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Semi-rational Engineering of Cytochrome P450sca-2 in A Hybrid System for

Enhanced Catalytic Activity: Insights into the Important Role of Electron Transfer

Lina Ba, Pan Li, Hui Zhang, Yan Duan, and Zhanglin Lin

From the Department of Chemical Engineering, Tsinghua University, Beijing 100084, China

Tables

Supplementary Table I.

Primers used for site-directed saturation mutagenesis

(SDSM) and iterative saturation mutagenesis (ISM) with the site for mutagenesis underlined.

Supplementary Table II

Coupling efficiency for selected mutants measured with a

P450sca-2:Pdx:Pdr concentration ratio of 1:20:2 for P450sca-2 variants (1.5 μM) in the presence of access substrate (500 μM). Rates were given in nmol/min/nmol P450.

Supplementary Figure Legends

Supplementary Figure 1.

Michaelis-Menten analyses of the overall apparent kinetics of mevastatin hydroxylation for

(A)

P450sca-2 wild-type, R8-5C, and SDSM mutants and

(B)

ISM mutants. The solid lines indicate the fittings of data to the

Michaelis–Menten equation by nonlinear regression. The standard deviation was calculated from three independent experiments.

1

Supplementary Figure 2.

Michaelis-Menten analyses of the apparent kinetics of

Pdx-dependent mevastatin hydroxylation for

(A)

P450sca-2 wild-type, R8-5C, and

SDSM mutants and

(B)

ISM mutants. The solid lines indicate the fittings of data to the Michaelis-Menten equation by nonlinear regression. The standard deviation was calculated from three independent experiments.

2

Supplementary Table I. Primers used for site-directed saturation mutagenesis (SDSM) and iterative saturation mutagenesis (ISM) with the site for mutagenesis underlined.

Primers

Pr

R77-for

a

R77-rev

b

T85-for

T85-rev

V194-for

Library

SDSM

SDSM

SDSM and ISM

SDSM

SDSM

ISM

ISM

ISM

Sequence

5'-TTTAAGCAGCGACNNKTTACATGCGGACTTCCCGGC-3'

c

5'-CATGTAAMNNGTCGCTGCTTAAACGCGGG-3'

c

5'-GGACTTCCCGGCCNNKTCTCCGCGCATCAAAGCATTCC-3'

5'-GAGAMNNGGCCGGGAAGTCCGCATGTA-3'

5'-TGCANNKGCGGCACGCGACGATTTTG-3'

V194-rev

T119-for

T119-rev

N363-for

N363-rev

Cast-T119-for

Cast-T119-rev

C ast

-N363

-for

SDSM and ISM

SDSM and ISM

SDSM and ISM

SDSM

SDSM

5'-ATCGTCGCGTGCCGCMNNTGCAGAATCTGCGTCAACGGCTTG-3'

5'-TCTGAGTTTNNKGTCAAACGCATCAAAGGCATGCGCCCAGATG-3'

5'-TGCGTTTGACMNNAAACTCAGAGATCGTCATG-3'

5'-TGTTTAGGCCAGNNKTTAGCACGCTTAGAACTGGT-3'

5'-GCTAAMNNCTGGCCTAAACACTGGTGAAC-3'

5'-TCTGAGTTTNDTGTCAAACGCATCAAAGGCATGCGCCCAGATG-3'

d

5'-TGCGTTTGACAHNAA ACTCAGAGAT CGTCATG-3'

d

5'-TGTTTAGGCCAGNDTTTAGCACGCTTAGAACTGGT-3'

C ast -N363 -rev ISM 5'-GCTAAAHNCTGGCCTAAACACTGGTGAAC-3'

a

for: forward primer for site-directed saturation mutagenesis.

b

rev: reverse primer for site-directed saturation mutagenesis.

c

NNK (MNN): degeneracy codon used in the forward (reverse) primer to introduce saturation mutagenesis; N stands for A+C+G+T, K stands for T+G, and

M stands for A+C.

d

NDT (AHN): degeneracy codon used in the forward (reverse) primer to introduce saturation mutagenesis; H stands for A+T+C.

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Supplementary Table II

Coupling efficiency for selected mutants measured with a

P450sca-2:Pdx:Pdr concentration ratio of 1:20:2 for P450sca-2 variants (1.5 μM) in the presence of access substrate (500 μM). Rates were given in nmol/min/nmol P450.

R8-5C

Variant I

Variant II

Background NADH consumption rate

a

11.94 ± 3.35

19.15 ± 3.44

14.46 ± 1.68

12.24 ± 4.14

NADH consumption rate

b

2.29 ± 4.16

13.48 ± 10.39

17.93 ± 11.86

Product formation rate

0.82 ± 0.01

4.60 ± 0.19

5.12 ± 0.52

5.25 ± 0.65

Coupling efficiency (%)

c

35.81

34.12

28.56

Variant III 13.87 ± 7.53 37.85

a

The background NADH consumption rate was measured in the absence of substrate.

b

The NADH consumption rate of each variant was calculated with the subtraction of the respective background NADH consumption.

c

Coupling efficiency was calculated as the percentage of NADH utilized for product formation over the total NADH consumption.

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Supplementary Figure 1

A

B

5

Supplementary Figure 2

A

B

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