Labeling Studies Clarify the Committed Step in Bacterial Gibberellin Biosynthesis

Article abstract of DOI:10.1021/acs.orglett.6b02569

Bacteria have evolved gibberellin phytohormone biosynthesis independently of plants and fungi. Through ¹³C-labeling and NMR analysis, the mechanistically unusual B ring contraction catalyzed by a cytochrome P450 (CYP114), which is the committed step in gibberellin biosynthesis, was shown to occur via oxidative extrusion of carbon-7 from ent-kaurenoic acid in bacteria. This is identical to the convergently evolved chemical transformation in plants and fungi, suggesting a common semipinacol rearrangement mechanism potentially guided by carbon-4α carboxylate proximity.

Full text of DOI:10.1021/acs.orglett.6b02569

Letter
pubs.acs.org/OrgLett
Labeling Studies Clarify the Committed Step in Bacterial Gibberellin
Biosynthesis
Ryan S. Nett,† Jeroen S. Dickschat,‡ and Reuben J. Peters*,†
†Roy J. Carver Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011, United
States
‡Kekule-
53121 Bonn, Germany
́
Institut fur Organische Chemie und Biochemie, Rheinische Friedrich-Wilhelms-Universitat Bonn, Gerhard-Domagk-Straße 1,
̈
̈
S
* Supporting Information
ABSTRACT: Bacteria have evolved gibberellin phytohormone biosynthesis independently of plants and fungi. Through 13C-
labeling and NMR analysis, the mechanistically unusual “B” ring contraction catalyzed by a cytochrome P450 (CYP114), which is
the committed step in gibberellin biosynthesis, was shown to occur via oxidative extrusion of carbon-7 from ent-kaurenoic acid in
bacteria. This is identical to the convergently evolved chemical transformation in plants and fungi, suggesting a common
semipinacol rearrangement mechanism potentially guided by carbon-4α carboxylate proximity.
ibberellins (GAs) are phytohormones that play important
roles in plant growth, development, and interactions with
oxygenases (CYPs) catalyzing this mechanistically unusual
and challenging reaction are termed ent-kaurenoic acid oxidases
(KAOs).
G
microbes.1,2 These diterpenoid-derived compounds are charac-
terized by a 6-5-6−5 fused ring structure, termed the ent-
gibberellane carbon skeleton. However, GAs are produced via
ent-kaurane precursors, which have a 6-6-6−5 carbon skeleton
(see Scheme 1 for numbering and ring nomenclature).
In addition to their endogenous production by plants, GAs
are also produced by certain plant-associated fungi and bacteria,
wherein the relevant biosynthetic pathways have independently
evolved.3,4 Plant and fungal GA biosynthesis has been
extensively studied, and it has been directly demonstrated
that the carbon extruded from the “B” ring is C-7 in fungal
biosynthesis,5−7 and convincing indirect evidence has been
presented that plants also extrude C-7 (i.e., retention of the C-
6α proton from 1 during the “B” ring contraction reaction).8,9
Moreover, 2 has been shown to be a bona fide intermediate in
both plant and fungal GA biosynthesis. By contrast, 6β,7β-
dihydroxy-ent-kaurenoic acid (4; ent-6α,7α-dihydroxy-kaure-
noic acid), which is also produced in the KAO-catalyzed
oxidation of 1 by plants and fungi, is not,10−14 thus, implicating
a mechanism in which C-7, but not C-6, is hydroxylated prior
to ring contraction.5,15
Scheme 1. Reaction Catalyzed by CYP114 + FdGA in
a
Bacterial GA Biosynthesis
a
1 (ent-kaurenoic acid) is representative of the ent-kaurane backbone,
and 3 (GA12-aldehyde) represents the ent-gibberellane backbone.
The GA biosynthetic pathway in bacteria has only recently
been elucidated.4,16 In particular, the role of each enzyme from
a CYP-rich gene cluster/operon in symbiotic rhizobia has now
been functionally identified, showing that they act to produce
GA9 (Scheme S2). While rhizobia only express these enzymes
Accordingly, the committed step in GA biosynthesis is
contraction of the “B” ring from a cyclohexane to cyclopentane.
This occurs via oxidation of ent-kaurenoic acid (1; ent-kaur-16-
en-19-oic acid), first to 7β-hydroxy-ent-kaurenoic acid (2; ent-
7α-hydroxykaurenoic acid), and then to GA12-aldehyde (3), the
latter of which involves oxidative extrusion of an endocyclic “B”
ring carbon (Scheme 1). The cytochrome P450 mono-
Received: August 27, 2016
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.6b02569
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Scheme 2. 13C Label from (2-13C)-5 Is Specifically
Incorporated into 1 via Metabolically Engineered Bacteria
and produce GA after differentiation into their nodule-residing
bacteroid form,17 it was possible to observe activity with the
individual enzymes upon recombinant expression.4,16,18,19
Notably, “B” ring contraction requires not only a CYP
(CYP114) but also the ferredoxin (FdGA) found within the
operon, which presumably acts as an electron donor.4 This is
distinct from plant and fungal KAOs, which simply utilize an
archetypical cytochrome P450 reductase for their activity.12,20,21
When expressed alone, CYP114 only converts 1 to 2, while
coexpression of CYP114 and FdGA enables full conversion of 1
to 3. This suggests that endogenous ferredoxins from the
recombinant host support partial CYP114 activity and indicates
a unique role for FdGA in facilitating full activity, presumably
through its interaction with CYP114. Although recombinantly
coexpressed CYP114 and FdGA are not able to convert 2 to 3,
nodule-extracted rhizobial bacteroids can use 2 as a GA
precursor, implicating this as an intermediate in bacterial GA
biosynthesis as well.4
a
a
13C-labeled 1 can then be incubated with bacteria recombinantly co-
expressing CYP114 and FdGA to produce 13C-labeled 3.
13C NMR analysis showed three enriched carbons with
chemical shifts between 30 and 50 ppm, indicative of alkyls,
while the fourth had a chemical shift of over 200 ppm,
representing a carbonyl carbon (Figure 1; Tables S3 and S4).
Though the intermediacy of 2 might be taken to suggest that
C-7 also will be extruded during the “B” ring contraction
reaction catalyzed by the convergently evolved bacterial
enzymes, it is still plausible that C-6 might be extruded instead
(e.g., via a pinacol ring rearrangement mechanism).5 The
extrusion of C-7 during fungal GA biosynthesis was shown by
feeding (2-13C)mevalonolactone (5; the δ-lactone form of
mevalonate) to cultures of Fusarium fujikuroi (the anamorph of
Gibberella fujikuroi), which leads to specific labeling of C-7 in 1,
followed by NMR analysis of the resulting GA3 final product.5,7
This approach was enabled, at least in part, by the high titers of
GA3 produced by fungal cultures. By contrast, rhizobia produce
only small amounts of GAs.17 Nevertheless, the recombinant
coexpression of CYP114 and FdGA, which carry out “B” ring
contraction with 1, provides a means to analyze this reaction in
more detail (i.e., via incubation with 13C-labeled 1).
Although bacteria usually produce isoprenoids/terpenoids via
the nonmevalonate pathway, Keasling and co-workers have
engineered incorporation of the mevalonate-dependent iso-
prenoid precursor pathway from yeast into E. coli.22 Of
particular relevance here, a single plasmid enables production of
the universal isoprenoid precursors isopentenyl diphosphate
(IPP) and dimethylallyl diphosphate (DMAPP) from 5. In
turn, a modular metabolic engineering system has been
developed that is compatible with this plasmid.23 This enables
the production of diterpenoids via incorporation of a (E,E,E)-
geranylgeranyl diphosphate synthase (producing this general
diterpene precursor from IPP and DMAPP), subsequently
acting diterpene cyclases/synthases, and even further down-
stream acting CYPs in conjunction with their requisite redox
partner.24 Thus, it was possible to produce 13C-labeled 1 by
simply feeding (2-13C)-5 to E. coli engineered to produce 1
from 5 (i.e., via coexpression of the necessary nine enzymes; see
Scheme S3, Figures S1 and S2). As expected, this enabled
isolation of 1 with 13C enrichment at four positions, as initially
confirmed by gas chromatography−mass spectroscopy (GC-
MS), with comparison to an authentic standard (Figure S2).
The expected incorporation of 13C at carbons 1, 7, 12, and 18
(Scheme 2)5,7 was verified by 13C NMR analysis with
comparison to unlabeled 1 (Figure S3; Tables S1 and S2).
To investigate the origin of the extruded carbon, 13C-
enriched 1 was fed to bacterial cultures recombinantly
coexpressing CYP114 and FdGA. This allowed isolation of 3
enriched at four positions, as confirmed by GC-MS comparison
to an authentic standard (Figure S4). Following purification,
Figure 1. Comparison of the 13C-labeled 3 13C NMR spectrum to that
of the unlabeled standard (800 MHz, CDCl3 for each) reveals that C-7
is extruded during the ring contraction from ent-kaurenoic acid 1 to
GA12-aldehyde 3. The 13C-enriched carbons in the labeled substrate
are indicated with asterisks (*).
These shifts were further verified by comparison to those
measured for unlabeled 3. Thus, it was demonstrated that C-7
of 1 was extruded and oxidized to the aldehyde of 3.
As with plant and fungal GA biosynthesis, 2 is observed and
seems to serve as an intermediate in bacteria as well,4 implying
C-7β hydroxylation prior to ring contraction. It is known for
plants and fungi that the 6β hydrogen of 1 is removed prior to
rearrangement/ring contraction, although 4 does not serve as
an intermediate,8,9,25 and seems to be a side product of the
corresponding CYPs in both kingdoms. Interestingly, closer
analysis of incubations of 1 in cells coexpressing CYP114 and
FdGA showed that a trace amount of 4 is produced (Figure S5).
However, feeding 4 to bacterial cultures recombinantly
coexpressing CYP114 and FdGA does not result in further
conversion (Figure S5), suggesting that 4 is a side product of
bacterial KAO activity. Thus, the KAOs from all three biological
kingdoms not only extrude C-7 in “B” ring contraction but also
exhibit a conserved order of chemical transformations, with
conversion of 1 to 3 via 2, but apparently not 4.
Although it is possible that the conversion of 1 to 3 could
proceed via transient formation of a C-19,6-γ-lactone ring,
which is used to achieve “B” ring contraction in the chemical
synthesis of GAs,26 this corresponds to the known kaurenolide
side products of GA biosynthesis in both plants and fungi.27
Notably, these compounds were not observed here, nor have
B
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these previously been reported in other investigations of
bacterial GA biosynthesis.4,16 To further evaluate the possibility
of the “B” ring contraction reaction proceeding through this
type of intermediate, kaurenolide and 7β-hydroxykaurenolide
(see Figure S6 for chemical structures) were fed to bacterial
conversion of 4 may argue against the classic pinacol
mechanism, it is possible that suboptimal expression of
CYP114 and/or FdGA prevented turnover here.
Consistent with a role for anchimeric assistance by the free
C-4α carboxylate, neither the methyl ester of 1 nor ent-kaurenal
(which in the predominant diol form sterically resembles 1) is
further transformed by recombinantly expressed CYP114
(either with or without coexpression of FdGA).4 Additional
support for the proposed mechanism stems from the use of
pinacol-like intermediates to achieve “B” ring contraction in the
chemical synthesis of GA.26 Thus, it seems likely that the
independently evolved CYPs catalyzing the characteristic “B”
ring contraction in gibberellin biosynthesis in all three
biological kingdoms may have converged on a common
(semi)pinacol rearrangement mechanism to selectively carry
out this unusual and challenging reaction.
cultures recombinantly coexpressing CYP114 and FdGA
.
However, these were not converted in this system (Figure
S6) and likely are not intermediates in bacterial GA
biosynthesis, similar to what has been reported for plants and
fungi.27
All three biological kingdoms have convergently evolved
KAOs that carry out this committed step in GA biosynthesis.4
In each case the KAO is a member of the CYP superfamily, but
falls within phylogenetically distinct families specific to each
biological kingdom, as bacterial KAOs come from the CYP114
family, the fungal KAOs from the CYP68 family, and those
from plants from the CYP88 family.14,28 However, the results
reported here show that these convergently evolved KAOs all
catalyze extrusion of the same carbon, using a conserved order
of central chemical transformations (i.e., from 1 to 3 via 2) in
each case.4 This suggests a physical/chemical restraint for the
“B” ring contraction reaction.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.6b02569.
Intriguingly, there appears to always be a free C-4α
carboxylate (C-19) present. While CYPs typically catalyze
radical based reactions, it has been previously suggested that
this “B” ring contraction reaction might proceed via a
carbocation based mechanism instead, with transfer of the
unpaired electron from the initially formed C-6 radical of 2 to
the heme-iron.29,30 Notably, the C-4α carboxylate is ideally
positioned to offer anchimeric assistance to formation of this
putative C-6 carbocation, potentially guiding the observed
oxidative extrusion of C-7 via a semipinacol rearrangement
mechanism (Scheme 3, path A).31 Alternatively, hydroxylation
Experimental methods, Supplemental figures and tables
(PDF)
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: rjpeters@iastate.edu.
ORCID
Ryan S. Nett: 0000-0002-2537-7010
Reuben J. Peters: 0000-0003-4691-8477
Notes
Scheme 3. Proposed Reaction Mechanisms for “B” Ring
Contraction during GA Biosynthesis That Proceed Either
through a Semipinacol (Path A) or Classic Pinacol (Path B)
Rearrangement
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank D. Bruce Fulton (Iowa State University) for expert
assistance with the NMR analysis and Dr. Peter Hedden
(Rothamsted Research, U.K.) for insightful discussion and for
the 6β,7β-dihydroxy-ent-kaurenoic acid (4), kaurenolide, and
7β-hydroxykaurenolide substrates. This work was supported by
grants from the National Institutes of Health (GM109773) and
National Science Foundation (CHE-1609917) to R.J.P. and by
the DFG Collaborative Research Center 813 “Chemistry at
Spin Centers” (J.S.D.).
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