Communication pubs.acs.org/biochemistry
Cite This: Biochemistry XXXX, XXX, XXX−XXX
Directed Evolution of Alcohol Dehydrogenase for Improved Stereoselective Redox Transformations of 1‑Phenylethane-1,2-diol and Its Corresponding Acyloin Emil Hamnevik, Dirk Maurer, Thilak Reddy Enugala, Thao Chu, Robin Löfgren, Doreen Dobritzsch,* and Mikael Widersten* Department of Chemistry-BMC, Uppsala University, Box 576, SE-751 23 Uppsala, Sweden S Supporting Information *
Table 1. Steady State Kinetic Parameters
ABSTRACT: Laboratory evolution of alcohol dehydrogenase produced enzyme variants with improved turnover numbers with a vicinal 1,2-diol and its corresponding hydroxyketone. Crystal structure and transient kinetics analysis aids in rationalizing the new functions of these variants.
enzyme/substrate wild type/(S)-1 wild type/(R)-2 wild type/3 wild type/4 C1/(R)-2 C1/4 C1B1/(R)-2 C1B1/4
E
nzymes can provide effective alternative routes as catalysts in synthetic chemistry,1−4 and the “green-ness” of biocatalysis can contribute to a more sustainable manufacturing of chemicals. An important chemical transformation in synthetic chemistry is the oxidation of secondary alcohols and the reduction of the corresponding ketones. Partial oxidation of 1,2-substituted (vicinal) diols can produce the corresponding αhydroxy ketones (acyloins) that are attractive building blocks for chiral auxiliaries, natural products, and pharmaceuticals.5−7 Enzyme-catalyzed production of acyloins has been demonstrated to be feasible using monooxygenases or alcohol dehydrogenases.8−10 Alcohol dehydrogenase A (ADH-A) from the bacterium Rhodococcus ruber DSM 44541 is an interesting candidate biocatalyst of redox reactions. ADH-A is unusually tolerant toward organic solvents, is highly regio- and enantioselective, and displays activity with a wide range of alcohols and ketones, including aryl-substituted vicinal diols.11−14The catalyzed oxidation of 1,2-diols by the wild-type enzyme is, however, relatively inefficient; the kcat/KM for oxidation of (R)-2 (Chart 1) into the corresponding hydroxy ketone 4 is approximately
a
80 0.73 36 2.0 1.9 19 5.5 8.8
± ± ± ± ± ± ± ±
kcat/KM (s−1 mM−1)
KM (mM)
20a 0.01a 0.8a 0.08a 0.05 2 0.4 1
0.63 17 1.2 3.7 37 36 120 20
± ± ± ± ± ± ± ±
0.05a 0.6a 0.09a 0.4a 2 6 20 4
130 0.044 30 0.55 0.050 0.53 0.050 0.44
± ± ± ± ± ± ± ±
30a 0.0008a 2a 0.03a 0.001 0.02 0.003 0.04
Data from ref 11.
Scheme 1. Kinetic Mechanism for ADH-A-Catalyzed Alcohol Oxidation
Table 2. Kinetic Rates enzyme/substrate
k3a (s−1)
k5a (s−1)
wild type/(S)-1 wild type/(R)-2 C1/(S)-1 C1/(R)-2 C1B1/(S)-1 C1B1/(R)-2
630 ± 40 42 ± 8 − 25 ± 2 340 ± 20 58 ± 10
± ± ± ± ± ±
b
51 51 76 76 93 93
c
6 6c 20 20 10 10
kcat (s−1) 80 0.73 110 1.9 120 5.5
± ± ± ± ± ±
20b 0.01b 7 0.05 6 0.4
a See the Supporting Information for a description of the estimation of k3 and k5. Example tracks recorded during the pre-steady state or preequilibrium phases and the model fittings to the observed transient rates are shown in Figures S3 and S4. bData from ref 14. cData from ref 23.
Chart 1. Substrate Alcohols and Ketones
to be independent of the alcohol substrate (or ketone product) because this step involves only the binary enzyme−nucleotide complex. ADH-A displays a kcat with (R)-2 of 0.73 s−1, which is 70-fold lower than the NADH release rate (Table 2). Assuming that oxidation of (R)-2 still obeys an ordered mechanism, the lower turnover rate is presumably due to nonproductive substrate binding,15 where the alcohol is bound in the ternary
3000-fold lower than that for the oxidation of (S)-1 into acetophenone (3) (Table 1). ADH-A follows an ordered sequential bi-bi mechanism with a low degree of accumulation of the ternary complex at the steady state.14 The rate-limiting step for the turnover of preferred substrates such as (S)-1 is NADH release (k5 in Scheme 1, Table 2). It is noteworthy that in a strictly ordered mechanism the NADH off rate is expected © XXXX American Chemical Society
kcat (s−1)
Received: January 16, 2018 Revised: January 31, 2018 Published: January 31, 2018 A
DOI: 10.1021/acs.biochem.8b00055 Biochemistry XXXX, XXX, XXX−XXX
Communication
Biochemistry complex, in a configuration(s) that does not facilitate its oxidation.16 In this work, we set out to improve the catalytic activity of ADH-A with the aryl-substituted vicinal diol (R)-2. More efficient enzyme variants can be directly employed in enzymecatalyzed reaction pathways such as those described in Scheme 2 in which racemic styrene oxide can be transformed into
Table 3. Selection of ADH-A Variants Scored as Top Hits during Library Screenings enzyme variant
substitution
relative activitya (variant/wild type)
C1
F43H
4.9 ± 3
C1B1
F43H/Y54L
9.0 ± 6
comments eight siblings two siblings
a
Measured with 10 mM (R)-2 and 1.6 mM NAD+ at pH 8.0. Activities were measured in crude bacterial lysates without normalization for enzyme expression levels (n ≥ 3).
Scheme 2. Enantioconvergent Hydrolysis of Styrene Oxide into (R)-2 Is Catalyzed by Epoxide Hydrolase20−22a
a
its crystal structure was determined (Figure S1 and Table S2). The increased activity with (R)-2 displayed by C1 can be attributed to a 2.6-fold increase in kcat (Table 1). However, because KM is also increased to the same degree, the overall catalytic efficiency expressed as kcat/KM is unchanged. C1 displays similar behavior in the reduction of ketone 4, with a 10-fold increase in both turnover rate and KM (Table 1). The parallel increases in kcat and KM suggest a lower degree of nonproductive binding.15 Furthermore, neither the rate of oxidation of (R)-2 into 4 nor the rate of NADH release is significantly changed by the F43H substitution in C1 (Table 2). Hence, the positive effect on kcat is likely due to destabilization of nonproductive alcohol binding, thus allowing for a higher frequency of formation of productive ternary complexes. The F43H substitution results in a noteworthy structural alteration. The imidazole side chain of H43 adopts a conformation different from that of the F43 benzyl group and is directed toward the C-2′ hydroxyl of the nicotinamide nucleotide ribose. In this conformation, the imidazole side chain flips away from the substrate-binding site, enlarging it. We have observed the same F43H substitution when searching for ADH-A variants with improved activities toward other alcohols.23 Interestingly, this substitution “restores” a hydrogen-bonding chain that has been proposed to be part of the catalytic machinery in the related horse liver ADH24−27 (Figure 2A). The significance of this change in enzyme−cofactor interactions is unclear but does obviously facilitate a higher rate of catalytic turnover in the case studied here. The C1 variant was subsequently employed as a parent scaffold for additional rounds of saturation mutagenesis. First, the A site was re-explored, but, again, without success. Revisiting the B site, however, now resulted in several improved variants (Table S1). The most active enzyme from the screen, C1B1 [F43H, Y54L (Table 3)], was purified and further characterized functionally and structurally. The apparent increase in the activity of C1B1 observed in the screen with (R)-2 as a substrate can be attributed to an additional 2.9-fold increase in kcat by the added Y54L substitution (Table 1 and Table S3). C1B1 also displays a parallel increase in KM, again resulting in an unchanged kcat/KM. Hence, without a decrease in the overall catalytic efficiency, the turnover number is increased by >7-fold with (R)-2. As described previously, the value of kcat is much lower than the rate of either (R)-2 oxidation or NADH release (Table 2). This indicates a fractional increase in the productively bound ternary enzyme−substrate complex as a result of the combined F43H and Y54L substitution effects. Because the positive effect of the Y54L mutation in the C1B1 variant did not penetrate without the prior introduction of the F43H substitution, the successful isolation of improved enzymes from this second-generation C1B library suggests synergistic (epistatic) coupling between these mutations.
(R)-2 can subsequently be oxidized into 4 by ADH-A.
acyloin 4. To achieve adequate flux through the coupled reaction steps, however, the catalytic efficiency of ADH-A with (R)-2 requires improvements. To optimize the turnover of alcohol oxidation in this case presumably requires that a relaxation of nonproductive binding be accomplished. We approached this by applying CASTing17−19 involving saturation mutagenesis of active-site residues predicted to interact with bound alcohol substrates. The targeted residues were grouped into three different sites, each comprising two residues: site A, Y294 and W295; site B, Y54 and L119; site C, F43 and I271 (Figure 1). Gene libraries encoding full unbiased codon sets at
Figure 1. Active-site residues subjected to saturation mutagenesis. The catalytic zinc ion is shown as a gray sphere and the bound coenzyme (NAD+) as blue sticks. The carbon atoms of the targeted residues are colored as follows: magenta for the A site, yellow for the B site, and green for the C site. The image was created with PyMOL 1.8.729 using the wild-type ADH-A coordinates (Protein Data Bank entry 3jv7).30
both positions were constructed (see the Supporting Information for experimental details), and the expressed proteins were challenged for activity with (R)-2. Screening >1200 enzyme variants of the A or B libraries did not produce any candidate hits. The C library, however, did present ADH-A variants with apparently improved oxidation activity with (R)-2 (Table S1). The most active variant identified, dubbed “C1” (F43H) (Table 3), was further characterized functionally, and B
DOI: 10.1021/acs.biochem.8b00055 Biochemistry XXXX, XXX, XXX−XXX
Communication
Biochemistry
substrate access is normally limited by only solubility, and possible inhibitory effects on the enzyme catalyst from high reactant concentrations can be alleviated if the binding affinity of the reactant is decreased, i.e., as reflected in a higher KM value. Also, under a high substrate load, the catalytic efficiency is more accurately measured by kcat than by kcat/KM because [ES] approaches [E]tot.28 The parallel increases in kcat and KM for (R)-2, while kcat/KM stays unchanged, indicate a larger number of accessible binding modes in the active site, increasing the likelihood of turnover at the cost of affinity. The degree of productive binding increases through the evolutionary trajectory, from the wild-type enzyme, via the F43H substitution in C1, and enhanced by the additional Y54L substitution in C1B1. This enzyme variant also shows excellent enantioselectivity in the oxidation and reduction of (R)-2 and 4, respectively.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.8b00055. Experimental details; supplementary tables of kinetic parameters, library screening outcomes, and crystallographic data and refinement statistics; and supplementary figures of kinetic data and product ratios (PDF) Accession Codes
Crystallographic models and data were deposited in the Protein Data Bank as entries 6ffx for C1 and 6ffz for C1B1.
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Figure 2. (A) Hydrogen bonding network involving the inserted H43 in the C1 variant (cyan). The side chain of F43 in wild-type ADH-A (pink) is oriented in a different direction. NAD+ and the Zn ligands are shown as sticks with blue and white carbon atoms, respectively. (B) The amino acid residue substitution in C1B1 frees the space occupied by the side chains of F43 and Y54 in the wild-type enzyme (pink), resulting in an expansion of the active site. The surface representation shows the dimensions of the active-site cavity in C1B1 (green).
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. ORCID
Thilak Reddy Enugala: 0000-0001-5915-1514 Mikael Widersten: 0000-0002-3203-3793 Funding
The work was supported by Stiftelsen Olle Engkvist Byggmästare (183-358).
As in C1, the side chain of H43 interacts with the 2′-hydroxyl of the bound coenzyme, which enlarges the active site to the same degree (Figure 2B). The inserted L54 is located perpendicular to H43, and the Y54L substitution further increases the active-site volume. The larger binding pocket may allow for conformational sampling of the substrate (alcohol or ketone) that increases the frequency of productive binding modes, observed as increases in kcat. C1B1 displays high enantioselectivity toward (R)-2 over (S)-2, with almost undetectable activity with (S)-2. The stereoselectivity is also reflected in the reduction of 4, producing exclusively (R)-2 as a product (Figure S2). Hence, the improved catalytic transformation of 4 also affords a more efficient production of enantiopure (R)-2. In conclusion, we have shown that it is possible to increase the rate of turnover of ADH-A with the aryl-substituted vicinal diol (R)-1-phenylethane-1,2-diol, and the acyloin 2-hydroxyacetophenone. However, the overall catalytic efficiencies with either of these substrates, if expressed as kcat/KM, did not increase due to parallel increases in KM. From an applied perspective, using the C1B1 enzyme as a biocatalyst for this transfomation, a higher value of kcat is favored over a low KM;
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The support from COST action 1303 Systems Biocatalysis is also gratefully acknowledged. The authors thank the Diamond Light Source for beam time (proposal mx11171) and the staff of beamline I04 for assistance with crystal testing and data collection. Access was supported in part by EU FP7 infrastructure grant BIOSTRUCTX (Contract 283570).
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DOI: 10.1021/acs.biochem.8b00055 Biochemistry XXXX, XXX, XXX−XXX
