Transcript Dioxygen Activation and Alkane Hydroxylation By The

Direct Evolution of P450 Enzyme to
Achieve the Controlled Oxidation of
Aliphatic Compounds or Methane
Steve S.-F. Yu
Institute 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
CF3
CHMe2
CMe3
CH=CH2
= 435 (104.0)
444 (106.0)
394 (94.2)
387 (92.5)
453 (108.3)
500 (119.5)
363 (86.7)
CCH
HC=O
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.
R
M + RH
[M
RH]
M
H
[O]
R
M + O
H
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.
R
R
Concerted
Radical
M
M
[O]
[O]
MO + RH
MO
+ RH
]
[MO
M
+
O
H
.
. R
M
OH
H
R
M +
O
H
The Enzymes that Involve in C-H Activation
R
H
C
H
C
H
H
R
R
[O]
H
C
C
H
H
OH
R
H
R
C
H
C
R
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.
Soluble Methane
Monooxygenase
from Methylococcus
capsulatus (Bath)
cytoplasm
periplasm
w-Hydroxylase from
Pseudomonas oleovorans
Cytochrome P-450cam
from Pseudomonas putida
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 stereoselectivity 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.
Cytochrome P450
• 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[a]pyrene
(found in cigarette smoke)).
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
Directed Evolution of Alkane Oxygenases
Target Gene Coding Sequence
pmo1 Promoter
pmo1 C
6xHis Tag & Termination Codon
Transcriptional Terminator Sequence
Octane hydroxylation activity
mutants
Surrogate
p-nitrophenyl octyl ether
Colorimetric identification
Frances H. Arnold et al., J. Am. Chem. Soc. (2003), 125, 13442.
Experimental Procedures
Cytochrome P450 BM-3
CH3CH2CH2 CH3 + “O”
CH3CH2CH(OH) CH3
Fatty acid monooxygenase from Bacillus megaterium
Glieder, A.; Farinas, E. T.; Arnold, F. H. Nat. Biotech. 2002, 20, 1135.
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
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
Cytochrome P450cam
HO
+ H2O
O
p450cam
O2, 2e-, 2H+
Bacterium: Pseudomonas putida
O
Cytochrome P450cam
Schlichting I, Jung C, Schulze H. FEBS Lett. 1997, 415, 253.
Production of alkane oxidation by wild-type cytochrome
P450cam and its mutants
F87W
Y96F
V247L
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.
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)-Nbenzyl-3-hydroxypyrrolidine
by the hydroxylation of Nbenzylpyrrolidine. (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-CoAreductase
• PhaC PHA synthase
References:
Polyhydroxylbutyrate, Wikipedia
Alexander Steinbüchel and Bernd Füchtenbusch, TIBTECH OCTOBER 1998 (VOL 16),
419.
Glossary
3HA 3-Hydroxyalkanoic acid
3HB 3-Hydroxybutyric acid
3HD 3-Hydroxydecanoic acid
3HDD 3-Hydroxydodecanoic acid
3HHx 3-Hydroxyhexanoic acid
3HO 3-Hydroxyoctanoic acid
3HTD 3-Hydroxytetradecanoic acid
3HV 3-Hydroxyvaleric acid
4HB 4-Hydroxybutyric acid
4HV 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.