Breaking Symmetry: Engineering Single-Chain Dimeric Streptavidin as Host for Artificial Metalloenzymes

Article abstract of DOI:10.1021/jacs.9b06923

The biotin-streptavidin technology has been extensively exploited to engineer artificial metalloenzymes (ArMs) that catalyze a dozen different reactions. Despite its versatility, the homotetrameric nature of streptavidin (Sav) and the noncooperative binding of biotinylated cofactors impose two limitations on the genetic optimization of ArMs: (i) point mutations are reflected in all four subunits of Sav, and (ii) the noncooperative binding of biotinylated cofactors to Sav may lead to an erosion in the catalytic performance, depending on the cofactor:biotin-binding site ratio. To address these challenges, we report on our efforts to engineer a (monovalent) single-chain dimeric streptavidin (scdSav) as scaffold for Sav-based ArMs. The versatility of scdSav as host protein is highlighted for the asymmetric transfer hydrogenation of prochiral imines using [Cp*Ir(biot-p-L)Cl] as cofactor. By capitalizing on a more precise genetic fine-tuning of the biotin-binding vestibule, unrivaled levels of activity and selectivity were achieved for the reduction of challenging prochiral imines. Comparison of the saturation kinetic data and X-ray structures of [Cp*Ir(biot-p-L)Cl]·scdSav with a structurally related [Cp*Ir(biot-p-L)Cl]·monovalent scdSav highlights the advantages of the presence of a single biotinylated cofactor precisely localized within the biotin-binding vestibule of the monovalent scdSav. The practicality of scdSav-based ArMs was illustrated for the reduction of the salsolidine precursor (500 mM) to afford (R)-salsolidine in 90% ee and >17 ?000 TONs. Monovalent scdSav thus provides a versatile scaffold to evolve more efficient ArMs for in vivo catalysis and large-scale applications.

Full text of DOI:10.1021/jacs.9b06923

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Article
Breaking Symmetry: Engineering Single-Chain Dimeric
Streptavidin as Host for Artificial Metalloenzymes
Shuke Wu, Yi Zhou, Johannes G. Rebelein, Miriam Kuhn, Hendrik
Mallin, Jingming Zhao, Nico V. Igareta, and Thomas R. Ward
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b06923 • Publication Date (Web): 11 Sep 2019
Downloaded from pubs.acs.org on September 12, 2019
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Breaking Symmetry: Engineering Single-Chain
Dimeric Streptavidin as Host for Artificial
Metalloenzymes
Shuke Wu,‡ Yi Zhou,‡ Johannes G. Rebelein,‡ Miriam Kuhn, Hendrik Mallin, Jingming Zhao,
Nico V. Igareta, and Thomas R. Ward*
Department of Chemistry, University of Basel, BPR 1096, Mattenstrasse 24a, CH-4058 Basel,
Switzerland
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ABSTRACT
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The biotin-streptavidin technology has been extensively exploited to engineer artificial
metalloenzymes (ArMs) that catalyze a dozen different reactions. Despite its versatility, the
homotetrameric nature of streptavidin (Sav) and the non-cooperative binding of biotinylated
cofactors, impose two limitations on the genetic optimization of ArMs: i) point mutations are
reflected in all four subunits of Sav and ii) the non-cooperative binding of biotinylated cofactors
to Sav may lead to an erosion in the catalytic performance, depending on the cofactor: biotin-
binding site ratio. To address these challenges, we report on our efforts to engineer a (monovalent)
single-chain dimeric streptavidin (scdSav) as scaffold for Sav-based ArMs. The versatility of
scdSav as host protein is highlighted for the asymmetric transfer hydrogenation of prochiral imines
using [Cp*Ir(biot-p-L)Cl] as cofactor. By capitalizing on a more precise genetic fine-tuning of the
biotin-binding vestibule, unrivaled levels of activity and selectivity were achieved for the reduction
of challenging prochiral imines. Comparison of the saturation kinetic data and X-ray structures of
[Cp*Ir(biot-p-L)Cl] · scdSav with a structurally related [Cp*Ir(biot-p-L)Cl] · monovalent scdSav,
highlights the advantages of the presence of a single biotinylated cofactor precisely localized
within the biotin-binding vestibule of the monovalent scdSav. The practicality of ScdSav-based
ArMs was illustrated for the reduction of the salsolidine precursor (500 mM) to afford (R)-
salsolidine in 90% ee and > 17'000 TONs. Monovalent scdSav thus provides a versatile scaffold
to evolve more efficient ArMs for in vivo catalysis and large-scale applications.
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INTRODUCTION
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Artificial metalloenzymes (ArMs) are hybrid catalysts combining attractive features of
organometallic catalysts (e.g., broad range of catalyzed reactions) and enzymes (e.g., genetically
evolvable and compatible with a cellular environment).1−12 They are often created by incorporation
of a synthetic metallocofactor within a protein scaffold. Thanks to the progress in biotechnology,
several scaffolds have proven versatile for the assembly and optimization of ArMs. These include,
among others, hemeproteins,13−16 prolyl oligopeptidase,17,18 transcriptional repressor LmrR,19,20
carbonic anhydrase,21,22 and streptavidin (Sav).23,24 Due to the high affinity of biotin for
(strept)avidin,25,26 this anchoring strategy had been exploited by several groups to produce ArMs
that catalyze over a dozen reactions.27−36 As pioneered by Whitesides, avidin (Avi) isolated from
egg-white was initially selected. For recombinant purposes however, streptavidin (Sav) is
preferred thanks to the following features: i) it can readily be expressed in high yields in E. coli,37
ii) it is less prone to aggregation and iii) its isoelectric point pISav = 6.4 compared pIAvi = 10.4.
Both Avi and Sav are homotetrameric proteins consisting of a dimer of dimer, where the two
neighboring subunits are related by a C2 axis. In the past fifteen years, more than twenty X-ray
structures of Sav-based ArMs have been reported.9,24,31–34 Inspection of the biotin-binding
vestibule, where the catalytic moiety resides, reveals a narrow distribution of biotinylated metals
in the immediate proximity of the C2 axis relating the two Sav monomers that make up the biotin-
binding vestibule, Figure 1a. Importantly, any point mutation is reflected in all four subunits of
homotetrameric Sav, Figure 1b. This symmetry-relationship imposes a significant limitation for
the genetic optimization of Sav-based ArMs. Indeed, inspection of the biotin-binding vestibule
(Figure 1a) highlights the proximity of biotinylated cofactors to the two symmetry-related S112
and K121 residues. Accordingly, any mutation of these residues is reflected in both Sav monomers
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(designated SavA and SavB hereafter), thus challenging a precise genetic fine-tuning of ArM's
performance: it is hard to decipher which of the two symmetry-related mutations (i.e., SavA or
SavB) affects (most) the catalytic performance.
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Figure 1. (a) Close-up view of the metal distribution of ArMs based on Sav. Homotetrameric Sav
is displayed as surface, residues S112 and K121 of adjacent Sav monomers forming the biotin-
binding vestibule are shown as sticks and labeled; the metals from the biotinylated cofactors are
represented by spheres: Ir (yellow, PDB codes 6GMI, 6ESS, 6ESU, 4OKA, and 3PK2), Ru
(raspberry, PDB codes 6FH8, 5F2B, 5IRA, 2QCB, and 2WPU), Cu (blue, PDB codes 5VKX,
5VL5, 5VL8, 5WBA, 5WBB, 5WBD, 6ANX, 5WBC, 5K67, 5K68, and 5L3Y), Pd (green, PDB
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code 5CSE) and Rh (orange, PDB codes 4GJV and 4GJS). For clarity, only the metal ions present
in the cofactors are shown (spheres, with only one metal per SAV dimer displayed). (b) Cartoon
representation of an ArM based on homotetrameric Sav with symmetry-related S112 and K121 in
the proximity of the biotinylated cofactor. (c) Close-up view of the X-ray structure of [Cp*Ir(biot-
p-L)Cl] ∙ Sav S112A (PDB code 3PK2) with all biotin-binding sites occupied. As can be
appreciated, severe steric clashes prevent the localization of adjacent cofactors to occupy the same
orientation.31 The homotetrameric Sav is displayed in surface representation and colored by protein
chains. The cofactors are represented as stick models and the Ir-atom as orange sphere; nitrogen,
blue; oxygen, red; sulfur, yellow; chloride, green. (d) Michaelis-Menten kinetics of [Cp*Ir(biot-p-
L)Cl] ∙ scdSav highlighting the marked influence of the cofactor loading on both the activity and
the stereoselectivity of the corresponding ATHase.33 The initial rates are displayed with respect to
the concentration of iridium. [Sav] = 25 μM (corresponding to [biotin-binding sites] = 100 μM)
was held constant, varying the Ir/Sav ratio from 1.0 (blue data points) to 2.0 (red data points), 3.0
(green data points), and 4.0 (magenta data points). For comparison, the initial rates for the free
[Cp*Ir(Biot-p-L)Cl] catalyst are displayed (50 μM, black data points). The positive ee values
correspond to (R)-salsolidine. The black solid lines correspond to the fit obtained using the
Michaelis−Menten or the Haldane equation. Reprinted in part with permission from ref 33.
Copyright 2014 ACS. (e) Design of scdSav relying on the linking of SavA and SavB via a peptide
linker (red line) and introducing a disulfide bond between two scdSav (yellow line).
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An additional challenge concerns the catalytic performance as a function of cofactor
occupancy within Sav. Indeed, some ArMs display markedly different catalytic performance as a
function of cofactor : Sav ratio. Upon varying the [Cp*Ir(biot-p-L)Cl] : Sav S112A ratio from one
to four, the enantioselectivity of asymmetric transfer hydrogenase towards the salsolidine
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precursor 1a erodes from 93 % ee (R)-2a to 45 % ee (R)-2a, Figure 1c-d. In stark contrast, for
[Cp*Ir(biot-p-L)Cl] : Sav S112K, the enantioselectivity remains by-and-large constant,
irrespective of the above ratio (e.g., 70 % ee (S)-2a vs. 78 % ee (S)-2a upon varying the ratio from
one to four).33 The complications are exacerbated by the non-cooperative binding of biotinylated
cofactors to Sav: adding two equivalents of biotinylated cofactor vs. homotetrameric Sav affords
a Poisson distribution of cofactor occupancy.38 With in vivo catalysis in mind, whereby the [Sav]
is unknown and highly variable, it is desirable to achieve a precise control of ArM's performance,
irrespective of the Sav : cofactor ratio.39−41 With these goals in mind, we present herein our efforts
towards the engineering of a single chain dimeric streptavidin (scdSav hereafter), enabling the
independent mutagenesis of the two neighboring Sav subunits.
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RESULTS AND DISCUSSION
Design of scdSav. Circular permutation strategies have been applied to fuse two subunits
of avidin while maintaining high biotin-binding affinity.42,43 Circularly-permutated Sav has also
been reported but with a significantly-reduced affinity to biotin.44 Initially, we evaluated a similar
approach to engineer circularly-permutated scdSav. However, these constructs expressed poorly
in E. coli (< 1 mg L-1 in culture medium), hampering thorough characterization and practical
implementation in biotechnology. We thus set out to fuse the C-terminus of SavA with the N-
terminus of SavB, Figure 1e. Inspection of the X-ray structure of mature Sav (PDB code 2BC3)45
reveals that the C- and N-termini are remote from each other in the quaternary structure. To
connect two Sav subunits, a 26 amino acid linker was thus introduced. In addition, the following
points were taken into consideration in the design of scdSav: i) the DNA sequence homology of
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the two linked-Sav subunits was minimized to facilitate mutagenesis of each Sav subunit
independently; ii) As the H127 residue forms a π-stacking interaction with its neighboring H127
residue, it was mutated to H127C in the SavB to favor the formation of a disulfide bond with an
adjacent scdSav. This ensures the precise assembly of two scdSavs into a single quaternary
structure (i.e., (scdSav)2) bearing one disulfide bond and one π-π stacking interaction between the
two remaining histidines H127 present in the (scdSav)2 quaternary structure, Figure 1e.43,46 With
these considerations in mind, scdSav was engineered using a 26aa linker between SavA and SavB,
with the H127C mutation in the SavB.
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To evaluate the suitability of the scdSav design in the context of ArMs, we selected the
asymmetric transfer hydrogenation of prochiral imines using [Cp*Ir(biot-p-L)Cl] as a test bed
(ATHase hereafter). In this context, the nature of the amino acid at position 112 and 121 was
repeatedly shown to significantly affect the ATHase performance, both in terms of activity and
selectivity.31,33,47,48 In contrast to other ArMs based on the biotin-streptavidin technology,9,23,24 the
catalytic performance of the evolved ATHases was shown to critically depend on the
cofactor/biotin-binding site ratio, Figure 1c-d. Building on this knowledge, we engineered 33
scdSavs with mutations at both of the S112 and K121 positions. The two S112 positions were
independently mutated to either alanine, lysine or arginine. The two K121 were independently
mutated to an alanine residue or kept as a lysine. To simplify the labeling of these variants, a 4-
letter code is used hereafter: the first 2 letters code for residues 112 and 121 within the SavA and
the last 2 letters code for residues 112 and 121 in the SavB, respectively. Accordingly,
scdSav(SKSK) represents the “wild type” scdSav, which includes the 26 aa linker and the H127C
mutation in the SavB, Figure 1e.
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The scdSav(SASK) gene was synthesized and introduced on the pRSFduet-1 plasmid. The
other 32 scdSavs were generated by site-directed mutagenesis using the above plasmid. All the 33
scdSavs were overexpressed in E. coli BL21(DE3) using an autoinduction medium and purified
by affinity chromatography on an iminobiotin-sepharose matrix (see SI Figure S1 for SDS-PAGE
analysis, Table S8 for MS data). For all 33 scdSav variants, 10-50 mg of purified protein was
obtained from 1 liter of culture medium. SDS-PAGE analysis of the purified scdSavs revealed the
formation of (scdSav)2 (= tetramer of Sav) as the major component, with higher oligomers of
scdSav as the minor byproducts (Figure S1). Importantly, all scdSavs maintain their "four
equivalents" binding capacity towards biotin-4-fluorescein (B4F).
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ATHase Performance of ArMs based on scdSav. Five prochiral imines 1a-e were
selected to evaluate the catalytic performance of the engineered [Cp*Ir(biot-p-L)Cl] ∙ scdSav.
Three isoquinoline derivatives (1a-1c), a 2H-pyrrole derivative (1d) and quinaldine (1e) were
selected as the substrates for the reduction, Scheme 1. With the exception of isoquinoline 1a, these
substrates have proven challenging to reduce enantioselectively with ATHases. The results of the
ATHase screening for substrates 1a-e are summarized in Scheme 1 (See also Table S1). While the
free cofactor [Cp*Ir(biot-p-L)Cl] affords the corresponding racemic amines 2a-e in low
conversion (1-18 %), incorporation within WT Sav leads to low to moderate conversion (9-68 %)
accompanied with modest ees (30-79 %). Upon substituting WT Sav by scdSav(SKSK), the
resulting ATHase activity is by-and-large remains, both in terms of conversion (14-70 %) and
enantioselectivity (42-79 %). Mutagenesis at positions 112 and 121 in both SavA and SavB
markedly altered the catalytic performance. To identify important trends, the results were grouped
and analyzed according to the amino acids at each specific position (Figure S2). For residue 112
in SavA, Ser and Ala clearly outperformed Lys and Arg in terms of conversion for all substrates
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(Figure S2a). The 112S/A in SavA is also more (R)-enantioselective for isoquinoline derivatives
(1a-1c) and (S)-enantioselective for 1e (Figure S2b). For residue 121 in SavA, Ala generally
improves the activity while largely maintains the enantioselectivity for 1a-1c and 1e (Figure S2c,
S2d). The general effects of 112S/A and 121A in SavA suggest that they are contributing to the
binding of [Cp*Ir(biot-p-L)Cl] in a more productive conformation, in line with a previous
computational study of ATHase based on Sav.33 On the other hand, no general trends could be
deduced for residue 112 and 121 in SavB (Figure S2e-2h). The substrate- and context-specific
effects of 112 and 121 in SavB suggest that they may contribute to interactions with the substrate
and thus fine-tune the activity and enantioselectivity. The best ATHases for each substrate are
discussed below. [Cp*Ir(biot-p-L)Cl] ∙ scdSav(SARK) reduced imine 1a to amine (R)-2a in 100%
conversion (400 TON) and 96% ee, and imine 1b to amine (R)-2b in 99% conversion (398 TON)
and 93% ee, respectively. In comparison with the ATHases based on homotetrameric Sav,
scdSav(SARK) is similar with the benchmark Sav S112A for (R)-2a (full conversion and 96% ee
at 5 °C).31 For amine (R)-2b, scdSav(SARK) is markedly more enantioselective than the
benchmark SavS112T (full conversion and 59% ee).49 Clearly, the fine-tuned scdSav(SARK)
bearing 112S and 121A in SavA and 112R and 121K in SavB outperforms all the homotetrameric
Savs tested to date. For the reduction of the bulky substrate 1c, none of the scdSavs exhibited high
stereoselectivity (i.e., ee < 70%). For the reduction of imine 1d, [Cp*Ir(biot-p-L)Cl] ∙
scdSav(SKAA) produced amine (R)-2d in 90% conversion (360 TON) and 98% ee, outperforming
the best ATHase with homotetrameric Sav (68% conversion and 77% ee with WT Sav). Such high
enantioselectivity (i.e. ee ≥ 95%) is challenging to achieve with ArMs. Again, scdSav(SKAA)
bearing 112S and 121K in SavA and 112A and 121A in SavB outperforms all homotetrameric Sav
tested to date. Quinaldine (1e) was only moderately reduced (conversion < 50%) by the ATHases
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under the experimental conditions. Nevertheless, chiral amine (S)-2e was produced in 91% ee with
[Cp*Ir(biot-p-L)Cl] ∙ scdSav(SARK). In recent years, a number of imine reductase enzymes
(IREDs) have been discovered and applied to reduce many cyclic imines to chiral amines with
high enantioselectivity.50-52 More recently, several IREDs are found to catalyze reductive
amination,53-55 i.e., first formation of imines and then reduction to amines, a breakthrough in the
enzymatic generation of amines. ATHases have widely been used as a testbed for the development
of ArMs,12,31,33,41,48,49 In comparison to evolved IREDS recently reported, ATHases typically
display more modest catalytic performances.
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Scheme 1. Asymmetric transfer hydrogenation of imines 1a-1e in the presence of [Cp*Ir(biot-p-
L)Cl] ∙ scdSav.a
R1
R1
R1
R1
[Cp*Ir(biot-p-L)Cl] 25 M
scdSav 50 M or Sav 100 M
NH
N
O
*
R2
R2
1a-1c
HN
H
2a-2c
NH
MOPS 0.6M, pH = 7
HCO2Na 3M, 25 oC, 24 h
H
H
N
S
*
Ir
N
H
N
Cl
S
NH2
O
1a 2a
,
: R1 = OMe, R2 = Me
: R1 = H, R2 = Me
: R1 = H, R2 = Ph
N
1b 2b
2d
,
1d
O
O
1c 2c
,
[Cp*Ir(biot-p-L)Cl]
*
N
H
N
1e
10 mM
2e
rac
0
ee (R) [%]
ee (S) [%]
Conversion (c) [%]:
60
40
10
40
60
>90
>90
-60
-90
20
80
80
20
-40
-80
-20
Protein
1a
1b
1c
1d
1e
Protein
1a
1b
1c
1d
1e
Protein
1a
1b
1c
1d
1e
scdSav(SKAA)
scdSav(AKAA)
scdSav(RKSA)
scdSav(SKRA)
scdSav(RKRA)
scdSav(SASA)
scdSav(AASA)
scdSav(SAAA)
scdSav(AAAA)
scdSav(RASA)
scdSav(RARA)
No prot.
scdSav(SASK)
scdSav(AASK)
scdSav(SAAK)
scdSav(AAAK)
scdSav(RASK)
scdSav(SARK)
scdSav(RARK)
scdSav(KASK)
scdSav(SAKK)
scdSav(KAKK)
scdSav(SKSA)
scdSav(AKSA)
Sav
scdSav(SKSK)
scdSav(AKSK)
scdSav(SKAK)
scdSav(AKAK)
scdSav(RKSK)
scdSav(SKRK)
scdSav(RKRK)
scdSav(KKSK)
scdSav(SKKK)
scdSav(KKKK)
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a
The size of the disk is proportional to the conversion and its color codes for the ee of the
corresponding product. Reaction conditions: 10 mM substrate, 25 μM [Cp*Ir(biot-p-L)Cl], 50 μM
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scdSav (= 100 μM biotin-binding sites), 0.6 M MOPS, 3 M formate, pH 7.0, 25 °C, 24 h.
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Engineering a Monovalent scdSav. In light of the high activity and enantioselectivity for
the imine reduction, we selected the double mutant scdSav(SARK) to engineer a monovalent
scdSav to investigate the influence of a neighboring cofactor on the catalytic performance of
ATHases based on scdSav. Binding of a second biotinylated cofactor in the neighboring Sav
subunit often leads to substantial erosion in rate and enantioselectivity.33 To determine the biotin-
binding stoichiometry of scdSav, a biotin-4-fluorescein (B4F hereafter) titration was performed,
relying on the intersection of the two linear segments of the titration curve to determine the binding
stoichiometry.56 scdSav(SASK) and scdSav(SARK) bind 1.9 ± 0.1 and 2.0 ± 0.1 B4F per scdSav
respectively (Figure S3a, S3b). To generate a monovalent scdSav, while maintaining the overall
topology within the biotin-binding vestibule, two reported sets of mutations were evaluated to
knockout the biotin-binding capacity in SavB. The following mutants were produced:
scdSav(SARK) with additional N23A/S27D in SavB (scdSav(SARK)mv1 hereafter) and
scdSav(SARK) with additional N23A/S27D/D128A in SavB (scdSav(SARK)mv2 hereafter). The
N23A/S27D and the D128A mutants have been reported to disrupt key H-bonding interactions
between Sav and the ureido oxygen and ureido nitrogen by Cantor57 and Salemme,58 respectively.
Gratifyingly, BF4 titration of the resulting scdSav(SARK)mv1 and scdSav(SARK)mv2 revealed
the monovalent character of these constructs: 1.1 ± 0.1 and 1.2 ± 0.1 biotin-binding sites were
determined for scdSav(SARK)mv1 and scdSav(SARK)mv2, respectively (Figure S3c, S3d).
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Saturation Kinetics of ATHases Based on scdSav. To gain further insight into the
catalytic efficiency of ATHases based on scdSav, we determined the saturation kinetics and
enantioselectivity for the reduction of imine 1a to chiral amine (R)-2a by [Cp*Ir(biot-p-L)Cl] ∙
scdSav(SARK) and its two monovalent variants with varying Ir : scdSav ratios. For all
measurements, the concentration of scdSav was kept at 50 μM. Whereas, 25, 50, 75, 100 μM
[Cp*Ir(biot-p-L)Cl] were used for scdSav(SARK) (Ir : scdSav ratio = 0.5, 1.0, 1.5, 2.0
respectively), and 25, 50 μM [Cp*Ir(biot-p-L)Cl] were used for the monovalent Savs
scdSav(SARK)mv1 and scdSav(SARK)mv2 (Ir : scdSav ratio = 0.5, 1.0 respectively). As
summarized in Figure 2 and Table 1, all the ATHases gave rise to high enantioselectivities and
pronounced rate-accelerations to afford up to 96 % ee (R)-2a and up to a tenfold higher kcat
compared to the free cofactor [Cp*Ir(biot-p-L)Cl]. For scdSav(SARK), capable of binding up to
two biotinylated cofactors per scdSav, the reaction rate (kcat) markedly decreased and the
enantioselectivity was moderately reduced upon increasing the Ir : scdSav beyond one. The erosion
of rate and enantioselectivity are in accordance with a previous study of [Cp*Ir(biot-p-L)Cl] ∙ Sav
S112A,33 suggesting that the binding of an additional [Cp*Ir(biot-p-L)Cl] in the neighboring Sav
subunit may cause a significant conformational change of the existing cofactor into a catalytically
less favorable conformation.33 Because scdSav(SARK)mv1 and scdSav(SARK)mv2 are
monovalent, the biotinylated cofactor cannot bind to the second Sav subunit. Thus, no detrimental
effects were observed upon increasing the cofactor to protein ratio up to full saturation. In addition,
when Ir : scdSav ≤ 1, a higher reaction rate was observed for monovalent scdSav (kcat = 16.0-23.6
min-1) than that of divalent scdSav (kcat = 10.0-12.5 min-1) and previous homotetrameric Sav
S112A (kcat = 11.4-14.1 min-1).33 The superiority of monovalent scdSav may be traced back to the
fact all the bound cofactor resides in a catalytically active conformation for the monovalent scdSav.
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In contrast and in light of the non-cooperative binding of the cofactor within Sav, even at Ir :
scdSav ≤ 1, a portion of the cofactor [Cp*Ir(biot-p-L)Cl] binds in an unproductive conformation,
leading to an erosion in rate.
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Figure 2. Saturation kinetic data for the reduction of imine 1a with [Cp*Ir(biot-p-L)Cl] ∙
scdSav(SARK), scdSav(SARK)mv1 and scdSav(SARK)mv2. The reaction rates are normalized
to the concentration of [Cp*Ir(biot-p-L)Cl]. The [scdSav] was set at 50 μM and [Cp*Ir(biot-p-
L)Cl] was varied: 25 μM (blue circles), 50 μM (red squares), 75 μM (green triangles), 100 μM
(purple triangles). For comparison, the kinetic profile of the free cofactor [Cp*Ir(biot-p-L)Cl] is
displayed (50 μM, black crosses). Solid lines correspond to the fitting to the Haldane equation
(ATHases) or Michaelis-Menten equation (free cofactor). Error bars represent ± standard
deviation.
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Table 1. Saturation kinetic parameters and enantioselectivity for the reduction of imine 1a with
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6
ATHases based on scdSav.a
7
8
9
[Cp*Ir(biot-p-L)Cl]
(R)-2a
ee (%)b
1
kcat
Km
Ki
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Protein
(μM)
50
(min-1)c
(mM)
(mM)
No protein
1.3 ± 0.1
10.0 ± 0.8
12.5 ± 1.2
4.6 ± 0.6
2.2 ± 0.3
16.0 ± 1.7
19.8 ± 2.9
23.6 ± 3.0
21.0 ± 2.7
57 ± 20
20 ± 5
32 ± 7
23 ± 8
38 ± 13
31 ± 7
36 ± 11
52 ± 12
42 ± 11
n.a.d
scdSav(SARK)
25
94
687 ± 189
359 ± 79
474 ± 158
614 ± 285
333 ± 77
294 ± 89
298 ± 74
513 ± 168
scdSav(SARK)
50
95
scdSav(SARK)
75
87
scdSav(SARK)
100
25
74
scdSav(SARK)mv1
scdSav(SARK)mv1
scdSav(SARK)mv2
scdSav(SARK)mv2
95
50
96
25
94
50
95
aThe reaction conditions are described in Figure 2. The kinetic parameters were obtained using the
Michaelis-Menten and Haldane equations for the free cofactor and the ATHases respectively. bThe
ee values were determined after 48 hours with [1a] = 25 mM. cThe kcat values are normalized to
d
the concentration of [Cp*Ir(biot-p-L)Cl]. Not-available. Error margins represent ± standard
deviation resulting from two independent experiments.
Structural Characterization of scdSav ArMs by X-ray Crystallography. To gain structural
insight into the ATHases based on the single chain dimeric streptavidin constructs, both
scdSav(SASK) and scdSav(SARK)mv2 were crystallized by sitting drop vapor diffusion (2.5 µL
26 mg/mL (scdSav)2 in 10 mM sodium phosphate buffer with 150 mM NaCl, pH 7 was mixed
with precipitation buffer 2.5 µL, 2 M (NH4)2SO4, 0.1 M Na-Acetate, pH 4). The resulting apo
crystals were soaked with excess [Cp*Ir(biot-p-L)Cl] (dissolved in DMSO). The soaked yellow
crystals were cryo-protected with 25 % glycerol and flash frozen in liquid nitrogen. The structure
was solved by molecular replacement using the PDB structure 3PK2 as a molecular model.
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Residual electron density in the Fo-Fc map was observed in the biotin-binding pocket and in the
biotin-binding vestibule. Anomalous dispersion density was observed in the biotin-binding
vestibule. Modeling of cofactor [Cp*Ir(biot-p-L)Cl] into the electron density projected the iridium
in the position of the anomalous density peak. The X-ray structures reveal, as anticipated, that
[Cp*Ir(biot-p-L)Cl] ∙ scdSav(SASK) and [Cp*Ir(biot-p-L)Cl] ∙ scdSav(SARK)mv2 differ in their
Ir : scdSav ratios. For the biological dimer of scdSav(SASK) (i.e. scdSav(SASK)2), both biotin-
binding sites present in the single chain dimer are occupied (i.e., amounting to four cofactors for
the functional dimeric assembly (scdSav)2), Figure 3a. In stark contrast, the biological dimer of
monovalent scdSav(SARK)mv2 (i.e. scdSav(SARK)mv2)2 contains only one [Cp*Ir(biot-p-L)Cl]
cofactor per single chain dimer (i.e., two cofactors for the functional dimeric assembly of
(scdSav)2), Figure 3b (See Figure S18 for the anomalous electron density of the iridium atoms).
To accommodate the two cofactors within the biotin-vestibule of scdSav(SASK), the neighboring
cofactors are forced to adopt two different confirmations CI and CII, each with a 50 % occupancy
and both in the (SIr)- configuration with a Cl- coordinated to Ir. Severe steric clashes prevent
neighboring cofactors of occupying two CI conformations simultaneously, Figure 3a. For
[Cp*Ir(biot-p-L)Cl] ∙ scdSav(SARK)mv2, only conformation CI is observed with 100%
occupancy with the (SIr)-configuration with a Cl- coordinated to Ir (see Figure S19 for an overlay
of the cofactors bound to scdSav(SASK) and scdSav(SARK)mv2 and the 2Fo-Fc electron density
map of the cofactor). The RMSD of the [Cp*Ir(biot-p-L)Cl] cofactor in conformation CI bound to
scdSav(SASK) and scdSav(SARK)mv2 is 0.576 Å (Table S7), suggesting that this is the same
conformation. Accordingly, we hypothesize that the first cofactor per scdSav adopts conformation
CI, for both scdSav(SASK) and scdSav(SARK)mv2. The second cofactor is forced to adopt
conformation CII. Conformation CI of cofactor [Cp*Ir(biot-p-L)Cl] observed for scdSav is similar
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to the binding mode to monomeric streptavidin Sav S112A (PDB: 3PK2)31 and Sav S112K (PDB:
4OKA)33 with an RMSD of 0.99-1.44 Å (See Table S7 and Figure S20). Interestingly, the biggest
RMSD deviation (1.51 Å, Table S7 and Figure S20) is observed for conformation CII of cofactor
[Cp*Ir(biot-p-L)Cl] in scdSav(SASK) compared to Sav S112K. This suggests that CII resides in
a shallower potential energy well, provided by the protein, than conformation CI.
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As can be appreciated in Figure 3a, the protein environment surrounding the two cofactor
conformations CI and CII for scdSav(SASK) is markedly different (See also Figure S21 for the
stereo view of the environment). As predicted by Maréchal and coworkers, the solvent-exposed
CII conformation leads to a decrease in activity and enantioselectivity, see Figure 2.33,59 As the
catalytically less efficient conformation CII is not observed in [Cp*Ir(biot-p-L)Cl] ∙
scdSav(SARK)mv2, the corresponding ATHase leads to higher TONs and enantioselectivity for
most substrates, Table 2. As modelled for Sav S112A- and Sav S112K-derived ATHases, we
speculate that the enantioselectivity is mainly determined by the absolute configuration at Ir.33,59
Accordingly, we believe that, as both CI and CII conformations have the same absolute
configuration at Ir (i.e., SIr with a Cl- coordinated to Ir), these preferentially lead to the same
enantioenriched amine, albeit with different levels of enantioselectivity.
Although the DNA sequence homology of the fused subunits was minimized to simplify
mutagenesis efforts, the amino acid sequence of both subunits is nearly identical: the size, the fold
and the corresponding electron density observed for each subunit of the scdSav are very similar.
Accordingly, in the crystal packing, each tetramer can occupy two orientations leading to a mixed
electron density. Nonetheless, we observed a preferential orientation of scdSav(SASK) and
scdSav(SARK)mv2 in the crystals resulting in differences for the electron density of
N23A/S27D/H127C/D128A and the cofactor binding of scdSav(SARK)mv2. We thus assigned
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the amino acid sequence according to the dominant electron density observed. The electron density
map (2Fo-Fc) of the H127 and C311 stabilizing the interaction of the homodimeric
(scdSav(SARK)mv2)2 is shown in Figure 3c, d. The statistical distribution of the tetramer
orientation and the resulting mixed structure is depicted in Figure 3e. The 26 amino acid linker
between the two subunits could not be modeled as no electron density was observed for these
amino acids.
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Figure 3. Structural characterization of scdSav(SASK) (a, PDB: 6S4Q) and scdSav(SARK)mv2
(b, PDB: 6S50) with bound [Cp*Ir(biot-p-L)Cl] cofactor. The scdSav proteins are displayed as
surface representation and colored by protein chains. The cofactors are represented as stick models
and the Ir-atom as orange sphere; nitrogen, blue; oxygen, red; sulfur, yellow. See Figure S21 for
stereo view. (a) The two cofactor conformations (CI and CII) of [Cp*Ir(biot-p-L)Cl] bound to each
binding site are depicted. Both conformations CI and CII are occupied at 50%. Selected distances
between the two cofactors are highlighted in yellow dashes. (b) For monovalent
scdSav(SARK)mv2, only one binding site per (scdSav)2 is occupied by [Cp*Ir(biot-p-L)Cl]. (c, d)
The electron density maps (2Fo-Fc at 1  of the residues stabilizing the interactions of a
homodimeric (scdSav(SARK)mv2)2; (c) the disulfide bond formed by the two C311-residues; (d)
the π-π stacking interaction of the two H127-residues. The protein is displayed in cartoon mode
and colored by protein chain. The interacting amino acids are shown as sticks; nitrogen, blue;
sulfur, yellow. (e) Schematic illustration of the (nearly) statistical orientation of crystal packing of
[Cp*Ir(biot-p-L)Cl] scdSav(SARK)mv2, reflected in the apparent occupation of both biotin-
binding sites, albeit with partial occupancy. The orientation (scdSav)2 highlighted in blue in the
crystal packing leads to an apparent partial occupation of all four biotin-binding sites, although
only the SavA site is effectively occupied.
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Reaction Optimization and Preparative Scale Synthesis. Having identified the best
(scdSav)2 scaffolds for the reduction of imines, we sought to optimize the conditions for the
ATHases. For this purpose, the effect of temperature, the pH and the substrate concentration were
evaluated. For the asymmetric reduction of imine 1a, pH = 7.0 proved best both in terms of
conversion and enantioselectivity (Figure S5). Upon increasing the temperature from 25 °C to
50 °C, higher turnover frequencies (TOF) and TONs were observed, at the cost of a slightly eroded
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ee (Table 2 and Figure S6). Next, the catalytic potential of [Cp*Ir(biot-p-L)Cl] ∙ scdSav(SARK)
and both its monovalent isoforms were tested (Table 2). At 50 °C and with [Cp*Ir(biot-p-L)Cl]
(5 µM), scdSav(SARK) (5 µM) and substrate 1a (100 mM), amine (R)-2a is produced in 78%
conversion (TON = 15'631) and 86% ee. With [Cp*Ir(biot-p-L)Cl] ∙ scdSav(SARK)mv2, the
conversion and the enantioselectivity increases to 88% (TON = 17'680) and 90% ee (R)-2a . This
TON is significantly higher than that of [Cp*Ir(biot-p-L)Cl] ∙ Sav S112A (TON = 4'000).31 High
substrate loading is often challenging for ATHases. Indeed, for the [Cp*Ir(biot-p-L)Cl] ∙
scdSav(SARK) isoforms, we determined substrate inhibition (Ki = 294-687 mM, Table 1). To
circumvent this challenge, we applied a slow feed of substrate 1a. To our delight, substrate 1a
(500 mM) was reduced to amine (R)-2a in 95% conversion and 87% ee with [Cp*Ir(biot-p-L)Cl]
∙ scdSav(SARK)mv2 (50 μM) (Table 2, and Figure S7 for the time course). This corresponds to a
product concentration of 98.5 g/L, which is similar to the current record of IRED (103.5 g/L) for
the same type of reaction.55 A preparative-scale experiment (reaction volume 10 mL) was carried
out with substrate 1a (200 mM) and [Cp*Ir(biot-p-L)Cl] ∙ scdSav(SARK)mv1 (50 μM) in a conical
flask: 408.2 mg of amine (R)-2a was isolated in 91% ee and 98.5% yield (see Figure S8 for the
time course). The same preparative reaction was also performed with [Cp*Ir(biot-p-L)Cl] ∙
scdSav(SARK)mv2 (50 μM) and 399.3 mg of amine (R)-2a was isolated in 92% ee and 96.3%
yield (Figure S8). Importantly, essentially pure (R)-salsolidine 2a could be obtained following a
simple extraction procedure (> 95 % purity by 1H NMR, See Figure S16 and S17).
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Table 2. Optimization of the reduction of dihydroisoquinoline 1a with ATHases based on
scdSav.a
[Cp*Ir(biot-p-L)Cl] 5-50 M
scdSav 5-50 M
MeO
MeO
MeO
MeO
N
NH
MOPS 0.6M, pH = 7
HCO2Na 3M, 25-50 oC
2a
(R)-
1a
10-500 mM
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[Cp*Ir(biot-p-
L)Cl] (μM)
1a conc.
(mM)
Temp.
(°C)
Conv. (R)-2a
Protein
TON
(%)
95
73
53
78
84
88
86
95
95
ee (%)
scdSav(SARK)
scdSav(SARK)
5
5
10
50
25
25
25
50
50
50
50
50
50
96
1'905
7'322
92
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scdSav(SARK)
5
100
100
100
100
500b
500b
500b
90
10'689
15'631
16'866
17'680
8'631
scdSav(SARK)
5
86
scdSav(SARK)mv1
scdSav(SARK)mv2
scdSav(SARK)
5
89
5
90
50
50
50
85
scdSav(SARK)mv1
scdSav(SARK)mv2
87
9'539
87
9'507
aReaction conditions: 10-500 mM substrate, 5-50 μM [Cp*Ir(biot-p-L)Cl], 5-50 μM scdSav (=
concentration of [Cp*Ir(biot-p-L)Cl]), 0.6 M MOPS, 3 M formate, pH 7.0, 25-50 °C, 48 h. 1a
(100 mM aliquots) was fed for five consecutive times at t = 0, 2, 5, 10 and 20 h.
b
The asymmetric reduction of other prochiral imines 1b, 1d and 1e was also investigated,
Scheme 2. By evaluating different loading of 1b and reaction temperatures (Table S2), we found
that imine 1b (100 mM) was quantitatively reduced to amine (R)-2b in 91% ee with [Cp*Ir(biot-
p-L)Cl] ∙ scdSav(SARK)mv2 (50 μM), corresponding to a TON of 2'000. Similarly, imine 1d
(100 mM) was reduced to amine (R)-2d in 99% conversion and 96% ee with [Cp*Ir(biot-p-L)Cl]
∙ scdSav(SKAA) (50 μM), corresponding to a TON of 1’976 (Table S3). Quinaldine (1e) proved
challenging to reduce: only 24% conversion to (S)-2e was obtained with [Cp*Ir(biot-p-L)Cl] ∙
scdSav(SARK) (Scheme 1). Upon increasing the reaction temperature to 50 °C and relying on
scdSav(SARK)mv2 (Table S4), 98% conversion (TON = 195) and 91% ee (S)-2e could be
achieved with 50 μM [Cp*Ir(biot-p-L)Cl] ∙ scdSav(SARK)mv2.
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Scheme 2. Optimized conditions for the reduction of prochiral imines 1b, 1d and 1e with
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ATHases based on scdSav variants.
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a)
[Cp*Ir(biot-p-L)Cl] 50 M
scdSav(SARK)mv2 50 M
1b
100 mM
N
NH
100% Conv.
91% ee
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MOPS 0.6M, pH = 7
HCO2Na 3M, 37 oC
2000 TON
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2b
(R)-
[Cp*Ir(biot-p-L)Cl] 50 M
scdSav(SKAA) 50 M
b)
c)
1d
100 mM
N
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H
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96% ee
1976 TON
MOPS 0.6M, pH = 7
HCO2Na 3M, 37 oC
2d
(R)-
[Cp*Ir(biot-p-L)Cl] 50 M
scdSav(SARK)mv2 50 M
1e
10 mM
98% Conv.
91% ee
195 TON
MOPS 0.6M, pH = 7
HCO2Na 3M, 50 oC
N
N
H
1e
2e
(S)-
CONCLUSION AND OUTLOOK
With the aims of expanding the genetic optimization potential as well as resolving the issue
related to multiple cofactor binding within the biotin-binding vestibule in ArMs based on the
biotin-streptavidin technology, we engineered a single-chain dimeric streptavidin. As both SavA
112 and SavB 112 residues as well as SavA 121 and SavB 121 lie in the immediate proximity of the
biotinylated metal moiety (Figure 1), the possibility of varying these four critical residues
individually (to potentially generate 204 = 160'000 scdSav mutants), significantly expands the
genetic diversity of ArMs based on scdSav.60-62 The potential of this strategy was highlighted by
evaluating the ATHase activity of 33 different scdSavs towards challenging substrates 1a-e.
Gratifyingly, the resulting ATHases outperformed that of homotetrameric Sav-based ATHases,
both in terms of activity (e.g., TON) and selectivity. Further improvements were observed in the
presence of monovalent scdSav(SARK)mv1 and scdSav(SARK)mv2. Enzyme kinetics revealed
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that the monovalent scdSavs outperform the related divalent scdSav for the reduction of substrates
1a, 1b and 1e, as the interference of neighboring cofactors is lifted, as highlighted in the X-ray
structure of [Cp*Ir(biot-p-L)Cl] ∙ scdSav(SASK) and [Cp*Ir(biot-p-L)Cl] ∙ scdSav(SARK)mv2.
The practicality of [Cp*Ir(biot-p-L)Cl] ∙ scdSav(SARK)mv2 was illustrated for the reduction of
imine [1a] = 500 mM to afford salsolidine (R)-2a in 90 % ee and > 17'000 TONs. In view of the
high recombinant Sav production yields obtained in fed-batch mode (e.g., > 8 g/L soluble
homotetrameric Sav),37 we hope that these findings will contribute to large scale applications of
this technology. With in vivo catalysis in mind,39-41, 63-65 the use of a monovalent scdSav may prove
versatile as the catalytic performance of the ArMs is independent of cofactor : Sav ratios. This may
also prove useful in other advanced biotechnological applications.66
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ASSOCIATED CONTENT
Supporting Information.
The Supporting Information is available free of charge on the ACS Publications website.
Experimental procedures, crystallographic data, spectroscopic data, supporting Figures and
Tables.
AUTHOR INFORMATION
Corresponding Author
*E-mail: thomas.ward@unibas.ch
ORCID
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Shuke Wu: 0000-0003-0914-9277
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Yi Zhou: 0000-0003-3467-193X
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Johannes G. Rebelein: 0000-0003-2560-716X
Jingming Zhao: 0000-0001-7104-8990
Thomas R. Ward: 0000-0001-8602-5468
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Author Contributions
‡S.W., Y.Z. and J.G.R. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
T.R.W. thanks the ERC advanced grant (the DrEAM, grant agreement No. 694424), the Swiss
National Science Foundation (Grant SNF 200020_182046) and the NCCR Molecular Systems
Engineering for generous support. S.W. thanks the Federal Commission for Scholarships for
Foreign Students for an ESKAS Scholarship, and J.G.R. thanks the European Molecular Biology
Organisation for a Long-Term fellowship (EMBO ALTF 194-2017). The authors thank Markus
Jeschek for preliminary experiments.
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