Subscriber access provided by University of Glasgow Library
Article
OleB from bacterial hydrocarbon biosynthesis is a #-lactone decarboxylase sharing key features with haloalkane dehalogenases James K Christenson, Serina L Robinson, Tiffany A Engel, Jack Eugene Richman, An N Kim, and Lawrence P. Wackett Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00667 • Publication Date (Web): 05 Sep 2017 Downloaded from http://pubs.acs.org on September 6, 2017
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Biochemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 38
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
1
Biochemistry
Journal: Biochemistry (ACS)
2 3 4
OleB from bacterial hydrocarbon biosynthesis is a β-lactone
5
decarboxylase sharing key features with haloalkane dehalogenases
6 7 8
James K. Christensona,b, Serina L. Robinsonb,c, Tiffany A. Engelb, Jack E. Richmana,b,
9
An N. Kimb, Larry P. Wacketta,b,d,*.
10 11
Department of Biochemistry, Molecular Biology, and Biophysics, University of
12
Minnesota, Minneapolis, Minnesotaa; Biotechnology Institute, University of Minnesota,
13
St. Paul, Minnesotab; Department of Microbiology, Immunology, and Cancer Biology,
14
University of Minnesota, Minneapolis, Minnesotac; Department of Biochemistry,
15
Microbial and Plant Genomic Institute, University of Minnesota, St. Paul, Minnesotad
16 17
Running Title: b-lactone decarboxylase
18 19
*Corresponding Author: L.P. Wackett
[email protected] 1 ACS Paragon Plus Environment
Biochemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 2 of 38
20
Abstract
21
OleB is an a/b-hydrolase found in bacteria that biosynthesize long-chain olefinic
22
hydrocarbons, but its function has remained obscure. We report that OleB from the
23
gram-negative bacterium Xanthomonas campestris performs an unprecedented β-
24
lactone decarboxylation reaction, to complete cis-olefin biosynthesis. OleB reactions
25
monitored by 1H-NMR spectroscopy revealed a selectivity for decarboxylating cis-β-
26
lactones and no discernable activity with trans-β-lactones, consistent with the known
27
configuration of pathway intermediates. Protein sequence analyses showed OleB
28
proteins were most related to haloalkane dehalogenases (HLDs) and retained the
29
canonical Asp-His-Asp catalytic triad of HLDs. Unexpectedly, it was determined that an
30
understudied subfamily, denoted as HLD-III, is comprised mostly of OleB proteins
31
encoded within oleABCD gene clusters, suggesting a misannotation. OleB from X.
32
campestris showed very low dehalogenase activity only against haloalkane substrates
33
with long alkyl chains. A haloalkane substrate mimic alkylated wild type X. campestris
34
OleB but not OleBD114A implicating this residue as the active site nucleophile as in
35
HLDs. A sequence-divergent OleB, found as part of a natural OleBC fusion and
36
classified as an HLD-III, from the gram-positive bacterium Micrococcus luteus was
37
demonstrated to have the same activity, stereochemical preference, and dependence
38
on the proposed Asp nucleophile. H218O studies with M. luteus OleBC suggested that
39
the canonical alkyl-enzyme intermediate of HLDs is hydrolyzed differently by OleB
40
enzymes, as 18O is not incorporated into the nucleophilic aspartic acid. This work
41
defines a previously unrecognized reaction in nature, functionally identifies some HLD-
42
III enzymes as β-lactones decarboxylases, and posits an enzymatic mechanism of β-
43
lactone decarboxylation.
2 ACS Paragon Plus Environment
Page 3 of 38
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
44 45
Biochemistry
Introduction The a/b-hydrolase enzyme scaffold is a very common fold, used to catalyze a
46
wide array of chemical reactions.1,2 The vast majority of a/b-hydrolases that have been
47
studied initiate catalysis via attack of a catalytic nucleophile to form an enzyme-
48
intermediate ester linkage. This species is then hydrolyzed by a water molecule that has
49
been activated by a conserved histidine residue, with subsequent release of the product
50
and a return to the resting enzyme.3,4 Despite their prevalence in nature, approximately
51
35% of enzymes annotated as a/b-hydrolases do not have a known substrate, thus their
52
cellular function remains unknown.2
53
One such a/b-hydrolase is encoded by the gene denoted as oleB, which resides
54
in the ole (olefin) gene cluster responsible for the biosynthesis of long-chain olefins in
55
bacteria.5,6 Earlier studies demonstrated that long-chain olefins are generated following
56
the Claisen condensation of two fatty acyl-CoA molecules.7 The olefins can be 19-31
57
carbons in length and contain an internal double bond at the site of C-C bond
58
formation.5,8,7 Genetic work in Shewanella oneidensis MR-1 concretely linked the four-
59
gene cluster, oleABCD, to hydrocarbon production, and the ole-genes have now been
60
identified in over 300 divergent bacteria.5,6 The a/b-hydrolase, OleB, is encoded from a
61
stand-alone gene or as part of a gene fusion with oleC. Recently, the OleB, OleC, and
62
OleD proteins from Xanthomonas campestris were found to associate in vivo to form an
63
active, multi-enzyme complex when recombinantly expressed and purified from
64
Escherichia coli, further suggesting an important function for OleB.9
65
Until recently, only OleACD were thought to be required for the generation of
66
long-chain olefins, leaving no apparent function for OleB (Figure 1).10 The roles of OleA
67
and OleD as the first two pathway steps had been previously established, with OleA
68
performing a Claisen condensation of two acyl-CoAs to form a b-ketoacid and OleD
69
catalyzing an NADPH-dependent reduction of the keto acid to produce a b-hydroxy acid
70
(Figure 1).7,11,12 The third enzyme, OleC, was initially thought to react with the b-
71
hydroxy acid in the presence of ATP to produce long-chain olefin thereby completing
72
the metabolic pathway. More recently, it was discovered that OleC generates a stable
73
b-lactone under physiological conditions.13 In the earlier work, the OleC reaction
74
product, the b-lactone, had been analyzed using gas chromatography at high 3 ACS Paragon Plus Environment
Biochemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 4 of 38
75
temperature, resulting in a spontaneous decarboxylation reaction to make the observed
76
olefin.10 The more recent report13 reserved a possible role for OleB, but the
77
decarboxylation of a b-lactone to an olefin was not demonstrated. O
O R2
OleA S-CoA
OH
O
R2
OH
OleD
O
R2
R1
OleC
OleB ?
OH R1
O
R2
R2
R1
R1
Δ S-CoA
78
O O
R2
R1
R1
79 80
Figure 1. Reactions previously established for the enzymes OleA, OleD, and OleC.
81
The b-lactone product of OleC spontaneously decomposes to olefin at temperatures
82
>100 °C. Preliminary evidence by Christenson et al. suggested that OleB performed
83
the equivalent enzymatic b-lactone decarboxylation, but this had never been
84
demonstrated.13 The R groups are typically linear alkanes between 12-14 carbons, but
85
can contain unsaturation or methyl branching.
86 87
Here, we report a reproducible purification scheme and new information that the
88
a/b-hydrolase, OleB, from Xanthomonas campestris selectively catalyzes a
89
decarboxylation of cis-b-lactones to yield cis-alkenes, the final step in bacterial long-
90
chain olefin biosynthesis. This is a previously unreported enzymatic activity and
91
represents a novel cellular route to hydrocarbons. Sequence comparisons to other a/b-
92
hydrolases revealed OleB proteins and OleBC fusion proteins are most related to
93
haloalkane dehalogenases (HLDs). Indeed, a previously designated group of
94
haloalkane dehalogenases, HLD-III14, includes proteins that function physiologically as
95
OleB enzymes. This connection to HLD proteins also provided testable hypotheses
96
regarding a possible reaction mechanism for OleB and specific residues were shown by
97
site directed mutagenesis to be critical for catalysis.
4 ACS Paragon Plus Environment
Page 5 of 38
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biochemistry
98
Methods
99
Chemical synthesis of b-hydroxy acids, b-lactones, and olefins.
100
All compounds, cis- and trans-3-octyl-4-nonyloxetan-2-one (b-lactones), 3-
101
hydroxy-2-octyldodecanoic acid (b-hydroxy acids), cis- and trans-9-nonadecene
102
(olefins) were chemically synthesized as described previously.13 Briefly, b-hydroxy
103
acids were synthesized from decanoic acid and decanal and recrystallization yielded a
104
1:1:1:1 ratio of diastereomers.15 The cis-b-lactone was synthesized from decanoic acid
105
via a ketene dimer that was subsequently hydrogenated to yield a cis-b-lactone.16
106
Trans-b-lactone was separated from a cis- and trans-b-lactone mixture generated from
107
the corresponding b-hydroxy acid triethylamonium salt and methanesulfonyl
108
chloride/Et3N.17 The cis-olefin was generated from the coupling of 1-decyne with 1-
109
bromononane precursors followed by hydrogenation with Lindlar catalyst.18,19
110
Photoisomerization of the cis-olefin generated the trans-olefin standard.20
111
Generating mutants of OleB and OleBC.
112
Site-directed mutations of OleB derived from the wild-type protein sequences
113
from Xanthomonas campestris ATCC 33913 (WP_012437021.1) and Micrococcus
114
luteus OleBC (WP_010078536.1) were made with New England Biolabs Q5 quick
115
change site directed mutagenesis kit following manufacturer's instructions. All primers
116
were ordered from Integrated DNA Technologies (IDT). To confirm each mutant, single
117
colonies were grown in 5 mL Luria-Bertani broth at 37 °C overnight under kanamycin
118
selection. Plasmids were isolated using a QIAGEN Miniprep kit and sent to ACGT Inc
119
for sequencing.
120
Purification of OleB and OleBC fusion.
121
The buffer for OleB purification contained 200 mM NaCl, 20mM NaPO4, 10%
122
glycerol, and 0.5% polyethylene glycol 400 (PEG 400) (Hampton Research) at pH 7.4.
123
E. coli BL-21 DE3 cells containing OleB with a 6x Histidine tag on the N-terminal were
124
grown, sonicated, and used to purify protein according to manufacturer’s protocol with a
125
GE HisTrap HP 5 mL column. Protein concentrations of purified OleB solutions were
126
measured by Bradford assay. Purified OleB solutions were routinely stored in elution
127
buffer at -80°C. OleBC and OleBD163AC fusion proteins from M. luteus were generated
5 ACS Paragon Plus Environment
Biochemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
128
with a 6x Histidine tag and purified as described previously without the addition of PEG
129
400.13
130
X. campestris OleB and M. luteus OleBC reactions with b-lactone followed by 1H-
131
NMR Spectroscopy.
132
Page 6 of 38
For enzyme reactions, the appropriate cis- or trans-b-lactones were dissolved in
133
100% ethanol at 0.17 mg/ml. Substrate for the M. luteus OleBC fusion protein was 0.33
134
mg/mL of b-hydroxy acid (1:1:1:1 racemic diastereomeric mixture) in ethanol.
135
Reactions were carried out in separatory funnels containing 1.0 mg X. campestris OleB
136
(or M. luteus OleBC fusion), 3.0 mL of the b-lactone or b-hydroxy acid substrate, 10 μL
137
of 10% 1-bromonaphthalene as an internal standard, and 97 mL buffer (200 mM NaCl,
138
20 mM NaPO4, pH 7.4), and incubated at room temperature overnight. The overnight
139
reaction mixtures were extracted with 10 mL and 5 mL methylene chloride,
140
consecutively. The organic extracts were pooled and back-extracted with 15 mL
141
double-distilled H2O. The organic fraction was dried, dissolved in CDCl3, and analyzed
142
in 5 mm NMR tubes with tetramethylsilane (TMS) as a reference. Spectra were typically
143
acquired using 1,024 pulses with a 3 second pulse delay on a Varian Inova 400 MHz
144
NMR spectrometer using a 5 mm Auto-X Dual Broadband probe at 20°C.
145
Bioinformatic analysis of OleB and haloalkane dehalogenases.
146
Sequences for representative members of the a/b-hydrolase protein superfamily
147
were retrieved from the Protein Data Bank (PDB) on 3/13/2017, filtered by SCOP
148
classification of a/b-hydrolases, and curated to include only representative bacterial
149
sequences for each protein family (Figure S1). To obtain a higher resolution phylogeny
150
for the relationship of OleB/OleBC sequences with haloalkane dehalogenases,
151
accession numbers for characterized HLD-I, -II, and -III accession numbers were
152
extracted from the National Center for Biotechnology Information (NCBI) Protein
153
database using accession numbers from Nagata et al. (2015). Accession numbers for
154
five experimentally characterized OleB and OleBC sequences were obtained from
155
Sukovich et al.5 Protein sequences were aligned and curated using the DECIPHER
156
package in R.21 Due to the length of OleBC fusion sequences interfering with proper
157
alignment, the last 550 residues were trimmed to eliminate the OleC region. Maximum-
158
likelihood phylogenies with 100 bootstrap replicates were inferred from alignment using 6 ACS Paragon Plus Environment
Page 7 of 38
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biochemistry
159
the JTT method using the phangorn R package.22 A structural homology model for the
160
X. campestris OleB sequence (WP_012437021.1) was built using default parameters in
161
Phyre2.23
162
OleB reactions with haloalkanes.
163
The following haloalkane substrates were dissolved (5 mM) in DMSO for testing
164
with X. campestris OleB: 1-iodobutane, 1,3-iodopropane, 1-bromopentane, 1-
165
chlorohexane, 1-chlorooctane, 1-bromooctane, 1-iodoundecane, and 7-
166
(bromomethyl)pentadecane. Reactions were carried out in glass GC vials containing
167
purified OleB (40 µg) and 10 µL of DMSO substrate solution in 500 µL of 200 mM NaCl,
168
20 mM NaPO4 at pH 7.4. Reactions and no enzyme controls were incubated overnight
169
at room temperature, followed by extraction with tert-butyl methylether (MTBE). The
170
MTBE extract in a clean GC vial was analyzed by gas chromatography/mass
171
spectrometry (GC/MS Agilent 7890a & 5975c with an Agilent J&W bd-ms1 column 30 m
172
length, 0.25 mm diameter, 0.25 µm film).
173
Mass spectroscopy of alkyl-enzyme intermediate with haloalkanes.
174
To identify an alkyl-enzyme intermediate, Matrix Assisted Laser Desorption
175
Ionization Time Of Flight (MALDI-TOF) mass spectrometry was carried out on wild type
176
OleB and OleBD114A proteins that had been reacted with 7-(bromomethyl)pentadecane
177
(Tokyo Chemical Industry). This substrate contained the reactive bromomethyl group in
178
the middle of two long alkyl chains, thereby mimicking the b-lactones that are in
179
physiological substrates of OleB. Reactions contained 500 µM substrate and 40 µg of
180
OleB in 100 µL of buffer (20 mM NaCl, 5 mM NaPO4, pH 7.4). Reactions were prepared
181
for MALDI-TOF using standard C4 ZipTip (Millipore) procedures and spotted on a plate
182
with sinapinic acid. Samples were analyzed on a Bruker Autoflex Speed MALDI-TOF
183
instrument.
184
M. luteus OleBC reactions in H218O.
185
The OleBC fusion protein from M. luteus was chosen over the X. campestris
186
OleB protein because the size of the aspartic acid containing peptide generated by a
187
trypsin digest was more amenable for MALDI-TOF. 75 µL of buffer (20 mM NaCl, 5 mM
188
NaPO4, pH 7.4) was added to glass vials and evaporated. The salts were dissolved in
189
72 µL of H218O (97%, Sigma Aldrich) and these reactions contained either b-hydroxy 7 ACS Paragon Plus Environment
Biochemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 8 of 38
190
acid (46 µM) or cis-b-lactone (24 µM), M. luteus OleBC (1.2 µM) and 3.0% ethanol for
191
substrate solubility. ATP and MgCl2 were added (both at 130 µM) to reactions with b-
192
hydroxy acid. Reactions were run for 5 hours before the addition of 6.0 mg of urea, 3.75
193
µL of acetonitrile, CaCl2 to 1.0 mM, and 0.5 µg of trypsin from bovine pancreas.
194
Reactions were digested overnight at 37 °C followed by C18 ZipTip (Millipore) adapted
195
from Millipore. Peptide elution from C18 ZipTips was facilitated by up to 90%
196
acetonitrile. Peptides were spotted with an α-cyano-4-hydroxycinnamic acid matrix and
197
analyzed on a Bruker Autoflex Speed MALDI-TOF instrument.
198 199
Results
200
High–yield purification of OleB.
201
The OleB protein from X. campestris had been purified previously as part of a
202
protein structure-focused study demonstrating that OleB, OleC, and OleD combine to
203
form large enzyme assemblies on the order of 2 MDa molecular weight.9 The individual
204
activity of the OleB protein was not demonstrated in that study. Moreover, in that
205
previous report, OleB purification required the presence of 0.05% Triton X-100 to
206
maintain the protein in a soluble form. In the present study, we found that the addition of
207
PEG 400 to purification buffers in place of Triton X-100 stabilized OleB, making it more
208
amenable to purification and concentration. Purification yields increased to 30 mg OleB
209
per liter of culture compared to 2 mg/L previously reported.9 OleB from X. campestris
210
purified with PEG 400 was used in these studies, although Triton-purified OleB was
211
shown to catalyze the same reaction.
212
OleB decarboxylates only cis-β-lactones.
213
Previous studies demonstrated that OleA, OleD, and OleC can act sequentially to
214
produce a b-lactone ring from acyl-CoA precursors, but the final step to the biologically-
215
relevant cis-olefin was undefined (Figure 1).7,12,13 To determine if X. campestris OleB
216
might complete long-chain olefin biosynthesis with a β-lactone decarboxylation reaction,
217
cis- and trans-3-octyl-4-nonyl-2-oxetanone (cis- and trans-β-lactone) were chemically
218
synthesized. 1H-NMR spectra demonstrated that both the cis- and trans-β-lactone
219
enantiomeric pairs contained 10,000 fold 15 ACS Paragon Plus Environment
Biochemistry
362
lower activity than other HLDs.29 No detectable activity was observed against the
363
following haloalkane substrates: 1-iodobutane, 1,3-iodopropane, 1-bromopentane, 1-
364
chlorohexane, 1-chlorooctane, and 1-bromooctane. However, we observed partial
365
conversion of 1-iodoundecane and 7-(bromomethyl)pentadecane to the dehalogenated
366
alcohol product during overnight reaction. The calculated specific activity for both of
367
these compounds is 0.6 nmol product × min-1 × mg enzyme-1 (~1.5 turnovers × enzyme-1 ×
368
hr-1). This low level of dehalogenase activity further links OleB with HLD proteins.
369 370
Haloalkane substrate mimic reacts to form a stable alkyl-enzyme intermediate.
371
Intensity (%)
100
Wt OleB No Substrate
80
60 40
+ 221 m/z
20
+ 223 m/z
0 35.6 Wt OleB 35.8 100 + Intensity (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 16 of 38
36
36.2
36.4
36.6
36.8
36.2
36.4
36.6
36.8
80
60 40 20
0 35.6
35.8
36
m/z (KDa)
372 373
Figure 5. OleB forms a stable alkyl-enzyme intermediate when reacted with 7-
374
(bromomethyl)pentadecane. OleB show a mass shift ~222 m/z consistent with the
375
nucleophilic attack and displacement of bromine with the substrate. The mass shift
376
expected with the loss of bromide is 225 m/z. OleBD114A did not show any mass shift
377
when reacted with the bromo-alkane substrate. This data is consistent with a
378
haloalkane dehalogenase-like mechanism. The two major peaks and one minor peak
379
appear in all OleB and OleB mutant MALDI-TOF experiments and are presumed to be
380
the result of an ion with a m/z of ~180. 16 ACS Paragon Plus Environment
Page 17 of 38
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biochemistry
381 382
The first half of the HLD reaction cycle generates an alkyl-enzyme intermediate
383
between the nucleophilic Asp and the dehalogenated substrate fragment, which is
384
subsequently resolved by hydrolysis of the alkyl-enzyme ester bond.4,29 To investigate
385
whether OleB might mimic the first half of the HLD reaction to form a covalent enzyme
386
intermediate, X. campestris OleB was reacted with 7-(bromomethyl)pentadecane and
387
subjected to MALDI-TOF mass spectrometry. MS/MS was initially attempted, but the
388
peptide of interest could not be identified. A mass shift in OleB corresponding to the
389
mass of the debrominated alkyl chain was observed (Figure 5). Parallel incubations
390
without substrate and with cis-b-lactone substrate were conducted as controls and gave
391
no mass shift via mass spectrometry. Another experiment was conducted with a mutant
392
OleB protein in which the putative nucleophilic residue, Asp114, had been mutated to an
393
unreactive alanine residue. The OleBD114A enzyme did not show a mass shift when
394
incubated with 7-(bromomethyl)pentadecane, further implicating Asp114 as the
395
nucleophilic aspartic acid as in HLDs. Collectively, these data indicated that the
396
nucleophilic attack by OleB mimics HLDs, but processing of the alkyl-enzyme
397
intermediate by OleB may proceed somewhat differently with β-lactones.
398
18
399
O does not incorporate into Asp163 of OleBC. HLD enzymes resolve the alkyl-enzyme intermediate by attack of water at the
400
carbonyl carbon ester formed between the active site aspartate and the substrate. With
401
multiple turnovers of an HLD enzyme, both carboxylate oxygens of aspartic acid are
402
exchanged with oxygens from solvent water.30 As such, we searched for a mass shift of
403
+4 m/z in the peptide containing Asp163 of M. luteus OleBC
404
(DLAATGHPLITLGHDWGGVVSLGWAAR) under multi-turnover conditions in H218O
405
solvent by MALDI-TOF (Figure 6). Every fragment of M. luteus OleBC showed
406
incorporation of a single 18O from the trypsin digestion and ~40% of fragments showed
407
the additional nonspecific incorporation of a second 18O. Figure 6A shows the expected
408
isotopic envelop from the fragment containing Asp163 starting at 2770.4 m/z with no 18O
409
incorporation. The actual data matches the simulated data and is shown in Fig S8. A
410
no substrate control of OleBC run in H218O showed the exact mass envelope of the
411
predicted fragment, but began at 2774.3 m/z (+3.9 m/z) due to digestion and a second 17 ACS Paragon Plus Environment
Biochemistry
non-specific incorporation of 18O (Figure 6B). Reactions of OleBC starting with b-
413
hydroxy acids show no additional incorporation of 18O compared with the control
414
reaction (Figure 6C). Reactions starting with b-lactone that bypass the OleC domain of
415
the OleBC fusion (Figure 6D) are similar to the no substrate control, but the distribution
416
of the isotopic envelope is altered, indicative of a population of fragments that have a +2
417
m/z shift. However, this distribution difference is significantly less than the theoretical
418
+4 m/z mass shift from the incorporation of two 18O atoms into Asp163 (Figure 6E).
419
Repeat experiments gave identical results. Every peptide fragment from M. luteus
420
OleBC fusion identified by MALDI-TOF gave identical shifts and envelope patterns
421
between all three samples indicating the nucleophilic aspartic acid was not simply on
422
another fragment. These data suggest the resolution of an acyl-enzyme intermediate
423
does not primarily occur at the carbonyl of the nucleophilic aspartic acid as in HLD
424
enzymes and are further addressed in the discussion. A
Simulated Peptide
B
OleBC nosubstrate control
C
OleBC + β-hydroxy acid
D
OleBC + cis-β-lactone
E
Simulated Peptide with 2x 18O incorporation
2000
DLAATGHPLITLGHDWGGVVSLGWAAR 2770.4
3000
1000 0 5000 4000 3000 2000
wack003_chri2625_16340_20170630_C 2774.3
Intens. [a.u.]
Intens. [a.u.]
412
0 6000 4000
wack003_chri2625_16340_20170630_B 2774.3
Intens. [a.u.]
1000
0 3000 2000
wack003_chri2625_16340_20170630_ 2774.3
Intens. [a.u.]
2000
0 3000 2000
2778.4
1000 Intens. [a.u.]
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 18 of 38
1000 0
425
2760
2765
2770
2775
2780
2785
2790
2795
2800
18 ACS Paragon Plus Environment
Page 19 of 38
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biochemistry
426
Figure 6. H218O does not significantly incorporate into Asp163 of OleBC during the
427
catalytic cycle. (A) Simulated peptide fragment containing the nucleophilic Asp163
428
demonstrating expected isotopic envelope. (B) OleBC fusion with no substrate
429
incubated and digested in H218O. (C) OleBC fusion run with b-hydroxy acid substrate
430
(which utilize both the OleC and OleB domains) and digested in H218O. (D) OleBC
431
fusion run with cis-b-lactone acid substrate (which utilizes only the OleB domain) and
432
digested in H218O. (E) Simulated peptide with incorporation of two 18O over the no-
433
substrate control.
434 435 436
Discussion To our knowledge, OleB is the first enzyme reported to catalyze the
437
decarboxylation of a b-lactone to form a cis-olefin. There are other known a/b-
438
hydrolase superfamily members from plants that perform decarboxylation reactions,
439
such as MKS1 from Solanum habrochaites (wild tomato), that decarboxylate b-keto
440
acids to methylketones.31 However, these show only ~12% sequence identity to X.
441
campestris OleB and do not retain a nucleophilic aspartate or serine, that is typical of
442
the vast majority of a/b-hydrolases.31 Additionally, a/b-hydrolase superfamily members
443
such as AidH from Ochrobactrum sp. are known to catalyze the hydrolytic opening of
444
the five-membered g-lactone rings of quorum sensing molecules.32 Similarly, these
445
lactonases show little sequence identity to OleB (~19%) and contain a serine as the
446
catalytic nucleophile.32
447
OleB appears to react preferentially with one enantiomer of the synthetic cis-b-
448
lactone pair. The low solubility of the b-lactone substrates (~25 µM in 3% ethanol) and
449
even greater insolubility of the olefin products prevented more quantitative assessments
450
of substrate selectivity and steady-state kinetic rate constants for OleB. Some solubility
451
issues are likely alleviated in vivo by the presence of the OleBCD multi-enzyme
452
complex.9 The preceding pathway enzymes, OleA and OleD, are known to generate a
453
b-hydroxy acid having a 2R,3S-configuration.12 OleC is believed to preserve the 2R,3S
454
stereochemistry during its ring closure reaction to the b-lactone. As such, it is likely that
455
OleB preferentially acts on the 2R,3S-b-lactone to produce a cis-olefin, but studies to
19 ACS Paragon Plus Environment
Biochemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 20 of 38
456
identify the absolute stereochemistry of the remaining lactone must be conducted. No
457
trans-olefin was ever observed, consistent with multiple literature reports that bacteria
458
expressing the Ole pathway enzymes exclusively produce cis-olefins.8,24 Additionally,
459
the observed retention of configuration, cis-b-lactone giving rise to cis-olefin, argues
460
against nucleophilic attack on the 4-position of the ring which would cause an inversion
461
of the stereocenter, leading to trans-olefins.
462
Our results demonstrate that OleB enzymes, encoded in oleABCD gene clusters,
463
make up 72% of haloalkane dehalogenase subfamily-III (HLD-III) as described by
464
Chovoncova et al.14 While HLD subfamilies I and II are defined by multiple proteins
465
having crystal structures, no structural data are available for HLD-IIIs. To our
466
knowledge, only three HLD-III members have been purified: DrbA, DmbC, and DmrB,
467
which share ~40-45% amino acid identity to OleB sequences.26,25 All were reported to
468
have poor dehalogenase activity with typical haloalkane substrates. Direct activity
469
comparisons between research groups are difficult; however, the poor dehalogenase
470
activity of OleB is similar to other HLD-IIIs, but three to four orders of magnitude lower
471
than the activity of DhlA (HLD-I) and LinB (HLD-II).25,26,29 The preference of OleB for
472
haloalkanes with long alkyl chains is consistent with the structures of its native b-lactone
473
substrates. Additionally, low purification yields were reported for DrbA, and DmbC, 0.1
474
mg protein/g of cell pellet and 0.07 mg protein/g of cell pellet respectively.12 The
475
purification of OleB with PEG 400, to aid solubility, was 30 mg/L of culture or
476
approximately 7.5 mg protein/g of cell pellet. Given the activity of the two divergent
477
OleB sequences tested here, we believe that HLD-IIIs encoded within oleABCD gene
478
clusters act primarily as b-lactone decarboxylases rather than haloalkane
479
dehalogenases. The native substrates of HLD-III sequences not contained within
480
oleABCD gene clusters remains unclear, and further analyses are needed to define
481
their physiological roles.
482
In both sequence and structural alignments, the canonical Asp-His-Asp/Glu
483
catalytic triad of HLDs is completely conserved in OleB enzymes. Aspartic acid 114
484
from X. campestris OleB aligns with the nucleophilic aspartic acid of haloalkane
485
dehalogenases and was demonstrated to be essential for activity in both the X.
486
campestris OleB and the M. luteus OleBC fusion proteins. The observation of an alkyl20 ACS Paragon Plus Environment
Page 21 of 38
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biochemistry
487
enzyme intermediate by MALDI-TOF in bromoalkane substrate reactions with wild-type
488
OleB but not OleBD114A further implicated Asp114 as the essential nucleophile. His277 in
489
the X. campestris OleB protein is conserved in all HLDs and OleB sequences and the
490
inactivity of OleBH277A was consistent with its important mechanistic role. The role of the
491
conserved Asp249 in OleB (Asp249 in HLD-I or Glu130 in HLD-II) in maintaining the correct
492
protonation state of His277 for the activation of water is consistent with our data that X.
493
campestris OleB is slightly slower when Asp249 is mutated to an Ala. The rate decrease
494
may be partially masked because the olefin product is extremely hydrophobic, and we
495
believe product release is the slowest step of the OleB catalytic cycle. Further work is
496
currently underway to develop better assays to measure the unstable and hydrophobic
497
substrates and products of this reaction.
498
The accumulation of an alkylated OleB when reacted with 7-
499
(bromomethyl)pentadecane suggested that the enzyme ester intermediate is processed
500
differently than for HLDs. Our H218O studies also support this conclusion, as there was
501
no significant evidence for a +4 m/z shift in the nucleophilic aspartic acid peptide when
502
reacted with b-lactones, as observed in equivalent HLD experiments.30 Additionally, the
503
more physiologically relevant reaction in which both domains of the OleBC fusion are
504
utilized shows no incorporation of 18O. It is possible that only a +2 m/z shift would be
505
observed if the nonspecific incorporation of 18O in the no substrate control occurred at
506
Asp163. However, our data do not support this limited mass shift as only two complete
507
turnovers should result in 75% of the population with a +2 m/z over the no substrate
508
control. While the isotopic envelope of OleBC reacted with cis-b-lactone demonstrates
509
a population of the aspartic acid peptide fragments have 18O is incorporated, this does
510
not appear to be the dominant pathway for ester hydrolysis. In fact, this minor
511
incorporation gives credence to our assignment of Asp163 as the nucleophile since this
512
was the only peptide fragment that showed any change between the control and
513
substrate reactions. Overall, the absence of incorporated 18O points to the dominance
514
of the pathway shown in Figure 7B.
515 516
21 ACS Paragon Plus Environment
Biochemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 22 of 38
A) Known Haloalkane Dehalogenase Mechanism Asp
O O
oxyanion hole
Asp
-
His: H
Cl
halide stabilizing residues
R
As p
O
O
O O H
O
-
Cl
R
Asp
-
O O
O
H
-
Cl
OH
-
Cl
R
-
R
B) Proposed OleB Mechanism Asp114
oxyanion hole Asp114
O O
-
O
O O R1
517
Asp114
O
O
R2
R1
halide stabilizing residues
O
-
His: H
Asp 114
O O
O H
-
H O
O
Asp114
O O
O -
O
O O
-
R2
R1
R2
-
HO
-
R2 R1
O
O
O
R1
R2
518
Figure 7. Proposed mechanism of OleB as a b-lactone decarboxylase vertically aligned
519
with known HLD mechanism. (A) General mechanism of HLDs as described by multiple
520
reviews.4,29 (B) Analogous mechanism using b-lactone as a substrate with X. campestris
521
OleB. The “oxyanion hole” and “halide stabilizing residues” were labeled according to
522
the HLD mechanism for consistency, but perform different roles in the OleB mechanism.
523
The putative anhydride intermediate allows two possible mechanisms for OleB. The
524
absence of 18O in the nucleophilic Asp suggests attack of the distal carbon. Protonation
525
of the acyl carbonate intermediate would facilitate hydroxide attack, but it is unknown if
526
this protonation occurs.
527 528
In light of the bioinformatic and biochemical evidence, Figure 7 shows the
529
canonical mechanism for HLDs vertically aligned with our analogous, proposed
530
mechanism for OleB. The nucleophilic Asp114 of OleB attacks the carbonyl carbon of the
531
b-lactone ring to generate a tetrahedral intermediate. In HLD reactions, the side chains
532
of Trp115 and Gln40 act to stabilize the displaced negative halide ion. In OleB, these
533
completely conserved residues could act to stabilize the oxyanion of a tetrahedral
22 ACS Paragon Plus Environment
Page 23 of 38
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biochemistry
534
intermediate. This first tetrahedral intermediate resolves to expel the olefin product and
535
generate an anhydride as the equivalent to the alkyl-enzyme intermediate of HLDs.
536
There are two possible centers for the attack of water activated by His277 rather
537
than the single center in the HLD mechanism: the carbonyl of aspartic acid and the
538
carbonyl originating from the b-lactone. Our experiments indicate water attacks the
539
distal carbonyl originating from the 2-position of the b-lactone ring, thereby placing the
540
second oxyanion intermediate in the same spatial position as the initial oxyanion formed
541
during aspartate attack on the b-lactone carbonyl carbon. In HLD reactions, the
542
residues that form the oxyanion hole near the nucleophilic aspartic acid by their
543
backbone nitrogens also form the halide stabilizing pocket with their side chain
544
nitrogens (X of the HGXP motif and Trp adjacent to the nucleophile). In OleB, the
545
backbone nitrogens of these residues may act to correctly position the nucleophilic
546
aspartic acid for initial substrate attack while the side chain nitrogens are positioned to
547
stabilize the two oxyanions generated during initial substrate attack and subsequent
548
bicarbonate release in OleB mechanism. The overall OleB reaction is proposed to
549
remove CO2 from a b-lactone in the form of bicarbonate to generate an alkene that
550
preserves the stereocenters from the original b-lactone.
551
The resolution at the distal carbonyl makes OleB different from other a/b-
552
hydrolase enzymes bearing an Asp nucleophile, such as HLDs and epoxide hydrolases.
553
These rely on the carboxylic acid group of Asp to provide the entire enzyme-
554
intermediate ester linkage, while other superfamily members like lipases and lactonases
555
require only a nucleophilic Ser since the substrate donates part of the ester linkage. It
556
is unclear why Asp is utilized by OleB when the resolved ester linkage appears to be
557
provided by the substrate. However, it is possible that the electronics of a carboxylate
558
are required over an alkoxide during initial substrate attack to promote decarboxylation
559
instead of ring opening of the b-lactone. Chemical literature provides little insight on the
560
matter since decarboxylation of the labile b-lactone ring is almost always done with heat
561
rather than a catalyst. Conversely, the Asp nucleophile could simply be the result of
562
evolution from the HLD active site that is properly arranged for an Asp nucleophile.
563
In summary, OleB, a member of the a/b-hydrolase superfamily, is concretely
564
defined as the final step of the long-chain olefin biosynthesis pathway by catalyzing a 23 ACS Paragon Plus Environment
Biochemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 24 of 38
565
decarboxylation reaction with the b-lactone formed by the protein OleC. OleB enzymes
566
show many similarities to haloalkane dehalogenases and comprise most of the
567
sequences reported in the HLD subgroup III, suggesting at least a partial misannotation
568
of this group of enzymes. OleB proteins contain the conserved Asp-His-Asp/Glu
569
catalytic triad of HLDs, and current evidence supports a variation of the HLD
570
mechanism.
24 ACS Paragon Plus Environment
Page 25 of 38
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biochemistry
571
Acknowledgements
572
We acknowledge the support of the Biotechnology Institute from the University of
573
Minnesota, and the MnDRIVE Initiative from the Office of the Vice President for
574
Research of the University of Minnesota (LPW). SLR was supported by an NSF
575
Graduate Research Fellowship (Grant no. 00039202). We would like to thank Matt
576
Jensen for his help with homology models and Dr. Carrie Wilmot for her insightful
577
discussion. We also want to thank Todd Markowski and Lee-Ann Higgins for their
578
guidance in our MALDI-TOF endeavors and Peter Christenson for his help in purifying
579
X. campestris OleB.
580
25 ACS Paragon Plus Environment
Biochemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
581
Page 26 of 38
References
582 583
1.
Lenfant, N., Hotelier, T., Velluet, E., Bourne, Y., Marchot, P., Chatonnet, A. (2013)
584
ESTHER, the database of the α/β-hydrolase fold superfamily of proteins: Tools to
585
explore diversity of functions. Nucleic Acids Res. 41, D423--D429.
586
2.
Rauwerdink, A., Kazlauskas, R. J. (2015) How the Same Core Catalytic
587
Machinery Catalyzes 17 Different Reactions: The Serine-Histidine-Aspartate
588
Catalytic Triad of α/β-Hydrolase Fold Enzymes. ACS Catal. 5, 6153-6176.
589
3.
590 591
Nardini, M., Dijkstra, B. W. (1999) alpha/beta hydrolase fold enzymes: The family keeps growing. Curr Opin Struct Biol. 9, 732-737.
4.
Nagata, Y., Ohtsubo, Y., Tsuda, M. (2015) Properties and biotechnological
592
applications of natural and engineered haloalkane dehalogenases. Appl Microbiol
593
Biotechnol. 99, 9865-9881.
594
5.
Sukovich, D. J., Seffernick, J. L., Richman, J. E., Gralnick, J. A., Wackett, L. P.
595
(2010) Widespread head-to-head hydrocarbon biosynthesis in bacteria and role of
596
OleA. Appl Environ Microbiol. 76, 3850-3862.
597
6.
Sukovich, D. J., Seffernick, J. L., Richman, J. E., Hunt, K. A., Gralnick, J. A.,
598
Wackett, L. P. (2010) Structure, function, and insights into the biosynthesis of a
599
head-to-head hydrocarbon in Shewanella oneidensis strain MR-1. Appl Environ
600
Microbiol. 76, 3842-3849.
601
7.
Frias, J. A., Richman, J. E., Erickson, J. S., Wackett, L. P. (2011) Purification and
602
characterization of OleA from Xanthomonas campestris and demonstration of a
603
non-decarboxylative claisen condensation reaction. J Biol Chem. 286, 10930-
604
10938.
605
8.
Albro, P. W., Dittmer, J. C. (1969) The biochemistry of long-chain, nonisoprenoid
606
hydrocarbons. I. Characterization of the hydrocarbons of Sarcina lutea and the
607
isolation of possible intermediates of biosynthesis. Biochemistry. 8, 394-404.
608
9.
Christenson, J. K., Jensen, M. R., Goblirsch, B. R., Fatuma, M., Zhang, W.,
609
Wilmot, C. M., Wackett, L. P. (2017) Active multienzyme assemblies for long-
610
chain olefinic hydrocarbon biosynthesis. J Bacteriol. 199, e00890--16.
611
10.
Kancharla, P., Bonnett, S. A., Reynolds, K. A. (2016) Stenotrophomonas 26 ACS Paragon Plus Environment
Page 27 of 38
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biochemistry
612
maltophilia OleC-Catalyzed ATP-Dependent Formation of Long-Chain Z-Olefins
613
from 2-Alkyl-3-hydroxyalkanoic Acids. ChemBioChem. 17, 1426-1429.
614
11.
Goblirsch, B. R., Jensen, M. R., Mohamed, F. A., Wackett, L. P., Wilmot, C. M.
615
(2016) Substrate trapping in crystals of the thiolase OleA identifies three channels
616
that enable long chain olefin biosynthesis. J Biol Chem. 291, 26698-26706.
617
12.
Bonnett, S. A., Papireddy, K., Higgins, S., Del Cardayre, S., Reynolds, K. A.
618
(2011) Functional characterization of an NADPH dependent 2-alkyl-3-
619
ketoalkanoic acid reductase involved in olefin biosynthesis in Stenotrophomonas
620
maltophilia. Biochemistry. 50, 9633-9640.
621
13.
Christenson, J. K., Richman, J. E., Jensen, M. R., Neufeld, J. Y., Wilmot, C. M.,
622
Wackett, L.P. (2017) β-Lactone Synthetase Found in the Olefin Biosynthesis
623
Pathway. Biochemistry. 56, 348-351.
624
14.
625 626
Chovancova, E., Kosinski, J., Bujnicki, J. M., Damborsky, J. (2007) Phylogenetic analysis of haloalkane dehalogenases. Proteins Struct Funct Genet. 67, 305-316.
15.
Mulzer, J., Brüntrup, G., Hartz, G., Kühl, U., Blaschek, U., Böhrer, G. (1981)
627
Additionen von Carbonsäure-Dianionen an α, β-ungesättigte
628
Carbonylverbindungen - Steuerung der 1,2-/1,4-Regioselektivität durch sterische
629
Substituenteneffekte. Chem Ber. 114, 3701-3724.
630
16.
Lee, M. J., Gwak, H. S., Park, B. D., Lee, S. T. (2005) Synthesis of mycolic acid
631
biosurfactants and their physical and surface-active properties. J Am Oil Chem
632
Soc. 82, 181-188.
633
17.
634 635
esters. J Org Chem. 35, 3195-3196. 18.
636 637
19.
642
Buck, M., Chong, J. M. (2001) Alkylation of 1-alkynes in THF. Tetrahedron Lett. 42, 5825-5827.
20.
640 641
Lindlar, H. (1952) Ein neuer Katalysator für selektive Hydrierungen. Helv Chim Acta. 35, 446-450.
638 639
Crossland, R. K., Servis, K. L. (1970) Facile synthesis of methanesulfonate
Thalmann, A., Oertle, K., Gerlach, H. (1985) Ricinelaidic acid lactone. Org Synth. 63, 192.
21.
Wright, E. S. (2015) DECIPHER: harnessing local sequence context to improve protein multiple sequence alignment. BMC Bioinformatics. 16, 322. 27 ACS Paragon Plus Environment
Biochemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
643
22.
644 645
Page 28 of 38
Schliep, K. P. (2011) phangorn: Phylogenetic analysis in R. Bioinformatics. 27, 592-593.
23.
Kelley, L. A., Mezulis, S., Yates, C.M., Wass, M.N., Sternberg, M. J. E. (2015)
646
The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc.
647
10, 845-858.
648
24.
649 650
Frias, J. A., Riehman, J. E., Wackett, L. P. (2009) C 29 olefinic hydrocarbons biosynthesized by arthrobacter species. Appl Environ Microbiol. 75, 1774-1777.
25.
Fung, H. K., Gadd, M. S., Drury, T. A., Cheung, S., Guss, J. M., Coleman, N. V.,
651
Mathews, J. M. (2015) Biochemical and biophysical characterization of haloalkane
652
dehalogenases DmrA and DmrB in Mycobacterium strain JS60 and their role in
653
growth on haloalkanes. Mol Microbiol. 97, 439-53.
654
26.
Jesenská, A., Monincová, M., Koudeláková, T., Hasan, K., Chaloupková, R.,
655
Prokop, Z., Geerloff, A., Damborský, J. (2009) Biochemical characterization of
656
haloalkane dehalogenases DrbA and DmbC, representatives of a novel subfamily.
657
Appl Environ Microbiol. 75, 5157-5160.
658
27.
Hesseler, M., Bogdanović, X., Hidalgo, A., Berenguer, J., Palm, G. J., Hinrichs,
659
W., Bornscheuer, U. T. (2011) Cloning, functional expression, biochemical
660
characterization, and structural analysis of a haloalkane dehalogenase from
661
Plesiocystis pacifica SIR-1. Appl Microbiol Biotechnol. 91, 1049-1060.
662
28.
Novak, H. R., Sayer, C., Isupov, M. N, Gotz, D., Spragg, A. M., Littlechild, J. A.
663
(2014) Biochemical and structural characterisation of a haloalkane dehalogenase
664
from a marine Rhodobacteraceae. FEBS Lett. 588, 1616-1622.
665
29.
Koudelakova, T., Chovancova, E., Brezovsky, J., Monincova, M., Fortova, A.,
666
Jarkovsky, J., Damborsky, J. (2011) Substrate specificity of haloalkane
667
dehalogenases. Biochem J. 435, 345-354.
668
30.
Pries, F., Kingma, J., Pentenga, M., van Pouderoyn, G., Jeronimus-Stratingh, C.
669
M., Bruins, A. P., Janssen, D. B. (1994) Site-Directed Mutagenesis and Oxygen
670
Isotope Incorporation Studies of the Nucleophilic Aspartate of Haloalkane
671
Dehalogenase Biochemistry. 33, 1242-1247.
672 673
31.
Auldridge, M. E., Guo, Y., Austin, M. B., Ramsey, J., Fridman, E., Pichersky, E., Noel, J. P. (2012) Emergent Decarboxylase Activity and Attenuation of α/β28 ACS Paragon Plus Environment
Page 29 of 38
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biochemistry
674
Hydrolase Activity during the Evolution of Methylketone Biosynthesis in Tomato.
675
Plant Cell. 24, 1596-1607.
676
32.
Gao, A., Mei, G. Y., Liu, S., Wang, P., Tang Q., Liu, Y. P., Wen, H., An, X. M.,
677
Zhang, L. Q., Yang, X. X., Liang, D. C. (2013) High-resolution structures of AidH
678
complexes provide insights into a novel catalytic mechanism for N-acyl
679
homoserine lactonase. Acta Crystallogr Sect D Biol Crystallogr. 69, 82-91.
680
29 ACS Paragon Plus Environment
Biochemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
681
Page 30 of 38
For Table of Contents Use Only: Haloalkane Dehalogenases (HLD) O
HLD-II
O
HLD-I
R1 0.1
OleB R2
CO2 +
R1
R2
β-lactone decarboxylase
682
HLD-III
683
OleB from bacterial hydrocarbon biosynthesis is a β-lactone decarboxylase sharing key features
684
with haloalkane dehalogenases
685
686
James K. Christenson, Serina L. Robinson, Tiffany A. Engel, Jack E. Richman, An N. Kim, Larry P.
687
Wackett.
30 ACS Paragon Plus Environment
O
O
Page 31 of 38
R2
S-CoA
1 O 2 3 S-CoA 4 5 R1
OleA
OH
O
R2
OH R1
Biochemistry OleD R2
O
OleC
O O
OleB ?
OH
R1
R2
R1
Δ ACS Paragon Plus Environment R2 R1
R2 R1
A cis-olefin R1
R2
cis-β-lactone trans-β-lactone Biochemistry PageO 32 of 38 O O R1
1 2 3 4 5 6 7 8 B 9 10 11 12 13 14 15 16 C 17 18 19 20 21 22 23 24 25 26
O
O
R2 R1
R2
O
O R1
O R2 R 1
ACS Paragon Plus Environment
R2
Page Aa33 of 38
B b
Biochemistry
D114 D249 H277 _________________ QDWGS . . . D . . . HF I Q
Mycobacterium avium
1 2 3 34 4 5 83 6 7 8 9 10100 11 12 13 14 83 15 16 17 18 C 19 20 21 22 23 24 25 26
75
HLD−I
Psychrobacter cryohalolentis
99
QDWGG . . . D . . . HF LQ QDWGG . . . D . . . HFVQ
Plesiocystis pacifica
HDWGS . . . G . . . HF LQ
Alcanivorax dieselolei 79
HLD−II
Rhodobacteraceae bacterium
100
HDWGS . . . G . . . HF I Q HDWGS . . . G . . . HFFQ
Strongylocentrotus purpuratus Mycobacterium bovis
QDWGG . . . D . . . HF I Q
Xanthomonas campestris
HDWGG . . . D . . . HYVL
Stenotrophomonas maltophilia
HDWGG . . . D . . . HYVL
100 59 63
Shewanella oneidensis Micrococcus luteus 100
Paenarthrobacter aurescens
OleB
HLD−III
OleBC
Rhodopirellula baltica
0.1
HDWGG . . . D . . . HY I L HDWGG . . . D . . . HL LV HDWGG . . . D . . . HLVG HDWGG . . . D . . . HYV I
ACS Paragon Plus Environment
Xc
cis-olefin
cis-β-lactone Biochemistry trans-β-lactone Page 34 of 38 O
O R1
1 2 A 3 4 5 6 7 8 9 10 11 B 12 13 14 15 16 17 18 19 20
R2
R1
O
O R2
R1
O
O R2
R1
O
O R2 R1
ACS Paragon Plus Environment (ppm)
R2
Wt38 OleB Biochemistry Page 35 of
Intensity (%)
100
No Substrate
80
36.8
Intensity (%)
1 2 60 3 4 40 + 221 m/z 5 20 + 223 m/z 6 7 0 35.6 Wt OleB 35.8 36 36.2 36.4 36.6 8 + 9 100 10 80 11 12 60 13 40 14 15 20 16 0 ACS Paragon Plus Environment 17 35.6 35.8 36 36.2 36.4 36.6 18 m/z (KDa)
36.8
Simulated Peptide
DLAATGHPLITLGHDWGGVVSLGWAAR 2770.4
A
Biochemistry
Page 36 of 38
2778.4
2774.3
2774.3
2774.3
1 2 3 OleBC noB 4 substrate control 5 6 7 8 C 9 OleBC + β-hydroxy acid 10 11 12 13 14 OleBC + D 15 cis-β-lactone 16 17 18 19 20 Simulated Peptide with E 21 2x 18O incorporation 22 23 ACS Paragon Plus Environment 24 25 2765 2770 2775 2780 26
2785
A) Known Haloalkane Dehalogenase Mechanism Page 37 of 38 Biochemistry Asp
O
oxyanion hole
Asp
O
As p
O
-
1 O O 2 O O His: H Cl H O 3 halide Cl Cl R 4 H R stabilizing R 5 residues 6 7 B) 8 Proposed OleB Mechanism oxyanion 9 Asp hole Asp114 Asp114 Asp 114 10 114 O O O O 11 O 12 O O O His: H O O 13 O H O O O 14 H -O O O O 15 R1 R2 R2 16 R1 17 ACS Paragon Plus Environment halide R1 R2 18 R1 R2 stabilizing 19 residues
Asp
O O
-
Cl
OH R
Asp114
O O
-
HO -
O
R1
O
R2
Haloalkane Dehalogenases (HLD)
Biochemistry
HLD-II
Page 38 of 38
O
1 HLD-I 2 3 4
O
R1
OleB R2
ACS Paragon Plus Environment 0.1
CO2 +
R1
R2
β-lactone decarboxylase
HLD-III
