Investigating: Saccharomyces cerevisiae alkene reductase OYE 3 by substrate profiling, X-ray crystallography and computational methods

Article abstract of DOI:10.1039/c8cy00440d

Saccharomyces cerevisiae OYE 3 shares 80% sequence identity with the well-studied Saccharomyces pastorianus OYE 1; however, wild-type OYE 3 shows different stereoselectivities toward some alkene substrates. Site-saturation mutagenesis of Trp 116 in OYE 3 followed by substrate profiling showed that the mutations had relatively little effect, opposite to that observed previously for OYE 1. The X-ray crystal structures of unliganded and phenol-bound OYE 3 were solved to 1.8 and 1.9 ? resolution, respectively. Both structures were nearly identical to that of OYE 1, with only a single amino acid difference in the active site region (Ser 296 versus Phe 296, part of loop 6). Despite their essentially identical static X-ray structures, molecular dynamics (MD) simulations revealed that loop 6 conformations differed significantly in solution between OYE 3 and OYE 1. In OYE 3, loop 6 remained nearly as open as observed in the crystal structure; by contrast, loop 6 closed over the active site of OYE 1 by ca. 4 ?. Loop closure likely generates a greater number of active site protein contacts for substrate bound to OYE 1 as compared to OYE 3. These differences provide an explanation for the differing stereoselectivities of OYE 3 and OYE 1, despite their nearly identical X-ray crystal structures.

Full text of DOI:10.1039/c8cy00440d

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M. P. Buteler, S. Lenka, M. Crotti, S. Santangelo, M. J. Burg, S. Bruner, E. Brenna, A. E. Roitberg and J. D.
Stewart, Catal. Sci. Technol., 2018, DOI: 10.1039/C8CY00440D.
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Investigating Saccharomyces cerevisiae Alkene Reductase OYE
3 by Substrate Profiling, XꢀRay Crystallography and
Computational Methods
Robert W. Powell, III, M. Pilar Buteler†, Sunidhi Lenka†, Michele Crotti†,‡, Sara
Santangelo†,‡, Matthew J. Burg, Steven Bruner, Elisabetta Brenna‡, Adrian E.
Roitberg and Jon D. Stewart*
Department of Chemistry, 126 Sisler Hall, University of Florida, Gainesville, FL
32611 USA
‡Dipartimento di Chimica, Materiali ed Ingegneria Chimica “Giulio Natta”
Politecnico di Milano, Via Mancinelli 7, Iꢀ20131, Milano, Italy.
Phone & Fax 352.846.0743, Eꢀmail jds2@chem.ufl.edu
*Author to whom correspondence should be addressed
†These authors contributed equally to this work
1

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Abstract
Saccharomyces cerevisiae OYE 3 shares 80% sequence identity with the wellꢀstudied
Saccharomyces pastorianus OYE 1; however, wildꢀtype OYE 3 shows different
stereoselectivities toward some alkene substrates. Siteꢀsaturation mutagenesis of Trp 116 in
OYE 3 followed by substrate profiling showed that the mutations had relatively little effect,
opposite to that observed previously for OYE 1. The xꢀray crystal structures of unliganded and
phenolꢀbound OYE 3 were solved to 1.8 and 1.9 Å resolution, respectively. Both structures were
nearly identical to that of OYE 1, with only a single amino acid difference in the active site
region (Ser 296 versus Phe 296, part of loop 6). Despite their essentially identical static xꢀray
structures, molecular dynamics (MD) simulations revealed that loop 6 conformations differed
significantly in solution between OYE 3 and OYE 1. In OYE 3, loop 6 remained nearly as open
as observed in the crystal structure; by contrast, loop 6 closed over the active site of OYE 1 by
ca. 4 Å. Loop closure likely generates a greater number of active site protein contacts for
substrate bound to OYE 1 as compared to OYE 3. These differences provide an explanation for
the differing stereoselectivities of OYE 3 and OYE 1, despite their nearly identical xꢀray crystal
structures.
2

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Introduction
Since its discovery as the first cofactorꢀcontaining enzyme,1 the Old Yellow Enzyme from
Saccharomyces pastorianus2 (OYE 1) has been an important testbed for discovering general
principles of flavoprotein structure and mechanism.3ꢀ5 Biocatalytic interest in OYE 1 and its
relatives grew rapidly after Massey’s discovery that it reduces electronꢀdeficient alkenes with
very high stereoselectivity.6 Over the past two decades, the substrate range of S. pastorianus
OYE 1 has been explored extensively; in parallel, many homologs have been cloned,
overexpressed and their synthetic abilities evaluated.7ꢀ9 In addition, a number of protein
engineering approaches have also been applied to OYE 1 and its homologs to improve substrate
acceptance, alter stereoselectivity, etc.10ꢀ14
OYE 1ꢀmediated alkene reductions proceed with net transꢀaddition of both hydrogen atoms,
with hydride from reduced FMN adding to the substrate βꢀcarbon lying above the cofactor and
protonation from the opposite face of the alkene mediated by a Tyr sideꢀchain (Scheme 1).6
Substrate binding orientation thus dictates which face of the π system lies proximal to the flavin
and thereby determines the absolute stereochemistry of the reduced product.
Fox and Karplus solved the xꢀray crystal structure of OYE 1 in 1994;15,16 since then, the
structures of several OYE 1 homologs have been determined.17ꢀ26 While all family members
share a common TIM barrel architecture and have similar active site structures, the observation
that some substrates bind in different orientations (and thus yield opposite stereoisomers)
underscores the importance of small structural differences (for an early example, see [Hall, 2008
#4544]).
The genomes of both bakers’ yeast (Saccharomyces cerevisiae) and brewers’ bottom yeast
(S. pastorianus) contain the OYE 1 gene.27 The former also includes two homologs (OYE 2 and
3

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OYE 3).28 All share very high mutual sequence identity.29 To determine whether these minor
sequence differences impacted stereoselectivity, we examined all published examples of alkene
substrates that had been reduced by both OYE 1 and OYE 3. Unsurprisingly, the large majority
of alkenes were reduced with the same stereopreferences; however, five cases differed (Table 1).
In each, the reaction outcomes suggested that all five bound to OYE 1 in the “normal”
orientation (Scheme 1, left), but to OYE 3 in the “flipped” orientation (Scheme 1, right).
Because three of these examples involve substrates of particular interest to our synthetic
efforts,30 we solved the xꢀray crystal structure of S. cerevisiae OYE 3. We also explored the
response of OYE 3 to mutations at Trp 116, a position that has proven highly influential in
dictating the stereoselectivity of OYE 1.31 Finally, we also carried out molecular dynamics
simulations of both OYE 1 and OYE 3 to uncover possible differences in active site structure and
dynamics that might help explain why reactions with OYE 3 sometimes differ from those of
OYE 1.
Experimental
General. Restriction endonucleases, Phusion Hot Start II HighꢀFidelity DNA Polymerase
and T4 DNA ligase were purchased from New England Biolabs and primers were obtained from
Integrated DNA Technologies. Crystallography screening kits (Classics Suite/ AmSO4 and
PEGRx HT) were purchased from Qiagen and Hampton Research, respectively. All other
reagents were obtained from commercial suppliers and used as received. Plasmids were purified
on small scales by Wizard® minicolumns (Promega Life Sciences) and on large scales using
CsCl density gradient ultracentrifugation. DNA sequencing was carried out by the University of
4

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Florida ICBR using capillary fluorescence methods using standard protocols. Growth media
recipes have been described previously.[Sullivan, 2013 #5001]
Alkene substrates 5 – 19 were purchased from commercial suppliers or prepared inꢀhouse
during previous studies.33 Conversions and stereoselectivities were assessed by GC. Reactions
of substrates 3 and 4 were analyzed on a 0.25 mm × 30 m HPꢀ5MS column (Agilent) coupled
with an MS detector to assess conversion, and on a 0.25 mm × 25 m Chirasil DEX CB column
(Chrompack) coupled with an FID to assess enantioselectivity. Reactions of substrates 5, 6, 10
and 13 ꢀ 19 were analyzed on a 0.25 mm × 30 m Beta Dex 225 column (Astec) coupled with an
FID. Reactions of alkenes 7 ꢀ 9 and 16 were analyzed on a 0.25 mm × 30 m DBꢀ17 column
(Agilent J&W) coupled with an MS detector. Temperature programs were tailored to provide
baseline separations of each starting material and the corresponding product stereoisomers.
Determining the stereochemical courses of OYE 3 and OYE 1 reductions. Substrates 3 and 4
were prepared according to the literature.34,35 Racemic samples of 4ꢀmethylhexanꢀ3ꢀone36 and 4ꢀ
methylheptanꢀ3ꢀone,37 used as reference compounds, were prepared by hydrogenation of 3 and 4
on Pd/C in Et2O. The absolute configurations of 4ꢀmethylhexanꢀ3ꢀone and 4ꢀmethylheptanꢀ3ꢀ
one, obtained by bioreduction, were established by comparison with optical rotation values
reported in the literature.38
1
(E)ꢀ4ꢀMethylhexꢀ4ꢀenꢀ3ꢀone 3. HꢀNMR (CDCl3, 400 MHz):39 δ (ppm) 6.73 (1H, q, J = 6.9
Hz, CH=), 2.66 (2H, q, J = 7.3 Hz, COCH2CH3), 1.85 (3H, d, J = 6.9 Hz, CH3CH=), 1.78 (3H,
s, CH3C=), 1.09 (3H, t, J = 7.3 Hz, CH3CH2); 13CꢀNMR (CDCl3, 100.6 MHz):39 δ (ppm) 202.5,
138.2, 136.7, 30.4, 14.8, 11.2, 9.0; GCꢀMS (EI) m/z (%) = 112 (M+, 25), 83 (83), 55 (100).
1
(E)ꢀ4ꢀMethylheptꢀ4ꢀenꢀ3ꢀone 4. HꢀNMR (CDCl3, 400 MHz):40 δ (ppm) 6.61 (1H, t, J = 7.2
Hz, CH=), 2.68 (2H, q, J = 7.3 Hz, COCH2CH3), 2.25 (2H, quintuplet, J = 7.3 Hz, CH2CH=),
5

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1.78 (3H, s, CH3C=), 1.13 – 1.03 (6H, m, CH3CH2CO and CH3CH2C=); 13CꢀNMR (CDCl3, 100.6
MHz): δ (ppm) 202.6, 143.4, 136.6, 30.4, 22.4, 13.2, 11.3, 8.9; GCꢀMS (EI) m/z (%) = 126 (M+,
20), 97 (100), 69 (90).
1
4ꢀMethylhexanꢀ3ꢀone. HꢀNMR (CDCl3, 400 MHz):36 δ (ppm) 2.51 – 2.40 (3H, m, COCH2
+ COCH), 1.75 – 1.60 (1H, m, CHHCH3), 1.45 – 1.33 (1H, m, CHHCH3), 1.06 (3H, d, J = 7.0
Hz, CH3CH), 1.05 (3H, t, J = 7.2 Hz, CH3CH2CO), 0.87 (3H, t, J = 7.6 Hz, CH3CH2CH);3 13Cꢀ
NMR (CDCl3, 100.6 MHz): δ (ppm) 215.4, 47.7, 34.4, 26.2, 16.1, 11.8, 7.9; GCꢀMS (EI) m/z
(%) = 114 (M+, 7), 86 (11), 57 (100).
1
4ꢀMethylheptanꢀ3ꢀone. HꢀNMR (CDCl3, 400 MHz):37 δ (ppm) 2.58 – 2.50 (1H, m, COCH),
2.45 (2H, qd, J = 7.4 and 2.5 Hz, CH2CH3); 1.68 – 1.55 (1H, m, CHCHHCH2), 1.38 – 1.20 (3H,
m, CHCHHCH2 + CH2CH3), 1.06 (3H, d, J = 7.0 Hz, CH3CH), 1.04 (3H, t, J = 7.1 Hz,
CH3CH2CO), 0.90 (3H, t, J = 7.6 Hz, CH3CH2CH2); 13CꢀNMR (CDCl3, 100.6 MHz):37 δ (ppm)
215.5, 46.0, 35.4, 34.3, 20.6, 16.5, 14.2, 7.9; GCꢀMS (EI) m/z (%) = 128 (5), 86 (49), 71 (74), 57
(100).
Enone 4 was submitted to bioreduction with NADPD using both OYE 3 and OYE 1 in D2O.
The substrate (50 ꢁmol) dissolved in iꢀPrOHꢀd8 (100 ꢁL) was added to a KPi buffer solution (5.0
mL, 50 mM in D2O, pH 7.0) containing NADP+ (15 ꢁmol), Thermoanaerobium brockii alcohol
dehydrogenase (4 U, 3 mg) and the required OYE (ca. 250 ꢁg, dissolved in 500–700 ꢁL H2O).
The mixture was incubated for 24 h in an orbital shaker (160 rpm, 30°C). The solution was
extracted with dichloromethane, centrifuging after each extraction, and the combined organic
solutions were dried over anhydrous Na2SO4. 2H spectra were recorded on a 400 MHz
spectrometer with proton broad band decoupling, in CHCl3 solutions, using CDCl3 as internal
reference for chemical shift scale.
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Siteꢀsaturation mutagenesis of Trp 116 in OYE 3. Plasmid pRP4 (a derivative of pET 22b
with an OYE 3 coding region flanked by NdeI and XhoI sites) was the template used to make
OYE 3 Trp 116 siteꢀsaturation mutants. A pair of mutagenic primers containing a single codon
replacement for Trp 116 was used to make each of the 19 mutants. PCR was performed in a total
reaction volume of 50 ꢁL, composed of 0.5 ꢁL of template (18 ng / ꢁL), 5 ꢁL of both forward
and reverse primers (5 mM), 1 ꢁL of dNTP mix (10 mM), 10 ꢁL of 5 × HF Phusion® Hot start
buffer, 28 ꢁL of sterile water, and 0.5 ꢁL of Phusion® Hot Start II DNA Polymerase (2 U / ꢁL).
PCR involved an initial denaturation step at 98 °C for 30 s, followed by 25 cycles of
denaturation at 98 °C for 10 s, annealing at 64 °C for 30 s, and an extension step at 72 °C for 3
min 30 s, followed by a final extension step at 72 °C for 7 min 30 s.
PCR products were purified by Wizard® Plus SV Gel PCR Clean up kits (Promega)
according to the manufacturer’s instructions. The first aliquot of DpnI (0.5 ꢁL of a 20 U / ꢁL
stock) was added; after incubating for 4 h at 37ºC, a second aliquot of DpnI was added and
incubation was continued overnight at 37ºC. Digested PCR products were purified as before,
then aliquots (4 ꢁL) were used to transform 50 ꢁL of E. coli ElectroTenꢀBlue cells (Agilent)
using 2.5 kV. Immediately following electroporation, 600 ꢁL of SOC medium was added and
cells were incubated without shaking at 37 °C for 1 h, then plated onto LB plates supplemented
with 200 ꢁg / mL ampicillin. Plates were grown overnight at 37ºC. Plasmids were isolated from
randomlyꢀchosen colonies and sequenced to verify that the desired mutation was present, then
aliquots (4 ꢁL) were used to transform 80 ꢁL of E. coli BL21ꢀgold(DE3) cells using
electroporation (2.5 kV). Immediately after electroporation, 600 ꢁL of SOC medium was added
and cells were incubated without shaking at 37 °C for 45 min prior to plating onto LB plates
supplemented with 200 ꢁg / mL ampicillin. Plates were incubated overnight at 37ºC. Plasmids
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were isolated from each transformant and mutations were again verified by sequencing. The
library was arrayed in a 96 well microtiter plate and cells were grown overnight at 37ºC in 600
ꢁL of LB medium supplemented with 200 ꢁg / mL ampicillin. Aliquots (120 ꢁL) of each
saturated culture were transferred to a new 96 well microtiter plate containing 30 ꢁL of 80%
glycerol (which yielded a final glycerol concentration of 15%). This master plate was stored at ꢀ
80ºC.
Screening Trp 116 siteꢀsaturation mutagenesis library. E. coli BL21 (DE3) Gold cells
harboring plasmids encoding wildꢀtype and Trp 116 siteꢀsaturation OYE 3 mutants were shaken
overnight at 250 rpm in a 96 well plate containing 600 ꢁL of LB supplemented with 200 ꢁg / mL
ampicillin at 37°C. Aliquots of each saturated culture (20 ꢁL) were used to inoculate 2 mL wells
of a square bottom 96 well plate that contained 600 ꢁL of ZYPꢀ5052 autoꢀinduction medium32
supplemented with 200 ꢁg / mL ampicillin. Cultures were shaken overnight at 350 rpm and 37ºC
in a locallyꢀdesigned and fabricated aeration case.41 Cells were harvested by centrifugation and
then resuspended in 600 ꢁL of reaction mixture (50 mM KPi, 100 mM glucose, 15 mM alkene,
pH 7.0). Reaction mixtures were shaken overnight at room temperature at 250 rpm prior to
quenching with 500 ꢁL of EtOAc. After thorough mixing, the organic phase was separated by
centrifugation and removed for GC analysis.
OYE 3 purification and crystallogenesis. OYE 3 protein was purified as described in the
Supporting Information. Fractions containing OYE 3 were pooled and concentrated by
ultrafiltration to a final concentration of 40 mg / mL (using A280 and an estimated value of ε280
(76,905 Mꢀ1cmꢀ1).43
Initial crystallization conditions were identified by screening with the PEGRX HT (Hampton
Research) as well as the Classics Suite and AmSO4 Suite (Qiagen) kits. Sitting drop vapor
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diffusion was used and wells contained 2 ꢁL of crystallization solution and 2 ꢁL of 20 mg / mL
OYE 3 (in 50 mM TrisꢀCl, 50 mM NaCl, pH 7.5). The best crystals were obtained after 14 days
from well A8 of the PEG RX HT screening plate at room temperature and no further
optimization of these crystallization conditions was required (50 mM MES monohydrate, 22%
v/v PEG 400, 25 mM Tris, 25 mM NaCl, pH 8.0). Individual crystals were mounted and briefly
soaked briefly in cryoprotection buffer (100 mM MES monohydrate, 22% v/v PEG 400, 15%
(v/v) glycerol, pH 6.0) prior to flash cooling in liquid nitrogen for data collection.
Crystals were also grown from the well A10 conditions (PEG RX HT screening kit) using
sitting drop vapor diffusion (2 ꢁL of 100 mM sodium citrate tribasic dihydrate, 30% (v/v)
polyethylene glycol monomethyl ether 550 crystallization solution and 2 ꢁL of 20 mg / mL OYE
3 in 50 mM TrisꢀCl, 50 mM NaCl, pH 7.5). Crystals grew after 10 days at room temperature.
They were mounted in appropriatelyꢀsized loops, then soaked briefly in cryoprotection buffer
(100 mM sodium citrate tribasic dihydrate, 30% (v/v) polyethylene glycol monomethyl ether
550, 2 15% (v/v) glycerol, pH 5.0) supplemented with with 2 mM pꢀhydroxybenzaldehyde.
Samples were flash cooled in liquid nitrogen prior to synchotron data collection.
Data collection and structure refinement. All data sets were collected on the 21ꢀIDꢀG
beamline at the Advanced Photon Source, Argonne National Laboratory. Unsoaked and soaked
crystals diffracted to maximum usable resolutions of 1.8 and 1.9 Å, respectively. Reflection data
were processed using XDS.44 Phases were obtained by molecular replacement (PHENIX)45
using a modified S. pastorianus OYE 1 structure as the search model (PDB code 1OYB). All
ligands and water molecules were removed prior to molecular replacement. The initial model
was well defined throughout the scaffold of the protein except at the Cꢀterminus, giving an Rfree
value of 0.37. Residues 398 and 399 (Lys and Asn, respectively) were poorly fit and these two
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positions were therefore initially deleted from the model. The initially calculated 2FoꢀFc and Foꢀ
Fc maps showed electron density patterns readily identifiable as FMN. After an initial simulated
annealing refinement the Rfree dropped to 0.31. Residues 398 and 399 were added to the model
after the first round of refinement. Further refinement as well as continued cycles of model
building using the structure validation tools in COOT46 produced a final model with an Rfree
value of 0.22. Adding a chloride ligand in the active site accounted for most of the observed
active site electron density in the unsoaked crystals. For the pꢀhydroxybenzaldehydeꢀsoaked
crystals, the ligand was readily identifiable by its electron density.
Molecular dynamics studies of OYE 3 and OYE 1. All simulations were performed using the
AMBER16 suite.47 The starting structure for OYE 1 was taken from the PDB (3TX9, 2.00 Å
resolution) and the OYE 3 starting structure was from this study. The Amber ff14SB force field
was used for the proteins and the parameters for the FMN cofactor were obtained from Bryce’s
group.48 Starting topologies were built using the tleap module of AmberTools 16.47 The systems
were solvated in a 10 Å buffer of TIP3P water49 using a truncated octahedral box under periodic
boundary conditions. The SHAKE algorithm50 was used to keep bonds involving H atoms at
their equilibrium length and an 8 Å cutoff was applied for nonbonded interactions. Newton’s
equations51 were integrated with a 4 fs time step, using hydrogen mass repartition52 and frames
were collected at 40 ps intervals. The collision frequency of the Langevin thermostat was 2 ps–1.
All simulations used different random seeds (ig = ꢀ1) to avoid synchronization artifacts.53
The initial structures were minimized for 5000 steps using steepest descent for the first 1000
cycles and switching to conjugate gradient, with 10 kcal mol–1Å–2 restraints applied to the
backbone. Heating was done using a linear temperature gradient from 10 to 300 K during 0.8 ns
at constant volume. Equilibration was performed at the final temperature during 8 ns using the
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same backbone restraints under NPT conditions. Finally, we performed 100 ns of free dynamics
at a temperature of 300 K under NPT conditions. The simulations were done using
GPU(CUDA) version of the pmemd.cuda module.54 Five independent replicates of the
molecular dynamics runs were done to gauge the reproducibility of the results obtained.
Results and Discussion
Stereochemical courses of OYE 3ꢀ and OYE 1ꢀmediated reductions
Our initial hypothesis for the stereochemical divergence between OYE 3 and OYE 1 was that
the two enzymes bound substrates in opposite orientations. Ketones with the general structure of
1 are reduced by both OYE 3 and OYE 1 with (S)ꢀselectivity; this might arise from a “flipped”
substrate binding orientation in the active sites of both enzymes (Figure 1A). When the αꢀ
substituent was lengthened by one methylene, e.g., ketones 2 and Table 1, entries 3 – 5,
reductions by OYE 3 and OYE 1 proceeded with divergent stereoselectivities (Table 1). This led
us to test 3 and 4, whose βꢀsubstituents (methyl and ethyl) are smaller than the aryl analogs
investigated previously. Would such compounds behave similarly to ketone 2, in spite of the
reduced steric hindrance of the βꢀgroup?
Trisubstituted alkenes 3 and 4 were reduced by wildꢀtype OYE 3 and OYE 1 with (S)ꢀ and
(R)ꢀstereoselectivities, respectively (Table 2), which imply “flipped” (OYE 3) and “normal”
substrate binding orientations (OYE 1). The alternative explanation – that OYE 3 and OYE 1
followed different stereochemical courses in H2 addition – was ruled out by reducing enone 4
with NADPD in D2O.55 In the 1H NMR spectrum of the racemic hydrogenation product from 4,
the two protons at C5 are characterised by different chemical shifts: one gives a multiplet in the
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range 1.67ꢀ1.25 (shown in blue), and the other occurs as a multiplet in the range 1.38 ꢀ1. 20
(shown in red), which overlaps with the CH2 in position 6 (Figure 1B).
The 2H NMR spectra of the two deuterated samples show that both OYE 3 and OYE 1
promote the addition of two deuterium atoms in the same stereospecific way, presumably anti in
both cases (Figure 1C). The deuterium atoms are characterized by the same chemical shift, 2.45
and 1.21 ppm, corresponding to the multiplets at 2.44 and 1.38ꢀ1.20 ppm observed in the 1H
NMR spectrum of racemic reduction product of 4. No deuterium atom is present at the chemical
shift corresponding to the proton shown in blue (Figure 1B). These data demonstrate clearly that
the reversal of stereoselectivity between OYE 3 and OYE 1 does not arise because of a change
between net antiꢀ and synꢀaddition of H2, but rather is a consequence of substrate binding
orientation in the active sites. Based on these results, we attempted to magnify differences in
enzymeꢀsubstrate interactions between OYE 3 and OYE 1 by mutagenesis of Trp 116, which has
a dramatic impact on binding orientation for some substrates in OYE 1.31,56 Would changes at
Trp 116 have the same impact on OYE 3? To answer this questions, all possible variants at
position 116 were created.57
Consequences of Trp 116 mutations in OYE 3 and OYE 1
We tested the Trp 116 mutant collections for both OYE 3 and OYE 1 (Table 2) against both
3 and 4. In general, replacements for Trp 116 had relatively little impact in OYE 3, the sole
exceptions being Phe for 3 and Leu, Met and Tyr for 4, which increased the amount of (R)ꢀ
product (although the overall stereoselectivity was nearꢀracemic in all four cases). By contrast, it
was possible to identify mutants with either good (R)ꢀ and (S)ꢀstereoselectivities in the Trp 116
replacement library of OYE 1. These results showed that OYE 1 is far more sensitive to active
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site changes than OYE 3, suggesting that the active site of OYE 3 may have fewer proteinꢀ
substrate contacts that dictate binding orientation.
In previous work, we observed that the “flipped” binding mode is most commonly observed
with open chain trisubstituted alkenes.58 Only when the G group is large relative to the αꢀ
substituent does the substrate adopt a “normal” binding orientation (Figure 1D). In the case of 3
and 4, however, the increased bulkiness of the ethyl (the G group) with respect to the methyl
group (the αꢀsubstituent) promotes the “normal” binding mode in OYE 1, but not in OYE 3, as it
was observed for ketone 2, thus confirming that the hindrance of the βꢀgroup has little influence.
Steric interactions between the G group and the residue at position 116 are much more important
in OYE 1; by contrast, OYE 3 strongly prefers a “flipped” substrate binding orientation for
trisubstituted alkenes, which cannot be overriden by changes to Trp 116.
To better define the importance of active site changes in OYE 3, the wildꢀtype and all Trp
116 mutants were screened against 15 alkene substrates that we had investigated in previous
studies of OYE 1 using the same series of mutants (Figure 2).31,33,56
Wildꢀtype OYE 3 was unable to reduce BaylisꢀHillman adduct 5; by contrast, wildꢀtype OYE
1 provides the reduction product from a “flipped” binding orientation with 60% ee (Table 3).56
For both OYE 3 and OYE 1, all Trp 116 variants reduced 5 from a “normal” substrate binding
orientation. A higher proportion of OYE 1 mutants retained catalytic activity toward 5 as
compared to OYE 3, although all stereoselectivities were consistently (S).
2ꢀ(Hydroxymethyl)cyclohexenone 6 provided an interesting contrast between OYE 3 and
OYE 1. While nearly all Trp 116 variants gave consistent (S)ꢀstereoselectivities, Glu at position
116 of OYE 3 and OYE 1 yielded very different results (Table 3). In the sequence context of
OYE 3, the Trp 116 Glu mutant provided a nearly racemic mixture from BaylisꢀHillman adduct
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6 (2% ee favoring the (R)ꢀenantiomer). The analogous OYE 1 mutant gave the (S)ꢀproduct in
90% ee, consistent with all other Trp 116 variants in the sequence context of OYE 1.
Reductions of (S)ꢀ and (R)ꢀcarvone demonstrated even more clearly that altering Trp 116 in
OYE 3 and OYE 1 did not always yield the same results (Table 3). In the case of (S)ꢀcarvone 7,
the majority of substitutions for Trp 116 in OYE 1 prompted the alternative, “flipped” substrate
binding mode.31 This was not the case for OYE 3. In fact, none of the Trp 116 mutants
triggered the “flipped” substrate binding mode. This was a highly unexpected result. (R)ꢀ
Carvone 8 reductions also highlighted the subtle differences in active site behavior between OYE
1 and OYE 3. Two OYE 1 variants (Trp 116 Ala and Trp 116 Val) resulted in a predominantly
opposite stereochemical course for (R)ꢀcarvone reduction;31 however, the same two changes in
an OYE 3 background led to a nearly 1 : 1 mixture of diastereomers, showing that Trp 116
mutations exert less control over substrate binding orientation in OYE 3 as compared to OYE 1.
Neither wildꢀtype OYE 3 nor any Trp 116 variant reduced (R)ꢀpulegone 9 or
cyclopentenones 10 or 11 to significant extents. 2ꢀSubstituted cyclohexenone 12 was reduced by
wildꢀtype OYE 3, but it was not examined as a substrate for the Trp 116 variants. As
expected,[Swiderska, 2006 #4350] sterically and electronically challenging 3ꢀsubstituted
cyclohexenones 13 and 14 were recalcitrant toward reduction by wildꢀtype or any of the OYE 3
Trp 116 variants. While many of the OYE 3 Trp 116 mutants gave reasonable conversion for
exocyclic enone 15 (with the Trp 116 Asn variant best), none yielded the opposite stereoisomer.
The series of 4,4ꢀdisubstituted cyclohexenones 16 – 19 probed the ability of OYE 3 to accept
bulky substituents in this portion of the substrate. Interestingly, while 16 and 17 were efficiently
reduced by wildꢀtype OYE 3 and all Trp 116 variants, none accepted 18 and 19. While
reductions of 16 and 17 might show kinetic resolutions, this point was not investigated since the
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starting enantiomers could not be separated by any of our available chiral GC columns;
moreover, the achiral nature of the products makes any kinetic resolution only marginally useful.
Crystallographic studies of OYE 3
To help understand these differences in stereochemical outcomes, we solved the xꢀray crystal
structure of S. cerevisiae OYE 3. The protein was overexpressed in E. coli and purified by a
phenol affinity resin42 followed by gel filtration chromatography. After screening approximately
300 conditions, viable crystals were only observed when low molecular weight PEG was the
main precipitant at near neutral pH values (pH 6). The most successful conditions yielded
crystals that diffracted to a maximum usable resolution of 1.8 Å. The unit cell measured 61.2 ×
107.8 × 141. 1 Å and the crystals belonged to space group P212121 (Table 4). The asymmetric
unit contained two protein molecules and a solvent content of 53.5%. A Matthews coefficient of
2.65 Å3/Dal was calculated.
The structure of OYE 3 was solved by molecular replacement (MR) using OYE 1 as the
search model.15 Successive rounds of refinement and model improvement yielded an Rfree value
of 0.22. The overall TIM barrel structure of OYE 3 is nearly identical to that of OYE 1 (overall
rmsd = 0.26 Å using the 1OYB structure).
Once the protein scaffold was established, nonꢀprotein moieties were addressed. FMN was
easily fit into the structure using the observed electron density. The flavin environment of OYE
3 parallels that of OYE 1, with amino acids Thr 37, Gly 72, Gln 114 and Arg 243 hydrogen
positioned to form hydrogen bonds with the cofactor. The electron density in the vicinity of His
191 and Asn 194 was tentatively assigned to chloride as the predominant species. To help
understand how substrates might interact with OYE 3, we solved the structure after briefly
soaking with pꢀhydroxybenzaldehyde (Figure 3A). This had essentially no change on the OYE 3
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structure (apart from displacement of the active site chloride ligand). The phenolate oxygen lies
between the sideꢀchains of His 191 and Asn 194 and distances between the phenol and protein
residues for OYE 3 are very close to those observed previously for the analogous OYE 1
complex.15
All active site residues were identical between OYE 3 and OYE 1, except for position 296
(Ser in OYE 3 and Phe in OYE 1), located within loop 6 (residues 289 – 309). In native
(unsoaked) OYE 3 crystals, the electron density for all residues in loop 6 is very wellꢀdefined
(Figure 3B). This is also the case for OYE 3 crystals soaked with pꢀhydroxybenzaldehyde
(Figure 3C), although three solventꢀexposed Glu sideꢀchains showed somewhat weak electron
density beyond Cβ.
Structural comparison of OYE 3 and OYE 1
As expected from their high sequence identity, the overall structures of OYE 3 and OYE 1
are highly similar. Even loop 6, which contains the only active site change (Ser 296 in OYE 3
and Phe 296 in OYE 1), was found in the essentially the same conformation for OYE 3 (in both
ligandꢀsoaked and unsoaked crystals) as well as in ligandꢀsoaked crystals of OYE 1 (Figure 4).
The only significant difference between OYE 3 and OYE 1 in crystallo is the rotameric
preference of the sideꢀchains at position 296. In OYE 1, Phe 296 extends into the active site; in
OYE 3, its sideꢀchain is directed outward toward the solvent (Figure 4).
Computational studies of OYE 3 and OYE 1
Since substrate binding orientation was the key to dictating stereoselectivity, we used the
relevant crystal structures to model Michaelis complexes for all five substrates in Table 1 into
the active sites of OYE 3 and OYE 1. Each was modeled separately in the “normal” and
“flipped” orientations. This approach had been successful in understanding the stereopreferences
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in OYE 1 and other homologs.41 For both enzymes, the “normal” substrate binding mode was
easily modeled by overlaying the dashed lines with the pꢀhydroxybenzaldehyde bound to the
active sites (Table 1). In the models of “flipped” substrate binding, the phenolic oxygen of pꢀ
hydroxybenzaldehyde and the metaꢀring carbon marked the approximate locations of the
carbonyl oxygen and βꢀcarbon of the substrates, respectively (Scheme 1). In all cases, the
substrate positions were consistent with angle and distance values observed for efficient hydride
transfer from reduced FMN to Cβ,59 supporting the notion that these modeled structures might
mirror catalytically productive complexes. Key proteinꢀsubstrate distances were measured to
identify possible steric interactions that would influence the choice “normal” versus “flipped”
substrate binding.
Unfortunately, the modeled Michaelis complexes utterly failed to uncover any explanations
for the differences in stereopreferences between OYE 3 and OYE 1. In fact, our models based
on the xꢀray structures predicted (incorrectly) that both enzymes would give entirely consistent
outcomes, contradicting the experimental observations.
Because the active sites of OYE 3 and OYE 1 differ at position 296, and this residue lies on
mobile loop 6, we considered the possibility that these proteins might possess different
conformational and / or dynamic properties in solution that were not apparent in the xꢀray
structures. Each crystal structure was used as the starting point for MD simulations in the
absence of substrate. Over the course of the simulations, variations in loop 6 conformations
were observed; however, the average structures were wellꢀdefined (Figures 5A and 5B). While
both proteins showed approximately the same level of conformational mobility during the
simulations, the average structure of loop 6 was more closed in OYE 1 as compared to OYE 3.
This can be seen in the distance distributions between the Cα of residue 296 and the site of
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hydride addition (N5 of the FMN) (Figure 5C). In the starting xꢀray structures of OYE 3 and
OYE 1, these atoms are 10.1 and 9.9 Å apart, respectively. The MD simulation shows that in
solution, loop 6 of OYE 1 closes significantly since the Cα,296 – N5,FMN distance decreased to
approximately 7 Å. By contrast, loop 6 in OYE 3 failed to close over the active site – in fact, the
C
α,296 – N5,FMN distance actually increased slightly to ca. 11 Å (Figure 5D). The more open
conformation of loop 6 over the active site of OYE 3 likely means that it has negligible contact
with bound substrates, which would lessen its impact on stereoselectivity. Based on these
results, we anticipate that the significantly altered loop 6 conformation in OYE 1, which is
largely absent in OYE 3, is the major reason for the stereochemical differences that have been
observed between these two enzymes (Table 1).
In the crystal lattices, loop 6 in both OYE 3 and OYE 1 faces a solvent channel, and there are
no close contacts with symmetryꢀrelated protein molecules. Thus, the nearly identical loop 6
conformations observed in crystallo for these two proteins are not likely to be due to crystal
packing forces. As noted above, the electron density for loop 6 is very wellꢀdefined in both the
OYE 3 and OYE 1 structures and Bꢀfactors in this region are nearly the same as for other surface
loops. Only by carrying out MD simulations in solution did the conformational differences
become apparent.
That loop 6 is more closed over the active site in OYE 1 versus OYE 3 is also consistent with
the stereochemical behavior of the two enzymes. In general, OYE 1 provides greater chiral
discrimination, and changes elsewhere in the active site, e.g., position 116, had large impacts on
stereoselectivity. By contrast, the lessened ability of loop 6 to close over the OYE 3 active site
yields a more open cavity with multiple opportunities for nonꢀproductive enzymeꢀsubstrate
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complexes to form. Moreover, this means that contacts with other amino acid sideꢀchains will
have lessened ability to influence substrate binding, as observed in this study.
Finally, the results from circular permutation studies of OYE 1 by the Lutz group are also
consistent with our observations here.13,60 The largest increases in catalytic efficiency (≥ 10ꢀ
fold) occurred when the new termini dissected loop 6, which created an effectively “permanently
open” loop 6. Interestingly, this group also found that mutating Trp 116 in the most catalytically
efficient circular permutants had little impact on the stereoselectivity of (S)ꢀcarvone reduction,
reminiscent of our observations on OYE 3. In both cases, loop 6 occupies a more open
conformation than that found in wildꢀtype OYE 1.
Conclusions
The conformation of loop 6 in S. pastorianus OYE 1 likely has a different structure in
solution than that observed in its crystal structure. In solution, loop 6 of OYE 1 closes down
over the active site, allowing it to interact closely with bound substrates and exert significant
control over their binding orientations. By contrast, loop 6 in S. cerevisiae OYE 3 largely retains
its crystallographically observed conformation in solution. Because the loop does not close over
the active site of OYE 3, there are fewer protein – substrate contacts and substrate binding is less
impacted by residues on this loop and elsewhere in the active site, e.g., Trp 116. The divergent
conformational preferences of loop 6 in OYE 1 and OYE 3 in solution may explain their
differing stereochemical preferences toward some substrates, despite the two proteins sharing
80% sequence identity and nearly the same crystal structures.
Whether the loop 6 conformations of OYE 3 and / or OYE 1 can be further impacted by
additional mutations must await future experimental and computational studies that are currently
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underway. While in principle, mutating Ser 296 to Phe in OYE 3 might be expected to yield a
variant with properties identical to OYE 1, a number of other residues differ between OYE 3 and
OYE 1, and a single amino acid change may not be sufficient to effectly turn one protein into
another. Only by examining a complete set of mutations and molecular dynamics trajectories
will conclusions emerge. We also note that such altered structures might have very useful
impacts on stereoselectivity, particularly when combined with additional active site changes.
What has been clearly established by this work, however, is that static xꢀray structures – while
extremely useful – are not always sufficient to understand OYEꢀmediated alkene reductions.
Conflicts of Interest
There are no conflicts of interest to declare.
Acknowledgements
We are very grateful for financial support from the National Science Foundation (CHEꢀ
1111791 and CHEꢀ1705918). We would also like to thank Blue Waters (National Center for
Supercomputing Applications) and the High Performance Computing Center (University of
Florida) for providing computational resources.
Electronic Supplementary Information
Complete NMR spectra for ketones 3 and 4 along with their reduced products.
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Figure Legends
Figure 1. Novel alkene substrates for OYE 3 and OYE 1. A. Trisubstituted alkenes 3 and 4
were designed as analogs of ethyl ketone 2 to test the sensitivity of OYE 3 and OYE to altered
1
steric demands in the βꢀposition. B. H NMR spectrum of the racemic hydrogenated product
2
from enone 4. C. H NMR spectrum of the (4S,5R)ꢀd2 product obtained by OYE 3ꢀmediated
reduction of ketone 4 in D2O with NADPD (top) and the (4R,5S)ꢀd2 product obtained by OYE 1ꢀ
mediated reduction of ketone 4 in D2O with NADPD (bottom). The peaks labelled by an asterisk
arise from acetoneꢀd6 and isoꢀpropanolꢀd8. .D. Steric demands of trisubstituted alkene substrates
of OYE 3 and OYE 1. The designations “L” and “S” refer to large and small substituents,
respectively.
Figure 2. Alkene substrates used to profile wild-type and Trp 116 site-saturation mutants
of OYE 3.
Figure 3. OYE 3 crystal structure. Electron density (2mFo – DFc) is shown at the 1.5 σ level.
A. Active site structure of crystals soaked with pꢀhydroxybenzaldehye. The positions of key
residues are shown. B. Loop 6 region (residues 289 – 309) of unsoaked OYE 3 crystals. Weak
electron density was observed for the sideꢀchain of Glu 303. C. Loop 6 region of OYE 3
crystals soaked with pꢀhydroxybenzaldehyde. Weak electron density was observed beyond the
Cβ carbons for Glu 301, Glu 303 and Glu 306.
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Figure 4. Structural alignment of loop 6 regions for unsoaked OYE 3 crystals (purple),
OYE 3 crystals soaked with p-hydroxybenzaldehyde (green) and OYE 1 crystals soaked
with p-hydroxybenzaldehyde (red). Distances from the Cα of residue 296 to N5 of FMN are ca.
10 Å in all cases.
Figure 5. MD simulation results for OYE 3 and OYE 1. A. Overlaid snapshots from a 100
ns simulation of OYE 3. Loop 6 is colored red and is the only portion of the protein structure
that undergoes significant movement. B. Overlaid snapshots from a 100 ns simulation of OYE 1
in the absence of substrate. Loop 6 is colored red. C. Distances between the Cα of residue 296
and N5 of FMN measured during MD simulations of OYE 3 (blue) and OYE 1 (red) in the
absence of substrate. D. Overlaid protein structures; only the loop 6 regions are shown: OYE 3
xꢀray structure (green), OYE 3 average coordinates after MD simulation (blue), OYE 1 xꢀray
structure (red), OYE 1 averaged coordinates after MD simulation (orange).
27

Catalysis Science & Technology
Page 28 of 43
View Article Online
DOI: 10.1039/C8CY00440D
Table 1. Alkenes reduced by S. pastorianus OYE 1 and OYE 3 with differing
stereoselectivities. All substrates are shown in the “normal” orientation and the phenol inhibitor
skeleton is indicated by dashed grey lines. Products derived from a “flipped” substrate binding
orientation are shown in red.
OYE 1
% ee
OYE 3
% ee
Entry
Substrate
Ref.
75% (R)
60% (S)a
59% (R)
59% (S)
68% (R)
89% (S)
61
1
62
30
2
3
20% (R)
60% (R)
95% (S)
81% (S)
30
30
4
5
aThe indicated binding orientations presume that the activating ester group was the same for both
OYE 1 and OYE 3; however, a change in activating ester group would also be consistent with
the observed data.
28

Page 29 of 43
Catalysis Science & Technology
View Article Online
DOI: 10.1039/C8CY00440D
Table 2. Reductions of trisubstituted olefins by OYE 3 and OYE 1. Reactions that yield
products from “flipped” substrate binding orientations are shown in red, and key results are
highlighted by grey shading. In entry the first number refers to fractional conversion and the
second to % ee.
%c a, % ee b
Amino
acid at
position
116
OYE 3
OYE 1
>98%, 44% (R)
37%, 82% (S)
40%, 70% (S)
---
OYE 3
OYE 1
>98%, 78% (S)
50%, 70% (S)
>98%, 56% (R)
W (wt)
82%, >99% (S)
---c
5%, 58% (S)
A
C
D
E
F
32%, 96% (S)
---
---
---
---
---
---
---
ꢀꢀꢀ
ꢀꢀꢀ
58%, >98% (R)
---
32%, 14% (R)
16%, 65% (R)
---
---
---
---
G
H
I
---
9%, 40% (R)
59%, 0%
---
8%, 80% (R)
64%, 50% (R)
---
---
---
---
---
>98%, 60% (S)
76%, 36% (S)
35%, 74% (S)
---
---
K
L
>98%, 50% (R)
57%, 67% (R)
33%, 34% (R)
9%, 88% (R)
20%, 82% (R)
---
45%, 0%
47%, 0%
10%, 46% (S)
---
14%, 78% (R)
78%, >99% (R)
11%, 64% (R)
---
M
N
P
Q
R
S
T
---
---
18%, >99% (R)
---
---
---
---
---
---
---
82%, >99% (S)
20%, >99% (S)
25%, 76% (S)
9%, 67% (S)
>98%, 86% (S)
11%, 78% (R)
6%, 99% (S)
31%, 96% (S)
30%, 4% (R)
---
>98%, 54% (S)
>98%, 60% (R)
V
Y
a Conversions were calculated on the basis of GC analysis of the crude mixture after 24 h
reaction time (three replicates).
b Optical purities were calculated from chiralꢀphase GC analysis (three replicates).
c≤ 5% substrate conversion after 24 hr was observed.
29