Supplementary information

Optimization of the turnover in artificial enzymes via directed evolution

results in the coupling of protein dynamics to chemistry

Joseph W. Schafer,†,‡ Ioanna Zoi,†,‡ Dimitri Antoniou,† and Steven D. Schwartz∗,†

†Department of Chemistry and Biochemistry, University of Arizona

‡Contributed equally to this work

E-mail: sschwartz@email.arizona.edu

S1

Table S1: Mutations for Variants I to V.

Variant Mutations

I K10E, F22V, E51V, K53E, S70A, L83T, K110S, E159L, N180S, L184F, L187G,E210K, S211L, G233S, F246L, L247E

II V51Y, E53S, T83K, M180F, R182M, D183NIII R23H, R43S, E53T, T95M, S110N, G178SIV S43R, F72Y, K135N, S178V, G212DV T53L, R75P, N90D, N135E, S151G, V178T, F180Y, A209P, K210L, I213F, S214F,

R216P, L231M

Table S2: Distances between the catalytic base and the inhibitor/substrate in the crystal and equi-librated structures for all five enzyme variants.

Distances Crystal structure (A) Equilibrated structure (A)

I. Glu53 OE1/OE2 – O11 7.47-7.90 6.5II. Tyr51 H–O11 4.90-6.85 5.9III. Tyr51 H–O11 5.15-5.93 5.7IV. Tyr51 H–O11 no crystal structure 9.1V. Tyr51 H–O11 1.64-1.71 4.5V. Tyr180 H–O11 3.93 3.90

Table S3: Average distances for variant I between the substrate and reaction coordinate residues at0 fs; at 100, 50, 10 fs before the TS; and at the TS.

0 fs TS − 100 fs TS − 50 fs TS − 10 fs TS

Glu53 OE1–O17 6.39 ± 0.3 6.73 ± 0.2 6.75 ± 0.4 6.90 ± 0.2 6.89 ± 0.4Phe89 CZ–C12 3.23 ± 0.5 3.51 ± 0.2 3.20 ± 0.5 3.54 ± 0.2 3.57 ± 0.2Leu131 CD1–O17 4.52 ± 0.2 4.00 ± 0.1 4.12 ± 0.1 4.10 ± 0.3 4.08 ± 0.3Leu159 CD2–O17 3.42 ± 0.4 3.27 ± 0.3 3.33 ± 0.2 3.31 ± 0.1 3.34 ± 0.3Met180 CE–O17 3.46 ± 0.3 2.85 ± 0.2 3.10 ± 0.6 3.10 ± 0.4 3.39 ± 0.2

S2

Table S4: Average distances for variant II between the substrate and reaction coordinate residuesat 0 fs; at 100, 50, 10 fs before the TS; and at the TS.

0 fs TS − 100 fs TS − 50 fs TS − 10 fs TS

Tyr51 OH–O17 5.34 ± 0.3 4.56 ± 0.2 5.37 ± 0.1 4.80 ± 0.2 4.70 ± 0.2Phe89 CZ–C12 6.04 ± 0.4 4.80 ± 0.3 4.78 ± 0.3 4.32 ± 0.1 4.26 ± 0.1Ser110 OG–O17 3.67 ± 0.1 3.40 ± 0.2 3.21 ± 0.1 3.99 ± 0.2 4.06 ± 0.2Ile133 CD–O17 5.60 ± 0.3 5.17 ± 0.3 4.66 ± 0.2 5.90 ± 0.2 6.00 ± 0.2Leu159 CD1–O17 5.35 ± 0.4 6.10 ± 0.2 5.45 ± 0.2 6.01 ± 0.2 6.11 ± 0.2

Table S5: Average distances for variant III between the substrate and reaction coordinate residuesat 0 fs; at 100, 50, 10 fs before the TS; and at the TS.

0 fs TS − 100 fs TS − 50 fs TS − 10 fs TS

Leu9 CD2–C4 5.83 ± 0.1 4.62 ± 0.07 4.08 ± 0.1 3.94 ± 0.2 3.74 ± 0.2Tyr51 OH–O17 4.28 ± 0.5 4.23 ± 0.4 4.02 ± 0.3 3.95 ± 0.3 3.90 ± 0.2Phe89 CE–C12 5.13 ± 0.4 3.98 ± 0.3 4.05 ± 0.2 3.62 ± 0.2 3.99 ± 0.1Asn110 ND2–O17 3.90 ± 0.5 4.14 ± 0.2 4.02 ± 0.2 3.54 ± 0.2 3.36 ± 0.2Phe180 CZ–O11 5.07 ± 0.4 4.84 ± 0.3 4.83 ± 0.3 4.57 ± 0.3 4.46 ± 0.3

Table S6: Average distances for variant IV between the substrate and reaction coordinate residuesat 0 fs; at 100, 50, 10 fs before the TS; and at the TS.

0 fs TS − 100 fs TS − 50 fs TS − 10 fs TS

Tyr51 OH–O11 7.32 ± 0.1 5.65 ± 0.1 5.98 ± 0.1 5.70 ± 0.1 5.79 ± 0.2Phe89 CE–C12 4.80 ± 0.2 4.40 ± 0.3 4.32 ± 0.3 4.46 ± 0.2 4.51 ± 0.1Asn110 ND2–O17 3.76 ± 0.2 3.80 ± 0.1 3.89 ± 0.2 3.49 ± 0.2 3.40 ± 0.1Phe112 CZ–C13 4.72 ± 0.3 5.00 ± 0.3 5.12 ± 0.3 5.02 ± 0.3 5.04 ± 0.3Leu159 CD1–O17 4.82 ± 0.2 4.53 ± 0.2 4.23 ± 0.3 4.63 ± 0.2 4.62 ± 0.2Phe180 CZ–O11 5.73 ± 0.2 4.30 ± 0.1 3.80 ± 0.2 3.51 ± 0.1 3.32 ± 0.3

S3

Table S7: Average distances for variant V (OBM) between the substrate and reaction coordinateresidues at 0 fs; at 100, 50, 10 fs before the TS; and at the TS.

0 fs TS − 100 fs TS − 50 fs TS − 10 fs TS

Leu9 CD2–C1 6.00 ± 0.3 5.70 ± 0.2 5.20 ± 0.2 5.58 ± 0.3 5.61 ± 0.2Leu53 CD1–C9 4.30 ± 0.3 4.44 ± 0.3 3.87 ± 0.2 3.70 ± 0.3 3.67 ± 0.3Ser56 CB–C10 3.39 ± 0.2 4.09 ± 0.3 3.80 ± 0.3 3.55 ± 0.2 3.45 ± 0.1Phe112 CZ–C13 4.39 ± 0.2 4.62 ± 0.1 4.85 ± 0.2 5.38 ± 0.2 5.32 ± 0.1Met182 CE–O11 3.59 ± 0.3 5.58 ± 0.3 4.86 ± 0.3 4.43 ± 0.3 4.47 ± 0.3Leu210 CD2–CE2 Tyr180 4.03 ± 0.2 3.74 ± 0.2 3.67 ± 0.1 3.58 ± 0.2 3.59 ± 0.2Ser233 CB–CZ Tyr51 4.13 ± 0.2 4.40 ± 0.3 4.35 ± 0.3 4.62 ± 0.1 4.60 ± 0.3Tyr180 OH–O11 4.46 ± 0.1 4.54 ± 0.1 4.20 ± 0.1 3.90 ± 0.1 3.83 ± 0.3Tyr51 OH–O11 3.87 ± 0.1 2.66 ± 0.1 2.97 ± 0.1 2.65 ± 0.1 2.64 ± 0.3

Table S8: Average distances for variant V (PRM) between the substrate and reaction coordinateresidues at 0 fs; at 100, 50, 10 fs before the TS; and at the TS.

0 fs TS − 100 fs TS − 50 fs TS − 10 fs TS

Leu53 CD1–C9 4.61 ± 0.2 4.20 ± 0.4 4.30 ± 0.5 3.86 ± 0.3 3.76 ± 0.4Phe89 CZ–C12 4.22 ± 0.2 4.73 ± 0.3 4.85 ± 0.2 4.60 ± 0.1 4.66 ± 0.1Leu159 CD1–CZ Tyr180 3.76 ± 0.2 4.43 ± 0.2 5.02 ± 0.2 5.54 ± 0.2 5.56 ± 0.1Leu210 CD2–CE Tyr180 4.69 ± 0.3 4.67 ± 0.3 4.54 ± 0.3 4.23 ± 0.3 4.24 ± 0.3Met231 CE–CE Tyr180 4.02 ± 0.2 3.87 ± 0.3 3.92 ± 0.3 3.78 ± 0.2 3.72 ± 0.2Met182 CE–O11 4.14 ± 0.2 3.58 ± 0.2 3.52 ± 0.3 3.99 ± 0.2 4.01 ± 0.3Thr178 CB–CD1 Tyr180 3.91 ± 0.2 3.97 ± 0.1 4.04 ± 0.1 4.18 ± 0.1 4.19 ± 0.3Asn110 NZ–O17 6.02 ± 0.2 5.06 ± 0.2 4.85 ± 0.3 4.30 ± 0.1 4.35 ± 0.3Tyr180 OH–O11 3.64 ± 0.2 2.89 ± 0.1 2.84 ± 0.1 2.39 ± 0.1 2.49 ± 0.1Tyr51 OH–O11 3.88 ± 0.1 2.96 ± 0.2 2.89 ± 0.2 2.63 ± 0.1 2.72 ± 0.1

Table S9: Number of the reaction coordinate residues in each variant that are hydrophobic andpolarizabile

Enzyme hydrophobic polar (polarizable)

variant I 4/5 0/5variant II 4/5 2/5variant III 4/5 2/5variant IV 5/6 2/6variant V OBM 7/9 4/9variant V PRM 8/10 4/10

S4

Figure S1: Cartoon representation of the protein scaffold with all mutations from variants I to Vhighlighted in dark blue and substrate bound in red.

0 0.2 0.4 0.6 0.8 1

Probability0

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Probability0

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F

Figure S2: Committor distribution histograms constraining only the substrate atoms for (a) vari-ant I, (b) variant II, (c) variant III, (d) variant IV, (e) variant V OBM, (f) variant V PRM.

S5

0 0.2 0.4 0.6 0.8 1

Probability0

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Figure S3: Successful committor distribution histograms for (a) variant I, (b) variant II, (c) vari-ant III, (d) variant IV, (e) variant V OBM, (f) variant V PRM.

N HN CA HA CBHB1

HB2

OG SER/CG A

SN

HG1 SER/OD1 A

SN

C SER/ND2 A

SN

O SER/HD1 A

SN

HD2 ASN

C ASN

O ASN

ATOMS

0

0.1

0.2

0.3

0.4

RM

SF

SER110 RA95.5

ASN110 RA95.5-5

Figure S4: RMSF comparison between residues Ser110 in variant II (black) and Asn110 in vari-ant III (red).

S6

N HN CA HA CBHB1

HB2 CG HGCD1

HD11HD12

HD13CD2

HD21HD22

HD23 C O

ATOMS

0

0.1

0.2

0.3

0.4

RM

SF

RA95.5

RA95.5-5

RA95.5-8

Figure S5: RMSF comparison of residue Leu159 in enzymes variant II (black), variant III (red)and variant IV (blue)

N HN CA HA HBHB1

HB2 OG HG1 C O

ATOMS

0

0.1

0.2

0.3

0.4

RM

SF

SER 56 IN OBM

SER56 IN PRM

0 0.2 0.4 0.6 0.8 1

Probability0

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Sli

ces

Bobserved mechanism with rc residues from proposed mechanism

Figure S6: Committor distribution for variant V when a) we applied the set of residues we identifiedfor the observed mechanism to the proposed mechanism and b) we applied the reaction coordinateresidues from the proposed to the observed mechanism.

N HN CA HA HBHB1

HB2 OG HG1 C O

ATOMS

0

0.1

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RM

SF

SER 56 IN OBM

SER56 IN PRM

NH

N CAH

ACB

HB1

HB2

CGCD

1H

D1

CE1H

E1CZ

HZ

CD2

HD

2CE2

HE2 C 0

ATOMS

0

0.1

0.2

0.3

0.4

RM

SF

PHE112 IN OBM

PHE112 IN PRM

Figure S7: Top: RMSF comparison of residue Ser56 in the most evolved enzyme for the observed(black) and proposed mechanism (red). Bottom: RMSF comparison of residue Phe112 in the mostevolved enzyme for the observed (black) and proposed mechanism (red)

S7

Figure S8: Protein sequence of the earliest and latest variants with mutations shown in red andreaction coordinate residues in bold.

S8

0 10 20 30Atom Number

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Figure S9: 4-hydroxy-4-(6-methoxy-2-napthyl)-2-butanone labeled corresponding to the Mullikencharge graphs x-axis. All charges have been calculated for the equicommittor point(eq), 10 fsbefore the equicommittor point (eq-10), 50 fs before the equicommittor point (eq-50), and 100 fsbefore the equicommittor point (eq-100). Top left corresponds to RA95.0, top right corresponds toRA95.5-5, bottom left corresponds to RA95.5-8F OBM and bottom right corresponds to RA95.5-8F PRM.

S9