Laboratory Evolution of Robust and Enantioselective Baeyer-Villiger Monooxygenases for Asymmetric Catalysis

Transcription

1 Laboratory Evolution of Robust and Enantioselective Baeyer-Villiger Monooxygenases for Asymmetric Catalysis Induced fit docking model Manfred T. Reetz* and Sheng Wu Max-Planck-Institut für Kohlenforschung Kaiser-Wilhelm-Platz 1, Mülheim an der Ruhr, Germany Supporting Information Figure S1. The putative substrate binding pocket of PAMO, FAD and phenylacetone are shown in cyan and gray, respectively. The amino acids in the range of 5 Å around phenylacetone are labeled. Designed Libraries Based on the induced fit docking model, five libraries were designed and generated separately by randomization at sites 441/442/443/444 (A), 445/446 (B), 152/153 (C), 338/339 (D) and 334/335 (E). Considering the limited throughput of GCscreening, a simplified codon degenracy based on protein sequence allignment of eight function-identified BVMOs was used to construct the librares. Later on, two libraries F and G were generated using a NNK codon degeneracy at site 440 and 437, respectively. Figure S2. Libraries constructed in PAMO evolution S 1

2 Table S1. Choice of codon degeneracies at each position selected for randomization Library Randomization of Codon Oversampling for 95% Encoded amino acids Codons amino acids degeneracy coverage Ser 441 KCA Ala, Ser Ala 442 KBG Ser, Ala, Leu, Val, Trp, Gly A Phe, His, Leu, Val, Tyr, Gly, Leu 443 BGC Asp, Arg, Cys Ser 444 NSC Ser, Ala, Pro, Thr, Arg, Gly, Cys B Asn 445 T Phe, Leu, Ile, Val, Tyr, His, Asn, Asp, Cys, Arg, Ser, Gly Met 446 T Phe, Leu, Ile, Val, Tyr, His, Asn, Asp, Cys, Arg, Ser, Gly Phe, Leu, Val, Ser, Pro, Gly Gln 152 BHK C Tyr, His, Glu, Gln, Asp Leu 153 KKG Leu, Val, Trp, Gly Leu 338 VYA Leu, Ile, Val, Pro, Thr, Gly D Phe, Leu, Ile, Val, Ser, Pro, Ile 339 NBC Thr, Gly, Gys, Arg, Gly E Gly 334 DDK All twenty amino acids except Gln, His, Pro, Thr Thr 335 DBC Phe, Ile, Val, Ser, Thr, Gly, Gys, F Pro 440 NNK All twenty amino acids G Pro 437 NNK All twenty amino acids Table S2. Primers used in generating the libraries A-G Library Primer A B C D E F G Sequences 5'- CAGGCCCGGGCAGCCCGKCAKBGBDCNSCAACATGCTGGTCTCTAT-3' 5'- ATAGAGACCAGCATGTTGSNGHVCVMTGMCGGGCTGCCCGGGCCTG-3' 5'- CCGTCTGCGCTCAGCTTCTGGTCTCTATCG-3' 5'- CCGTCTGCGCTCAGCTTCTGGTCTCTATCG-3' 5'- CATCATGGCCAGCGGCBHKKKGTCCGTCCCGCAGCTC-3' 5'- GAGCTGCGGGACGGACMMMDVGCCGCTGGCCATGATG-3' 5'- CCGTTCGGCACCAAGCGCVYANBCCTGGAAATCGACTACTAC-3' 5'- GTAGTAGTCGATTTCCAGGVNTRBGCGCTTGGTGCCGAACGG-3' 5'- CCAAGGGCTACCCGTTCDDKDBCAAGCGCCTCATCCTGGA-3' 5'- TCCAGGATGAGGCGCTTGVHMHHGAACGGGTAGCCCTTGG-3' 5'- GCAGGCCCGGGCAGCNNKTCTGCGCTCAGC-3' 5'- GCTGAGCGCAGAMNNGCTGCCCGGGCCTGC-3' 5'- CTTCATCGCAGGCNNKGGCAGCCCGTCTGCGC-3' 5'- GCGCAGACGGGCTGCCMNNGCCTGCGATGAAG-3' S 2

3 Table S3. The primers used in saturation mutagenesis at position 440 Mutation Primer P440A P440V P440S P440M P440D P440E P440Q P440Y P440W Sequence 5'-GGCCCGGGCAGCGCGTCTGCGCTCAGCAAC-3' 5'-GCTGAGCGCAGACGCGCTGCCCGGGCCTGC-3' 5'-GGCCCGGGCAGCGTCTCTGCGCTCAGCAAC-3' 5'-GCTGAGCGCAGAGACGCTGCCCGGGCCTGC-3' 5'-GGCCCGGGCAGCAGCTCTGCGCTCAGCAAC-3' 5'-GCTGAGCGCAGAGCTGCTGCCCGGGCCTGC-3' 5'-GGCCCGGGCAGCATGTCTGCGCTCAGCAAC-3' 5'-GCTGAGCGCAGACATGCTGCCCGGGCCTGC-3' 5'-GGCCCGGGCAGCGACTCTGCGCTCAGCAAC-3' 5'-GCTGAGCGCAGAGTCGCTGCCCGGGCCTGC-3' 5'-GGCCCGGGCAGCGAATCTGCGCTCAGCAAC-3' 5'-GCTGAGCGCAGATTCGCTGCCCGGGCCTGC-3' 5'-GGCCCGGGCAGCCAGTCTGCGCTCAGCAAC-3' 5'-GCTGAGCGCAGACTGGCTGCCCGGGCCTGC-3' 5'-GGCCCGGGCAGCTACTCTGCGCTCAGCAAC-3' 5'-GCTGAGCGCAGAGTAGCTGCCCGGGCCTGC-3' 5'-GGCCCGGGCAGCTGGTCTGCGCTCAGCAAC-3' 5'-GCTGAGCGCAGACCAGCTGCCCGGGCCTGC-3' Figure S3. Purification of WT PAMO and its mutant enzymes. Lane 1. purified Pro440Leu, Lane 2. purified Pro440Ile, Lane 3. purified Pro440Phe, Lane 4. purified Pro440Tyr, Lane 5. purified Pro440Trp, Lane 6 purified Pro440Asn, Lane 7. purified Pro440His, Lane 8. purified WT PAMO, M, protein molecular mass marker. S 3

4 Table S4. Chiral GC separation conditions. Ketone 1 Lactone 2 Comp Column Temp/ t R / min t R / min a 30 m BGB-175/SE-52 G/ / /min /min min iso b 30 m BGB-176/BGB-15 G/ /80 1.2/min /min min iso b 25 m LIPODEX-G G/ / min iso 50/min min iso 18/min c 29.5 m BGB-176/SE-52 G/ / /min min iso d 220/60 1.2/min 85 2/min /min e 220/60 1.2/min f 220/80 1/min 95 2/min /min g 25 m HYDRODEX BTBDAC G/ /60 1.2/min 100 2/min /min h 220/60 1.2/min 120 2/min /min i 220/80 1/min 95 2/min /min j 29.5 m BGB-176/SE-52 G/ /80 1.2/min /min min iso k 30 m BGB-176/BGB-15 G/ / /min 150 2/min /min min iso l 30 m G-TA G/ /120 1/min min iso Note: Pressure of H 2 is 0.5 bar except for compound l (0.9 bar); Speed of record: 0.5 cm/min. S 4

5 Table S5. Enantioselectivity of kinetic resolution catalyzed by PAMO mutants (ee s = ee of substrate 1; ee p = ee of product 2). Substrate Pro440Leu Pro440Ile Pro440Asn Pro440His Pro440Phe Pro440Tyr Pro440Trp Conv ee s ee p Conv ee s ee p Conv ee s ee p Conv ee s ee p Conv ee s ee p Conv ee s ee p Conv ee s ee p (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) rac-1a rac-1b rac-1c rac-1d 5 3 > > > > rac-1e > > > rac-1f > > > rac-1g > > > rac-1h > > rac-1i ,6 > > >99 rac-1j rac-1k rac-1l > : not determined, -: low conversion S 5