Carbon-carbon bond cleavage by cytochrome P450Biol (CYP107H1)

Article abstract of DOI:10.1039/b311652b

Cytochrome P450_Biol (CYP107H1) is believed to supply pimelic acid equivalents for biotin biosynthesis in Bacillus subtilis: we report here that the mechanistic pathway adopted by this multifunctional P450 for the in-chain cleavage of fatty acids is via consecutive formation of alcohol and threo-diol intermediates, with the likely absolute configuration of the intermediates also reported.

Full text of DOI:10.1039/b311652b

Carbon–carbon bond cleavage by cytochrome P450BioI (CYP107H1)†
Max J. Cryle and James J. De Voss*
Department of Chemistry, University of Queensland, St. Lucia, Brisbane, Australia 4072.
E-mail: j.devoss@uq.edu.au; Fax: +61 7 3365 4299; Tel: +61 7 3365 3825
Received (in Cambridge, UK) 22nd September 2003, Accepted 29th October 2003
First published as an Advance Article on the web 17th November 2003
Cytochrome P450BioI (CYP107H1) is believed to supply pimelic
acid equivalents for biotin biosynthesis in Bacillus subtilis: we
report here that the mechanistic pathway adopted by this
multifunctional P450 for the in-chain cleavage of fatty acids is
via consecutive formation of alcohol and threo-diol inter-
mediates, with the likely absolute configuration of the inter-
mediates also reported.
decanoic acid (1) is oxidised to mainly 11-, 12- and 13-hydroxyte-
tradecanoic acids along with a small amount of pimelic acid. The
lack of specificity observed in the oxidation of the free fatty acids
is unsurprising, given that it appears likely the natural substrate is
an ACP thioester of a fatty acid.10 However, we believed that as
pimelic acid was indeed produced from free fatty acids, the route by
which this was formed would likely be the same as that by which
the pimeloyl ACP was produced. We thus set out to determine the
pathway by which P450BioI catalysed the C–C bond cleavage of
fatty acids to produce pimelic acid using tetradecanoic acid 1 as the
illustrative substrate.
P450scc provides the only delineated example of oxidative bond
fission of an unactivated alkyl chain.‡ However, it was clear that
other pathways might be followed as direct P450 catalysed C–C
bond cleavage has also been observed in simple alcohols7 and
ketones.2 Thus, we planned to synthesise a range of possible
intermediates and determine the relative level of pimelic acid
production, reasoning that more advanced intermediates would lead
to greater pimelic acid production.
The cytochromes P450 (P450s) comprise a superfamily of
oxidative hemoproteins that catalyse an impressive array of
oxidative transformations. Typically, this involves oxidation at an
unactivated carbon centre to yield the corresponding alcohol.1
However, the oxidative potential of these enzymes is perhaps best
illustrated by their ability to catalyse the cleavage of C–C bonds.2
We report here that the mechanistic pathway adopted by the
multifunctional P450BioI for the in-chain cleavage of fatty acids is
via alcohol and threo-diol intermediates. This represents the first
prokaryotic P450 capable of catalysing C–C bond cleavage in an
unfunctionalised substrate.
A number of mechanistically different reaction types can be
distinguished for P450 catalysed C–C bond scission, but each type
is represented by only a few examples.2 Additionally, almost all of
the described reactions are restricted to eukaryotes; CYP51, which
Therefore, compounds 2–6 were synthesised and incubated with
a catalytically active P450BioI system consisting of purified E. coli
flavodoxin reductase, cindoxin and P450BioI and the level of
10
pimelic acid production quantified by GC analysis.
occurs in both eukaryotes and prokaryotes, is the only prokaryotic
The results (Fig. 1) show clear differences in the levels of pimelic
acid production and delineate the pathway by which P450BioI
cleaves the fatty acid chain. The C7 alcohol 2 is a better substrate
than 1 but the C7 oxo fatty acid 4 does not improve pimelic acid
production. Neither the C8 alcohol 3 nor the C8 oxo fatty acid 5 is
processed at all. The erythro 7,8-diol (rac 6a) is a poorer substrate
than the threo diastereomer (rac 6b) which leads to an approx-
imately 15-fold increase in C–C bond cleavage relative to 1. These
results are consistent with an oxidation pathway that is initiated by
C7 hydroxylation (Scheme 1). Subsequent C8 oxidation forms the
threo diol that then undergoes oxidative cleavage. The threo diol
would be predicted to arise from the enzyme acting upon one face
of the extended fatty acid chain.
3
example apart from P450BioI
.
Most P450 C–C bond scissions
occur a to an existing functional group,2,4–8 but perhaps more
impressive are those catalysed by multifunctional P450s which
oxidatively cleave an unactivated alkyl moiety. One such reaction
that has been fully described is the transformation of cholesterol
into pregnenolone and 4-methylpentanal catalysed by the eukar-
yotic P450scc.2‡ In this, two hydroxylation reactions lead specifi-
cally to 20(R),22(R)-dihydroxycholesterol and the C–C bond
between the two oxygenated carbons is then oxidatively cleaved. A
similar process occurs in the metabolism of the antimicrobial agent
olanexidine, but it is unclear whether a single P450 is responsible
for all of the oxidative steps.9
We have recently reported the over-expression and character-
isation of a Bacillus subtilus enzyme, P450BioI, that is involved in
biotin biosynthesis.10 Analysis of mutant phenotypes had suggested
that it played a role in the formation of pimelic acid (heptanedioic
acid) which provides the majority of the carbon backbone of
biotin.11 Expression of P450BioI in E. coli resulted in the isolation
of the enzyme alone and in complex with an acyl carrier protein
acylated with C14 to C18 fatty acids (acyl ACP).10 It was
demonstrated that turnover of this complex resulted in the
formation of a pimeloyl ACP, via cleavage of the alkyl chain of the
acyl moiety. Free fatty acids also serve as substrates for a
catalytically active P450BioI system.10,12,13 Initially, it was postu-
lated that w-functionalisation of the fatty acid may occur, providing
an alternative route for the production of pimelic acid via
subsequent chain shortening reactions.13 However, we have
recently demonstrated that the major positions of free fatty acid
oxidation are exclusively non-terminal.12 For example, tetra-
† Electronic Supplementary Information (ESI) available: 2, R 2, S 2, 4, SS
6b, RR 6b characterisation and synthesis. Enzymatic oxidation and analysis
protocols. See http://www.rsc.org/suppdata/cc/b3/b311652b/
Fig. 1 Pimelic acid production by P450BioI from racemic substrates
(substrate concentration = 1 mM).
86
C h e m . C o m m u n . , 2 0 0 4 , 8 6 – 8 7
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4

Unambiguous identification of the products of C–C bond
cleavage was necessary, as a mechanism involving diol cleavage
would predict formation of two aldehyde fragments. The increased
turnover of threo-7,8-dihydroxytetradecanoic acid (rac 6b) re-
vealed the presence of heptanal and 7-oxoheptanoic acid in
comparable amounts at short reaction times. Both compounds,
either as synthetic standards or produced via P450 catalysed bond
scission, were converted to the corresponding acids over time
( > 70% after 24 h) under enzyme turnover conditions via aerial
oxidation, independent of NADPH. All of these results are
due to the instability of the uncomplexed enzyme.10 However, it
was clear from the estimated association constants10 that the
hydroxylated fatty acids bound significantly (at least 4 fold) more
tightly to the enzyme than the starting fatty acid, but both alcohol
and diol appeared to bind equally well. This is analogous to the
situation seen with P450scc in which the hydroxylated intermediates
were bound equally, but more tightly than cholesterol.2 This is
presumed to increase the efficiency of the reaction: the inter-
mediates are not released or displaced by the initial substrate but
continue on to product. The fact that the C7 alcohol and the 7,8-diol
both facilitate and direct subsequent oxidation reactions is clearly
reflected in the analysis of the enzymic turnovers. While the 11-
and 12-hydroxytetradecanoic acids are the major products isolated
from P450BioI catalyzed hydroxylation of 1, they are not substrates
for further oxidation.12 2 does not accumulate in enzymic
oxidations of 1, nor does 6 appear in turnover of 1 or 2. Finally, the
only alternative oxidation pathway apart from C–C bond cleavage
seen for 2 is conversion into the corresponding ketone 4.
Interestingly, this pathway was much more significant for R 2 than
S 2 ( > 10 fold, quantified by GC), which produces less pimelic acid.
This suggests that the orientation of the substrate in the active site
is determined by the stereochemistry at C7 and this in turn directs
subsequent oxidation reactions.
consistent with C–C bond cleavage via a route corresponding to
2
that seen for P450scc
.
The absolute configurations of the preferred enzymic substrates
were investigated by the synthesis and evaluation of scalemic
samples of the 7-hydroxy (2) and threo 7,8-dihydroxytetradecanoic
acids (6b). The former was available in ~ 70% ee as determined by
enantioselective HPLC (Chiracel OD), with the key step in the
syntheses being a CBS borane reduction of the appropriate
acetylenic ketone.14 The enantiomerically pure threo diols (R,R and
S,S 6b) were synthesised from chiral, non-racemic tartaric acid,
utilizing methodology developed by Seebach for differentiating
either end of the starting diacid.15 Incubation of these substrates
with a catalytically active system indicated that the S 2 and the
corresponding R,R diol were preferentially processed by P450BioI
(Fig. 2). The enantioselectivity of the enzyme was low, with less
favored R 2 and S,S 6b still being good substrates for the enzyme.
A plausible explanation for this lack of selectivity stems from the
fact that the enantiomers of 6b differ only in the location of a
carboxyl or a methyl group five methylene groups removed from
the diol moiety. In the putative natural substrate, an acyl ACP, the
carrier protein would presumably dictate the binding orientation.
However, with the free fatty acids, two binding orientations are
possible that present the same apparent stereochemistry of the diol
at the active site differing only in the location of a distal methyl or
carboxylate.
In conclusion, we have identified the pathway by which the
multifunctional, prokaryotic P450BioI catalyses the cleavage of a C–
C bond in a fatty acid (Scheme 1). By analogy we predict cleavage
of an acyl ACP, its natural substrate, will occur in the same way.
The mechanism involves the consecutive formation of an alcohol
and a vicinal diol and subsequent C–C bond cleavage. This is
analogous to that seen with P450scc and P450BioI is thus only the
second P450 identified as being capable of such bond fission, and
the first from a prokaryote.
MJC is grateful for an Australian Postgraduate Research
Award.
Accurate dissociation constants for the enzyme–substrate com-
plexes were difficult to determine spectrophotometrically, probably
Notes and references
‡ Whilst C–C bond cleavage is observed with CYP19 and CYP51 in steroid
biosynthesis, both proceed via aldehyde intermediates which are in-
accessible to P450BioI
.
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Fig. 2 Pimelic acid production by P450BioI from scalemic substrates
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C h e m . C o m m u n . , 2 0 0 4 , 8 6 – 8 7
87