Engineering a Hyper-catalytic Enzyme by Photoactivated

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Letter pubs.acs.org/JPCL

Engineering a Hyper-catalytic Enzyme by Photoactivated Conformation Modulation Pratul K. Agarwal,*,† Christopher Schultz,‡ Aristotle Kalivretenos,§ Brahma Ghosh,∥ and Sheldon E. Broedel, Jr.‡ †

Computer Science and Mathematics Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, United States AthenaES, 1450 South Rolling Road, Baltimore, Maryland 21227, United States § Aurora Analytics, 1450 South Rolling Road, Baltimore, Maryland 21227, United States ‡

S Supporting Information *

ABSTRACT: Enzyme engineering for improved catalysis has wide implications. We describe a novel chemical modification of Candida antarctica lipase B that allows modulation of the enzyme conformation to promote catalysis. Computational modeling was used to identify dynamical enzyme regions that impact the catalytic mechanism. Surface loop regions located distal to active site but showing dynamical coupling to the reaction were connected by a chemical bridge between Lys136 and Pro192, containing a derivative of azobenzene. The conformational modulation of the enzyme was achieved using two sources of light that alternated the azobenzene moiety in cis and trans conformations. Computational model predicted that mechanical energy from the conformational fluctuations facilitate the reaction in the active-site. The results were consistent with predictions as the activity of the engineered enzyme was found to be enhanced with photoactivation. Preliminary estimations indicate that the engineered enzyme achieved 8−52 fold better catalytic activity than the unmodulated enzyme. SECTION: Biophysical Chemistry and Biomolecules

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can be altered by an external input. Previous studies from other groups have shown that peptide conformations can be changed very rapidly (at picosecond time-scales) by attaching a photoactivatable bridge and exposing the modified peptide to light sources of appropriate wavelengths.6,7 Nonspecific attachment of photoactivatable molecular bridges have previously been used to down-regulate and even inhibit enzyme activity.8−12 More recently, rapid photodynamics of vitamin B6 coenzyme has also been explored.13 Therefore, the experimental platform for testing our idea is to introduce a light-activated molecular switch across two surface loop regions of enzyme Candida antarctica lipase B (CALB), identified by computational modeling as part of the dynamically important enzyme network. A molecular switch, containing an azobenzene derivative, was covalently attached onto the lipase via crosslinking reagents. Using this approach, our preliminary work with CALB suggests that such a technique of introducing a compound that undergoes a light-inducible conformational change onto the surface of the protein can be used to manipulate enzyme conformation. This in turn resulted in an enhancement of the catalytic rate of the enzyme. Computational Modeling. A network of residues promoting catalysis in CALB was identified using computational modeling.

anipulation of biomolecules for achieving control on their activity has wide implications. Enzymes are biomolecules of particular interest, as they have high substrate specificity and exhibit high catalytic efficiency. Engineering of enzymes for improved catalytic efficiency has been widely sought for industrial applications. Recently, conformational flexibility has been proposed to be a contributing factor to the catalytic efficiency of enzymes.1−5 It has been hypothesized that enzyme catalysis involves the use of conformational fluctuations in the polypeptide structure of the protein to control the structural environment in the active site to facilitate the targeted chemistry.1 Several models have shown the presence of networks of dynamically important residues that connect flexible surface loop regions to catalytically important residues in the active site.5 These dynamic models particularly emphasize on the slow and global conformational fluctuations occurring at the time-scale of the enzyme reaction. Therefore, it should be possible to improve the catalytic efficiency of enzyme reactions by controlled manipulation of enzyme conformational fluctuations that are associated with the targeted chemical reaction. The objective of this study was to design and test, using a model enzyme, whether the controlled modulation of enzyme conformation leads to an increase in the catalytic rate of the enzyme (see Figure 1). Our strategy for implementing a modulation mechanism is to introduce chemical elements on the surface of the enzyme such that the enzyme conformations © 2012 American Chemical Society

Received: December 21, 2011 Accepted: April 11, 2012 Published: April 11, 2012 1142

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Figure 1. Strategy for enhancing enzyme catalytic rate. Enzyme conformation is modulated using a photoactivated ligand bridge. Under activation by light of suitable wavelengths, the bridge alters the enzyme conformation by activating the dynamically important regions that promote catalysis. A = active site, D = dynamically important regions for catalysis, N = network of residues, B = chemically conjugated bridge containing azobenzene, S = substrate.

Figure 2. Identification of dynamically active residues in CALB. (a) Network of protein motions promoting catalysis. Yellow arrows show the coupled motions in the network, and catalytic residues are labeled in green. Enzyme is colored based on the degree of flexibility, with dark blue corresponding to rigid regions and red/orange showing largest flexibility. (b) Enzyme flexibility associated with the four states along the hydrolysis reaction was identified by plotting root-mean-square fluctuations (RMSF) from the top 10 modes. RMSF was only plotted for Cα and was normalized by averaged fluctuations for entire enzyme. Excluding the free ends, five regions of high flexibility were identified. On the basis of geometry considerations, regions indicated by an asterisk (*) were selected for chemical conjugation of a photoactivatable bridge between Lys136 (magenta in panel a) and Pro192 (orange in panel a).

tional fluctuations that connect the enzyme landscape associated with the intermediate states along the reaction pathway. More details of the computational methodology and results are available in the SI. Figure 2 depicts the network pathway in CALB consisting of residues and interactions that connect surface regions to the active site catalytic residues. Several surface loop regions show considerable flexibility associated with the four states along the enzyme mechanism. In particular, the loop regions 136−152 and 185−192 show high dynamical correlations with the catalytic triad Ser105, Asp187, and His224. Additionally, the enzyme network is mediated by interactions between Thr138− Val190 (dynamical correlation 0.53) and Thr103−Phe131 (dynamical correlation 0.39). See Figures S2 and S3, Table S1, and related text in the SI for more information. It has previously been hypothesized that the surface loop regions provide a mechanism for coupling with the surrounding solvent, and the thermodynamical energy of the solvent is transferred to the active site through the network of residues and facilitates the reaction.1 Therefore, we hypothesized that selective addition of energy in these regions, by mechanically moving the peptide loops, could improve the catalytic rate of CALB. The region 185−192 contains the catalytic residue Asp187, and the nearby residue Glu188 (with its side-chain extending in the solvent) was initially selected as the site of bridge attachment, with Lys136 on the other loop. Azobenzene Bridge for Photoactivation. The transition of azobenzene between the cis and trans forms by light activation changes the end-to-end molecule length by about 3 Å. If

This methodology has previously been used to discover networks in other enzymes.14−16 The computational methodology consisted of sampling conformations along the four states of the proposed catalytic cycle of hydrolysis by CALB17 (see Supporting Information (SI), Figure S1) using molecular dynamics simulations. The model system consisted of CALB with substrate in explicit water based on the X-ray coordinates from the Protein Data Bank (code 1LBS). The conformations collected from these four simulations were used to characterize the slow conformational fluctuations associated with the reaction pathway. Structural analysis indicated that the important hydrogen bonds between catalytic residues and substrate were maintained during the simulations. Computational characterization of enzyme flexibility and analysis of dynamical cross correlation between enzyme residues were performed using the enzyme−substrate conformations collected from four simulations along the reaction pathway. Protein flexibility was identified using quasi-harmonic analysis (QHA) as described previously for other enzymes.16 In another study, we validated that our approach of sampling conformations along a limited number of states along the reaction can provide qualitative but yet important insights into enzyme flexibility linked to the reaction mechanism.18 For analysis of motions and conformational fluctuations, 4000 conformations of CALB collected along the intermediate states were used, and the QHA modes and dynamical correlations were analyzed to characterize a network of motions that connects flexible surface loops with the active site region. The computational modeling allowed identification of conforma1143

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Figure 3. Scheme for conjugating the azobenzene bridge on the enzyme.

evaluated. The scheme shown in Figure 3 proved to be the most effective. A meta-substituted benzyl azobenzene (2 μmol; 20 μL 100 mM stock) was coupled with cysteine (2 μmoles; 20 μL 100 mM stock) in a water-soluble carbodiimide-assisted reaction (EDC 2 μmol; 20 μL 100 mM stock) by mixing the components in DMA for 2 h. To the foregoing cys-azobenzene was added with 2 mg of CuSO4 (Cu/O2 redox pair to couple the cys-azobenzene to Cys192 via a disulfide bond) and 5 mg of CALBP192C in 1 mL of 50 mM sodium borate pH 8.3, and the resultant mixture was incubated at 4 °C for 64 h. The chemically modified CALBP192C was purified by adding 200 μL Ni-NTA agarose, washing the resin with borate buffer three times, and eluting the bound protein with 500 μL 50 mM sodium borate (100 mM EDTA pH 8.0). Note, EDTA was used instead of imidazole to avoid the presence of unwanted reactive nitrogen groups that could affect the next step in the conjugation chemistry. For the same reason, linkers with amide groups were not used. The amino-terminus of the linked cysazobenzene was coupled to Lys136 by adding 300 nmol (30 μL, 10 mM stock) of bis-(sulfosuccinimidyl)-glutarate (BS2G). After incubating for 2 h, the reaction was quenched with 150 μL 1 M Tris-Cl pH 8.0. The mixture was dialyzed to remove excess Tris and BS2G. The final protein concentration was 33 μg/mL. A 10 μg aliquot of enzyme gave a reaction rate of 8.2 nmol/min using 250 μM p-nitrophenyl butyrate (PNPB) as the substrate. The reaction rate was linear over the initial 3 min of the reaction. Enzyme Activity Modulation by Photoactivation. The activity of the chemically modified enzyme mixture was measured in duplicate 0.2 mL reactions composed of 1 μg of enzyme and 50 nmol of substrate PNPB (250 μM) in 50 mM Tris-Cl pH 8.0. PolySorp (Nunc) eight-well microtiter strips were used for the reactions. The reactions were initiated by the addition of 0.1 mL substrate (500 μM) and stopped by adding 0.1 mL of 10% SDS at 1 min intervals. The absorbance at 405 nm was measured after all of the reactions were completed. One set of reactions was incubated in the dark, while the other was exposed to long wavelength UV light (peak at 360 nm; for trans

introduced within a bridge between the two identified surface loops, light-activated switching between the two forms should move the polypeptide loops by about 3 Å. However, there are several design considerations that are required for the attachment of the switch to selected residues. The direct distance between the reactive side groups of two candidate amino acids, Lys136 and Glu188, on the respective loops is about 18 Å. The arc length between the loops is, therefore, about 29 Å (assuming a perfect semicircular arc). The azobenzene molecule is 9 Å in the trans form (relaxed), which leaves a distance of about 10 Å on either side of the azobenzene for conjugation of the bridge component. A number of different commercially available amino, carboxyl, and sulfhydryl cross-linking reagents were explored for the bridge conjugation (see SI for details). A significant challenge arose due to the total number of potential reactive sites. Thus, the reaction mixtures were likely to be very heterogeneous. To increase the specificity of the bridge coupling, it was decided to introduce a Cys residue into one of the loops such that the reactive sulfhydryl group is accessible.11 Pro192, in proximity to Asp187, was selected as the site for introduction of Cys mutation. The Pro192 residue in wild-type enzyme is about 10 Å from Lys136, and appeared to be in direct alignment across the surface of the protein, and a bridge would have an arc length of about 15 Å. Molecular modeling indicated that the mutation Pro192Cys would not alter the protein structure, and testing showed no change in the catalytic activity relative to wild-type. The coding sequence for CALB was chemically synthesized with the Pro192Cys substitution, yielding CALBP192C. The modified lipase sequence was introduced into pAES81 such that the expressed protein has an amino-terminal His tag. The protein was produced in Escherichia coli strain BL21 and purified by Ni-affinity chromatography. A total of 227 mg of protein was recovered (16.25 mg/mL, ∼492 μM). Conjugation of the Azobenzene Bridge to CALBP192C. The azobenzene was synthesized as described in the literature.19,20 Several schemes for introducing the azobenzene bridge were 1144

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to cis transformation of the molecular bridge) above the wells therefore simultaneously with blue light (peak at 420 nm from cool white fluorescent lights for cis to trans relaxation) from below the wells. The motivation for simultaneous exposure to these two light sources is to induce rapid and repeated modulation of enzyme conformation.6,7 The reaction rates are shown in Figure 4, and the catalytic rates from the two independently run assays are given in Table

0.035. Therefore, we estimate that on average 38.3% of the enzyme molecules carry an azobenzene conjugate. Moreover, there are three additional lysine residues (208, 290, 308) present within the cross-linker length for reaction with BS2G. Assuming 25% of the enzyme molecules have a bridge on the targeted lysine (Lys136) and that there is a 5−35% yield for homobifunctional ester-based protein cross-linking reaction21 provides us with an estimate of 0.48−3.35% enzyme molecules having the desired bridge. The preliminary estimate based on 0.48−3.35% enzyme molecules with the correctly placed photoactivatable bridge indicates specific activity to be in the range of 15.3−95.6 μmol/ min/mg (see SI for details). Thus, the light stimulation appeared to enhance catalytic activity 8−52-fold. It is important to note that we expect this to be a lower bound of the estimate, as we have ignored the cases where enzyme molecules are cross-linked to each other and enzyme down-regulation occurs due to conjugation of the bridge in the locations that unfavorably alter the conformation.8 Improvements in the cross-linking chemistry scheme would provide further engineering opportunities. Additionally, we hypothesize that the identified network (see Figures 1 and 2) could be a pathway to channel energy from the surface to the active site.1 Therefore, improvements in efficiency of energy transfer could also provide further improvement in enzyme activity. Note that the activity of the wild-type and CALBP192C mutant enzyme without the bridge had specific activities of 7.4 μmol/ min/mg. The catalytic activity of the chemically modified enzyme was reduced to 0.82−1.89 μmol/min/mg, and the estimated increase in catalytic activity due to light stimulation of 15.3 to 95.6 μmol/min/mg suggests that not only does the light stimulation of the bridge recover the lost activity due to the chemical modification but that activity is 2−13-fold higher than the wild-type enzyme. Overall, this study has demonstrated that it is possible to combine computational and experimental engineering approaches to manipulate biomolecular activity. Here we have shown that it is possible to improve the catalytic efficiency of enzymes through conformational modulation. Further, the molecular switch designed to modulate enzyme conformations distal to active site provides an enhancement of enzyme activity. This approach has interesting applications for developing hyper-catalytic enzymes as well as new biomolecular applications for nanotechnology.

Figure 4. Enzyme assay under photoactivated (light) and unmodulated (dark) conditions. Enzyme modulation was induced by simultaneous exposure of the azobenzene bridged CALBP192C to UV and blue light. Product formation was measured at intervals in the linear portion of the rate curves obtained from control experiments. Data is the average of two independent assays.

Table 1. Reaction Rates (nmol/min) of Azobenzene Bridged CALBP192C in the Presence of UV and Blue Light Sourcesa condition

assay 1

assay 2

averageb

SDc (CVd)

dark light

1.89 2.50

1.82 2.09

1.84 2.29

0.05 (3.0%) 0.29 (13%)

a

PNPB was used as substrate. bCalculated from averaged data points of assay 1 and 2. cStandard deviation. dCumulative variance.

1. The light exposed reactions showed an average increase in catalytic activity of 24% (2.29 versus 1.84 nmol/min, respectively). Note that in control experiments, nonmodified CALBP192C showed no change in catalytic activity in the presence or absence of light. These results suggest that the photoswitchable bridge enhances catalytic activity when exposed to both UV and blue light. The modulation of the CALB conformation through photoactivation of the azobenzene switch shows enhanced enzyme activity. The modulation of carefully selected dynamically important regions (136−152 and 185−192) coupled to enzyme reaction provides a unique mechanism to control enzyme activity. Control experiments show that native enzyme does not show change in activity with light exposure. Additionally, the modified enzyme with untargeted conjugation of azobenzene also does not show enhancement in activity when activated by light (see SI). Accurate estimation of the increase in enzyme activity through conformational modulation is difficult to obtain because of the nature of the chemical scheme used for crosslinking of the photoactivatable switch. The scheme is expected to provide a mixture of modified and unmodified enzyme. Preliminary estimate indicates that the absorbance at 280 nm of 100 μg of azobenzene-modified CALB was 0.183, and the corresponding meta-azobenzene absorbance at 322 nm was

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ASSOCIATED CONTENT

* Supporting Information S

Details of the computational protocol and results, alternate chemical scheme used for conjugation on to the enzyme, and calculation of the enhanced enzyme activity. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Address: Oak Ridge National Laboratory, P.O. Box 2008, MS 6016, Oak Ridge, TN 37831. Phone: (865) 574-7184; Fax: (865) 576-5491; E-mail: [email protected]. Present Address ∥

Pfizer Inc., 1 Burtt Road, Andover, Massachusetts 01810.

Notes

The authors declare no competing financial interest. 1145

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(17) Raza, S.; Fransson, L.; Hult, K. Enantioselectivity in Candida antarctica Lipase B: A Molecular Dynamics Study. Protein Sci. 2001, 10, 329−338. (18) Ramanathan, A.; Agarwal, P. K. Computational Identification of Slow Conformational Fluctuations in Proteins. J. Phys. Chem. B 2009, 113, 16669−16680. (19) Priewisch, B.; Ruck-Braun, K. Efficient Preparation of Nitrosoarenes for the Synthesis of Azobenzenes. J. Org. Chem. 2005, 70, 2350−2352. (20) Aemissegger, A.; Hilvert, D. Synthesis and Application of an Azobenzene Amino Acid As a Light-Switchable Turn Element in Polypeptides. Nat. Protoc. 2007, 2, 161. (21) Giedroc, D. P.; Keravis, T. M.; Staros, J. V.; Ling, N.; Wells, J. N.; Puett, D. Functional Properties of Covalent β-Endorphin Peptide/ Calmodulin Complexes. Chlorpromazine Binding and Phosphodiesterase Activation. Biochemistry 1985, 24, 1203−1211.

ACKNOWLEDGMENTS This work was in part funded by Technology Maturation Funds from Battelle Memorial Institute. The authors acknowledge Jennifer Caldwell and Michael Bauer for stimulating discussions.

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ABBREVIATIONS BS G, Bis-(sulfosuccinimidyl)-glutarate; CALB, Candida antarctica Lipase B; CALBP192C, CALB Pro192Cys mutant; DMA, dimethylamide; EDC, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride; EDTA, ethylenediaminetetraacetic acid; Ni-NTA, nickel-nitrilotriacetic acid; PNPB, p-nitrophenyl butyrate; SDS, sodium dodecyl sulfate.

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