C-P-C bond motif and the

biotechnological applications of PT has spurred a large

number of studies on its biosynthesis [8,70,71]. The PTT

biosynthetic pathway of S. hygroscopicus was largely solved

by the Seto group using an elegant combination of in vivo

labeling, in vitro biochemistry, genetics and gene cloning.

Subsequent studies in the laboratories of Thompson and

Wohlleben using S. viridochromogenes bolstered the

understanding of the biosynthetic pathway, especially with

regard to synthesis of the tripeptide. In combination, these

studies led to a proposed pathway that is substantially similar

to that shown in Fig. 18, but involving fewer enzymes and

intermediates [72].

Fig. 18. The most recent proposed biosynthetic pathway of phosphinothricin (From Wikipedia, the free encyclopedia). Three highly unusual transformations are

highlighted in blue, and the steps with similarities with glycolytic transformations are depicted in red.

The complete PTT gene cluster from S. viridochromogenes

was recently sequenced and contains 24 genes in a contiguous

33 kbp segment of the chromosome [73,74]. The complete S.

hygroscopicus gene cluster has also recently been sequenced


and is nearly identical (Metcalf and Blodgett, unpublished

data). No other unique genes are required for the synthesis of

PTT as shown by the ability of the cloned gene cluster to

confer antibiotic production on heterologous hosts [74]. The

availability of these genes allowed genetic and biochemical

experiments that led to the revised pathway presented in

Fig.18, which includes several unprecedented biochemical

transformations [72]. The revised pathway involves synthesis

of HEP via PEP mutase, PnPy decarboxylase and PnAA

reductase using genes and enzymes that are homologous to

those discussed in the preceding sections. HEP is then

converted to phosphinoalanine (PPA) by a series of

unprecedented reactions, some of which are analogous to

those of the Embden-Meyerhoff-Parnas pathway for

glycolysis, Fig. 18, inset. The complete biosynthetic pathway

for the preparation of phosphinothricin was discussed in

details in many literatures [73-90].

The potent herbicidal activity of both PTT and PT have led

to the development of the pat gene as a selectable marker for

genetic engineering of plants and it has been widely used in

this capacity. Further, the availability of crops carrying this

resistance allele has led to a recent boom in the paired use of

PT with recombinant plants in agriculture.

4.5. Biosynthesis of FR900098

FR900098 is one of a group of

N-hydroxypropylphosphonic acids that also includes

fosmidomycin, FR33289 and FR32863, which are produced

by strains of Streptomyces rubellomurinus and Streptomyces

lavendulae [91-93]. Both FR900098 and fosmidomycin have

very low toxicity in animals and humans and excellent

antibacterial activity against most Gram-negative bacteria, but

neither compound has achieved widespread clinical use.

Renewed interest in these antibiotics came with the genome

sequence of Plasmodium falciparum (the causative agent of

malaria), which revealed the unexpected presence of the target

pathway in this eukaryote. Fosmidomycin and FR900098 are

effective anti-malarial agents in animal models and have

shown promise in early human trails, including against

drug-resistant strains [94-96].

Fig. 19. Proposed biosynthetic pathway of FR900098 based on gene homologies and in vitro biochemistry (From Wikipedia, the free encyclopedia).


The FR900098 biosynthetic gene cluster was identified by

screening a large insert fosmid library from S. rubellomurinus

with degenerate PCR primers designed to amplify PEP

mutase-encoding (ppm) genes [97]. Further screening of

ppm-positive clones led to the identification of a clone that

conferred production of FR900098 to the heterologous host

Streptomyces lividans and, therefore, contains all of the

unique genes needed for synthesis of the antibiotic. Sequence

analysis of the clone revealed the presence of genes encoding

a putative PEP mutase and a number of genes related to those

encoding enzymes of the TCA cycle. The predicted functions

of these genes, in combination with in vitro biochemistry and

mass spectrometric identification of intermediates provide

strong support for the proposed biosynthetic pathway shown

in Fig. 19.

Unlike other phosphonate biosynthetic pathways, PnPy

decarboxylase does not provide the thermodynamic driving

force needed to pull the unfavorable PEP mutase reaction.

Instead, a homolog of homocitrate synthase, FrbC, catalyzes

the exergonic condensation of acetyl-CoA and

phosphonopyruvate to form 2-phosphonomethylmalate. This

activity has been verified in vivo using synthetic PnPy as

substrate, and also in a coupled reaction with the S.

rubellomurinus PEP mutase (FrbD) using PEP as the starting

material [97]. Subsequent steps are predicted to convert this

intermediate into 2-oxo-4-phosphonobutyrate, an analog of

2-oxo-glutarate, although this has yet to be demonstrated. An

unknown host encoded transaminase is thought to convert this

intermediate to 2-amino-4-phosphonobutyrate. FrbH is a

two-domain protein containing discrete

nucleotidyl-transferase and PLP-dependent

decarboxylase/aminotranserase domains that was predicted to

convert 2-amino-4-phosphonobutyrate into

3-aminopropylphosphonate. In vitro decarboxylation activity

of FrbH requires CTP and produces

5′-CMP-3-aminopropylphosphonate. The biological rationale

for this nucleotide modification is unclear; however, the

CMP-modified product is competent as a substrate for in vitro

acetylation with FrbF and N-hydroxylation by FrbG, whereas

free 3-aminopropylphosphonate is not. Removal of the CMP

is catalyzed by FrbI in vitro, although this gene is not required

in vivo, suggesting that other cellular phosphodiesterases can

fulfill this function as well.

Interestingly, the FR900098 gene cluster also includes a

gene (dxrB) that encodes a homolog of DXR, the target of the

antibiotic. The dxrB gene was proposed to encode an

FR900098-insensitive allele of DXR, and thus to be involved

in self-resistance to the antibiotic [97]. This idea has yet to be

tested experimentally, but if true, the structure of this protein

may lend insight into the nature of the interaction of the

antibiotic and the enzyme allowing design of more effective

derivatives.

4.6. Biosynthesis of

Phosphatidylethanolamine

Phosphatidylethanolamines (sometimes abbreviated PE)

are a class of phospholipids found in biological membranes. It

can mainly be found in the inner (cytoplasmic) leaflet of the

lipid bilayer [98].

Phosphatidylethanolamines are found in all living cells,

composing 25% of all phospholipids. In human physiology,

they are found particularly in nervous tissue such as the white

matter of brain, nerves, neural tissue, and in spinal cord, where

they make up 45% of all phospholipids [99]. In humans [100],

metabolism of PE is thought to be important in the heart.

When blood flow to the heart is restricted, the asymmetrical

distribution of PE between membrane leaflets is disrupted,

and as a result the membrane is disrupted. Additionally, PE

plays a role in the secretion of lipoproteins in the liver. This is

because vesicles for secretion of VLDLs coming off of the

Golgi have a significantly higher PE concentration when

compared to other vesicles containing VLDLs [101]. PE has

also shown to be able to propagate infectious prions without

the assistance of any proteins or nucleic acids, which is a

unique characteristic of it [102]. PE is also thought to play a

role in blood clotting, as it works with phosphatidylserine to

increase the rate of thrombin formation by promoting binding

to Factor V and Factor X, two proteins which catalyze the

formation of thrombin from prothrombin. PEs play a role in

membrane fusion and in disassembly of the contractile ring

during cytokinesis in cell division. Additionally, it is thought

that PE regulates membrane curvature. PE acts as an important

precursor, substrate, or donor in several biological pathways

[99].

As a polar head group, PE creates a more viscous lipid

membrane compared to phosphatidylcholine (PC). For

example, the melting temperature of di-oleoyl-PE is -16 °C

while the melting temperature of di-oleoyl-PC is -20 °C. If the

lipids had two palmitoyl chains, PE would melt at 63°C while

PC would melt already at 41 °C [100]. Lower melting

temperatures correspond, in a simplistic view, to more fluid

membranes.

Where phosphatidylcholine is the principal phospholipid in

animals, PE is the principal one in bacteria. One of the primary

roles for PE in bacterial membranes is to spread out the

negative charge caused by anionic membrane phospholipids.

In the bacterium E. coli, PE plays a role in supporting lactose

permease's active transport of lactose into the cell, and may

play a role in other transport systems as well. PE plays a role

in the assembly of lactose permease and other membrane

proteins. It acts as a 'chaperone' to help the membrane proteins

correctly folds their tertiary structures so that they can

function properly. When PE is not present, the transport

proteins have incorrect tertiary structures and do not function

correctly [103]. PE also enables bacterial multidrug

transporters to function properly. PE allows the formation of

intermediates that are needed for the transporters to properly


open and close.

Fig. 20. Biosynthesis of phophatidylethanolamine in bacteria (From Wikipedia, the free encyclopedia).


Phosphatidylethanolamines in food break down to form

PE-linked Amadori products as a part of the Maillard reaction

[104,105]. These products accelerate membrane lipid

per-oxidation, causing oxidative stress to cells that come in

contact with them [106]. Oxidative stress is known to cause

food deterioration and several diseases. Significant levels of

Amadori-PE products have been found in a wide variety of

foods such as chocolate, soybean milk, infant formula, and

other processed foods. The levels of Amadori-PE products are

higher in foods with high lipid and sugar concentrations that

have high temperatures in processing [105]. Additional

studies have found that Amadori-PE may play a role in

vascular disease [106,107], act as the mechanism by which

diabetes can increase the incidence of cancer, and potentially

play a role in other diseases as well. Amadori-PE has a higher

plasma concentration in diabetes patients than healthy people,

indicating it may play a role in the development of the disease

or be a product of the disease [108].

They are synthesized by the addition of Cytidine

diphosphate (CDP)-ethanolamine to diglycerides, releasing

Cytidine monophosphate (CMP). S-Adenosyl methionine can

subsequently methylate the amine of

phosphatidylethanolamines to yield phosphatidylcholines.

The phosphatidylserine decarboxylation pathway and the

CDP-ethanolamine pathways are used to synthesize PE.

Phosphatidylserine decarboxylase (PSD) is the enzyme that is

used to decarboxylate phosphatidylserine in the first pathway.

The phosphatidylserine decarboxylation pathway is the main

source of synthesis for PE in the membranes of the

mitochondria. PE produced in the mitochondrial membrane is

also transported throughout the cell to other membranes for

use. In a process that mirrors phosphatidylcholine synthesis,

PE is also made via the CDP-ethanolamine pathway, using

ethanolamine as the substrate. Through several steps taking

place is both the cytosol and endoplasmic reticulum, the

synthesis pathway yields the end product of PE [104]. PE is

also found abundantly in soya or egg lecithin and is produced

commercially using chromatographic separation (Fig. 20).

5. Conclusion and Prospective

Natural products continue to play a pivotal role in

drug-discovery efforts and in the understanding of human

health.

Phosphonate containing natural products constitute very

important part of biomolecules such as: phospholipids, nucleic

acids, proteins and polysaccharides, as well as nucleotide

cofactors that involved in energy transport and catalysis of

many cell processes.

The wide occurrence of phosphonates among biogenic and

abiogenic (natural and man-made) organophosphorus

compounds makes the question about the catabolism of these

compounds topical and at the same time potentially resolvable.

The diversity of phosphonate structures, occurrence among

these natural compounds with activated C-P bond

(aminophosphonates, acetylphosphonates); and other

compounds with non-activated, more stable C-P bond

(alkylphosphonates) determine the great number of pathways

of their catabolism.

A wide range of microorganisms, mainly bacteria, was

shown able to degrade different phosphonates. The

fundamental knowledge of the mechanism of biodegradation

of organophosphorus compounds with the C-P bond will serve,

in turn, as a basis of biotechnology for environmental

protection and biodegradation of toxic organophosphorus

compounds with stable C-P bond, which uncontrollably enter

the environment as pesticides, herbicides, and other products

of economic activity. However, only the pathways of activated

C-P bond degradation have been characterized rather

completely, and enzymes catalyzing this degradation have

been identified and characterized.

The most problematic aspect of phosphonate

biodegradation -from both fundamental and biotechnological

points of view- is the degradation of the most stable C-P bond

of alkylphosphonates. Neither the mechanism of degradation

of this bond nor the appropriate enzyme (or polyenzyme

complex) C-P-lyase has been finally determined and

characterized so far.

In summary, this article provides a brief survey on the

classification and biosynthesis of some natural products

containing C-P bond.

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