Direct Evolution of P450 Enzyme to Achieve the Controlled Oxidation of Aliphatic Compounds or Methane

Steve S.-F. YuInstitute of Chemistry, Academia Sinica

2009/04/15

Methane is a green-house gas. It is an abundant fossil fuel. It is excreted from the earth’s mantle via vents in the ocean floor. There is considerable methane trapped as methane cathrates in the ocean floor at the high pressures that exist there. There is sufficient methane in the earth to serve as a source of fuel for 200 years at the present rate of energy consumption, if we know how to harness the gas.

One of the Holy Grails of Organic Chemistry

R1R2R3CH + “O” R1R2R3OH

Energetics of C-H Activation

Calculated bond dissociation energies for the reaction

R- H R + H

R DR-H/KJ mol-1 (kcal mol-1)

CH3 = 435 (104.0)

CF3 444 (106.0)

CHMe2 394 (94.2)

CMe3 387 (92.5)

CH=CH2 453 (108.3)

CCH 500 (119.5)

HC=O 363 (86.7)

C-H activation by Metal Complexes in Organometallic Chemistry

Organometallic catalyzed C-H activation usually involves the activation of the C-H bond first, then reacts with dioxygen to

form the alcohol.

M + RH [M RH] M

H

R

O

H

R

M[O]

+

C-H activation by Metal Complexes in Biology

In the enzymatic pathway, the dioxygen is usually activated at the metal site first, and then followed by oxo-transfer chemistry with the substrate, either by a two step radical process (hydrogen abstraction followed by rebound chemistry), or by concerted "oxene" insertion.

MO + RH [MOR

O

H

R

M

+ RH O

H

R

MMO M

R

OH

M[O]

M[O]

H] +

..+

Concerted

Radical

RC C

RH

H

HH

RC C

RH

H

HOH

[O]

RC C

R

H

H

The Enzymes that Involve in C-H Activation

1.Alkane Monooxygenase:Soluble Methane Monooxygenase, C1-C8 alkanes or aromatic compounds; Particulate Methane Monooxygenase, C1-C5 alkanes, Iron Heme Cyt. P-450, fatty acids or bridge compounds, i.e. camphor; Butane Monooxygenase from Pseudomonas butanovora (ATCC 43655);Alkane monooxygenase from Pseudomonas oleovorans (ATCC 29347).

2. Fatty Acid Desaturases.

Cytochrome P-450cam

from Pseudomonas putida

cytoplasm

periplasm

-Hydroxylase from Pseudomonas

oleovorans

Soluble Methane Monooxygenase

from Methylococcus capsulatus (Bath)

Cellular metabolism

In all forms of life, many important and difficult chemical transformations are catalyzed by enzymes. Many of these enzymes are involved in the metabolism of the cell, particularly, the biosynthesis of key metabolites.

Nature has adapted modular design to allow the evolution of these enzymes to accommodate different substrates. Enzymes belonging to a given type of chemical transformation are designed to consist of a “module” that is specific to a given type of chemistry, for example, the transfer of “O” to the C-H bond of an organic substrate, and a different module that could be tuned to accommodate specific substrates and the specific C-H bond to be oxidized.

The modular design makes sense if the same chemistry is to be utilized for different substrates. The substrate module could be tuned to control the regio-specificity and stereo-selectivity of the chemical transformation. In this manner, the wheel is not reinvented every time a new substrate comes on to the scene. Instead, Nature responds to a specific need by taking advantage of what is already there and adapting the design of the second module to the accommodate the new substrate.

For many years, the paradigm of the enzymes that mediate controlled

biological oxidations was

Cytochrome P450.

• A family of closely similar enzymes.

• Several hundred members of this protein family are known, each with a different substrate specificity.

• In the adrenal cortex, a specific cytochrome P450

participates in the hydroxylation of steroids to yield the adrenocortical hormones.

• Mitochondrial cytochrome P450 converts cholesterol to the sex hormones progesterone, testosterone and estradiol in the human reproductive glands (gonads and placenta).

• Eukaryotic microsomal cytochrome P450 is also important in the hydroxylation of many different drugs, such as barbiturates and other xenobiotics (substances that are foreign to the body, e.g., the carcinogen benzo[pyrene (found in cigarette smoke)).

• In the adrenal cortex, a specific cytochrome P450

participates in the hydroxylation of steroids to yield the adrenocortical hormones.

• Mitochondrial cytochrome P450 converts cholesterol to the sex hormones progesterone, testosterone and estradiol in the human reproductive glands (gonads and placenta).

• Eukaryotic microsomal cytochrome P450 is also important in the hydroxylation of many different drugs, such as barbiturates and other xenobiotics (substances that are foreign to the body, e.g., the carcinogen benzo[pyrene (found in cigarette smoke)).

Cytochrome P450

Typically, the substrates are hydrophobic, so many of these enzymes are membrane proteins.

There are a few exceptions, for example cytochrome P450cam from the bacterium Pseudomonas putida, and the fatty acid monooxygenase from Bacillus megaterium.

Hydroxylation Reaction Mediated by Cytochrome P450 BM-3

pmo1 Promoter

pmo1 C

Target Gene Coding Sequence

6xHis Tag & Termination CodonTranscriptional Terminator Sequence

pmo1 Promoter

pmo1 C

Target Gene Coding Sequence

6xHis Tag & Termination CodonTranscriptional Terminator Sequence

Directed Evolution of Alkane Oxygenases

Octane hydroxylation activity

Surrogatep-nitrophenyl octyl ether

Colorimetric identification

mutants

Frances H. Arnold et al., J. Am. Chem. Soc. (2003), 125, 13442.

Experimental Procedures

CH3CH2CH2 CH3 + “O”

CH3CH2CH(OH) CH3

Fatty acid monooxygenase from Bacillus megaterium

Cytochrome P450 BM-3

Glieder, A.; Farinas, E. T.; Arnold, F. H. Nat. Biotech. 2002, 20, 1135.

Fasan, R.; Meharenna, Y. T.; Snow, C. D.; Poulos,T. L. and Arnold, F. H. J. Mol. Biol. 2008, 383, 1069

Glieder, A.; Farinas, E. T.; Arnold, F. H. Nat. Biotech. 2002, 20, 1135.

Comparison of the specific activities of various monooxygenases toward their substrates

Comparison of the specific activities of the wild type cytochrome P450 BM-3 and its 139-3 variant for various alkane substrates

Production distribution for alkane oxidation by wild-type cytochrome P450 BM-3 and its 139-3 variant

Bacterium: Pseudomonas putida

Cytochrome P450cam

O O

HO

p450cam

O2, 2e-, 2H+

+ H2O

Schlichting I, Jung C, Schulze H. FEBS Lett. 1997, 415, 253.

Cytochrome P450cam

S. G. Bell, J.-A. Stevenson, H. D. Boyd, S. Campbell, A. D. Riddle, E. L. Orton, L.-L. Wong, Chem. Commun. 2002, 490.

F. Xu, S. G. Bell, J. Lednik, A. Insley, Z. Rao, L. L. Wong, Angew. Chem. Int. Edit. 2005, 44, 4029.

Production of alkane oxidation by wild-type cytochrome P450cam and its mutants

F87W

Y96F

V247L

The above analysis should pertain to the hydroxylation of any hydrocarbon that show a reasonable binding affinity for the active site of cytochrome P450.

What about small alkanes, e.g., methane, ethane, propane, and so on, which have larger C-H bond energies (and large barriers for C-H activation), but which are expected to bind to the hydrophobic pocket of cytochrome P450 with only a very low sticking coefficient.

Production of synthetically useful alcohols by enzymatic hydroxylation. Sites of enzymatic hydroxylation of (a) Trimegestrone ® and (b) codeine are indicated by open arrows. (c) The conversion of a-pinene to the mint flavour ingredient verbenol. (d) The production of (S)-N-benzyl-3-hydroxypyrrolidine by the hydroxylation of N-benzylpyrrolidine. (e) The prodution of diastereomerically define vicinal diol derivatives of fatty acids by the hydroxylation of hydroxymyristic acids using cytochrome (cyt P450-BM3).

Taxol Biosynthetic

Pathway

Addition of Radicals to Alkenes: Polymers

• A polymer is a very large molecule consisting of repeating units of simpler molecules, formed by polymerization

• Alkenes react with radical catalysts to undergo radical polymerization• Ethylene is polymerized to poyethylene, for example

Free Radical Polymerization of Alkenes

• Alkenes combine many times to give polymer– Reactivity induced by formation of free radicals

Free Radical Polymerization: Initiation

• Initiation - a few radicals are generated by the reaction of a molecule that readily forms radicals from a nonradical molecule

• A bond is broken homolytically

Polymerization: Propagation

• Radical from intiation adds to alkene to generate alkene derived radical

• This radical adds to another alkene, and so on many times

Polymerization: Termination

• Chain propagation ends when two radical chains combine• Not controlled specifically but affected by reactivity and concentration

Other Polymers• Other alkenes give other common polymers

Polymer Synthesized by Microorganism

• PHA Poly(hydroxyalkanoic acid)

• ACP Acyl-carrier protein

• PhaA b-Ketothiolase

• PhaB NAD(P)H-dependent acetoacetyl-CoA-reductase

• PhaC PHA synthase

References:Polyhydroxylbutyrate, Wikipedia

Alexander Steinbüchel and Bernd Füchtenbusch, TIBTECH OCTOBER 1998 (VOL 16), 419.

Glossary

3HA 3-Hydroxyalkanoic acid3HB 3-Hydroxybutyric acid3HD 3-Hydroxydecanoic acid3HDD 3-Hydroxydodecanoic acid3HHx 3-Hydroxyhexanoic acid3HO 3-Hydroxyoctanoic acid3HTD 3-Hydroxytetradecanoic acid3HV 3-Hydroxyvaleric acid4HB 4-Hydroxybutyric acid4HV 4-Hydroxyvaleric acid

Polyhydroxybutyrate (PHB)

• Polyhydroxybutyrate (PHB) is a polyhydroxyalkanoate (PHA), a polymer belonging to the polyesters class that was first isolated and characterized in 1925 by French microbiologist Maurice Lemoigne. PHB is produced by micro-organisms (like Alcaligenes eutrophus or Bacillus megaterium) apparently in response to conditions of physiological stress.

• Microbial biosynthesis of PHB starts with the condensation of two molecules of acetyl-CoA to give acetoacetyl-CoA which is subsequently reduced to hydroxybutyryl-CoA.

• This latter compound is then used as a monomer to polymerize PHB.

• Water insoluble and relatively resistant to hydrolytic degradation. This differentiates PHB from most other currently available biodegradable plastics, which are either water soluble or moisture sensitive.

• Good oxygen permeability. • Good ultra-violet resistance but poor resistance to acids and

bases. • Soluble in chloroform and other chlorinated hydrocarbons. • Biocompatible and hence is suitable for medical applications.

•Melting point 175°C., and glass transition temperature 15°C. •Tensile strength 40 MPa, close to that of polypropylene. •Sinks in water (while polypropylene floats), facilitating its anaerobic biodegradation in sediments. •Nontoxic.

• PHB is everywhere. Trace amounts in short chains of only about 150 units have been found in the cells of yeast, carrots, spinach, sheep, pigs, cattle and even in humans. It exists in the cells of a staggering variety of different organisms. In fact, it seems that you can find PHB in any cell that you care to choose, if you look hard enough. And nobody knows what it's there for. Surely, for something to be so ubiquitous, it must have some function. It's inconceivable that it's just an accident that PHB is present in so many places. Some scientists have even claimed that PHB could be as important as proteins and that HB units (hydroxy butyrate) might have been present in the primordial soup on earth, before amino acids and proteins. These claims may be extravagant, but whatever the real story is, watch this space; PHB must do something significant in cells.

White PHB blobs inside a cressleaf

The PHB formed 14% of the dry weight of the leaves.

The future for PHA

• Owing to a number of novel features of poly(3HB) and poly(3HB-co-3HV), these PHAs were initially used mainly in the manufacture of bottles, films and fibres for biodegradable packaging materials and as mulch films for agriculture.

• A latex of PHAs may be applied to paper or cardboard to form a waterresistant layer and to produce a completely biodegradable compound material that requires relatively low amounts of the currently expensive PHAs;

• PHAs can also be applied as a matrix in retardant materials for the slow release of drugs, hormones, herbicides, insecticides, flavours and fragrances in medicine, pharmacy, agriculture and the food industry.

• In addition to the production of PHA by microbial fermentation for special biotechnological applications, the production of some PHAs as commodity chemicals in transgenic plants will most probably be feasible in the future.