Supplementary information
Fungal Carbonyl Sulfide Hydrolase of Trichoderma harzianum Strain
THIF08 and Its Relationship with Clade D β-Carbonic Anhydrases
Yoshihito Masaki1†#, Ryuka Iizuka1#, Hiromi Kato2, Yuka Kojima3, Takahiro Ogawa1,$, Makoto
Yoshida1, Yasuhiko Matsushita4, and Yoko Katayama1,5*
1Graduate School of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho,
Fuchu, Tokyo 183-8509, Japan; 2Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira,
Sendai 980-8577, Japan; 3Institute of Global Innovation, Tokyo University of Agriculture and
Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan; 4Gene Research Center, Tokyo
University of Agriculture and Technology, 3–5–8 Saiwai-cho, Fuchu, Tokyo 183–8509, Japan;
5Independent Administrative Institution, Tokyo National Research Institute for Cultural Properties,
13-43 Ueno-Park, Taito-ku, Tokyo 110-8713, Japan
# These authors contributed equally to this work.
†Present address: Chemical Management Center, National Institute of Technology and Evaluation,
2-49-10 Nishihara, Shibuya-ku, Tokyo 151-0066, Japan
$ Present address: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2–24–16 Naka-cho, Koganei, Tokyo 184–8588, Japan Table S1. COS-degrading activity among various enzymes and associated enzyme kinetics* -1 -1 -1 Enzyme** Kingdom Organism name k cat (s ) K m (µM) k cat/K m (s M ) Reference COSase Bacteria Thiobacillus thioparus strain THI115 58 60 9.6×105 Ogawa et al ., 2013 7 CS2 hydrolase Archaea Acidianus sp. strain A1-3 1800 22 8.2×10 Smeulders et al ., 2011 7 CS2 hydrolase Bacteria Acidithiobacillus thiooxidans strain S1p 810 74 1.1×10 Smeulders et al ., 2013 8 CS2 hydrolase Bacteria Acidithiobacillus thiooxidans strain G8 2200 14 1.6×10 Smeulders et al ., 2013 β-CA Plantae Pisum sativum 93 39 2.4×106 Ogée et al ., 2016 α-CA Animalia Bos taurus 41 1.9×103 2.2×104 Haritos et al ., 2005 Nitrogenase Bacteria Azotobacter vinelandii 0.16 3.1×103 52 Seefeldt et al ., 1995 CO dehydrogenase Bacteria Rhodospirillus rubrum ATCC11170 0.52 2.2 2.4×105 Ensign, 1995 RuBisCO Bacteria Rhodospirillus rubrum 6.3 5.6×103 1.1×103 Lorimer and Pierce, 1989 RuBisCO Plantae Spinacia oleracea 3.8 1.8×103 2.2×103 Lorimer and Pierce, 1989 * Modified from Ogawa et al ., 2013. Adapted with permission from (J. Am. Chem. Soc. 2013, 135, 3818-3825). Copyright (2013) American Chemical Society.
** COSase, carbonyl sulfide hydrolase; CS2 hydrolase, carbon disulfide hydrolase; CA, carbonic anhydrase; RuBisCO, ribulose-1,5-bisphosphate carboxylase/oxygenase. Table S2. Oligonucleotides for cloning and sequencing Name Sequence (5' to 3') ITS5_primer GGAAGTAAAAGTCGTAACAAGG ITS4_primer TCCTCCGCTTATTGATATGC TEF1_primer GCCATCCTTGGAGATACCAGC TEF728_primer CATCGAGAAGTTCGAGAAGG 20kd_F01 GGAACCGTTTGCTCCAAGTGAGG 20kd_F02 CATTGCATTTCTCTTTTCACACAGC 20kd_R02 CTGAGATGAACTGGTGGTATTAATCTAC 20kd_R03 TTAAACATTAACCTTGTTAATCTTGCCCGTC 20kd_F06_EcoRIGCGAATTCATCGAAGGTCGTATGACCGTCGCCAGC 20kd_int1_R AGTCAAGACAAAAACCTTTCGGCCGGGAGGCATAG 20kd_int1_F CTATGCCTCCCGGCCGAAAGGTTTTTGTCTTGACT 20kd_int2_R GCATACCGCAGTCGGTGTGATGGATAACAACGAC 20kd_int2_F GTCGTTGTTATCCATCACACCGACTGCGGTATGC 20kd_R09_XhoI GCGCCTCGAGTTAAACATCAACCTTGTTAATCTTG 60kd_F01 CTCTAATCAAGCATCTTCCCTC 60kd_F02 AACATGGCCCCCGCAAACACTC 60kd_F04 GACCCTCCACTTCGTCTCCAAC 60kd_F05 CGCATTATCGTGAGGCATTTCGAACC 60kd_F06 TCCGCCTGCTGCATCAATCCATCACC 60kd_F07 GTTCCTTGCTGGTGCTCACGCCATGG 60kd_R01 CAACCAGGCGAGTCTGGGCCTG 60kd_R02 CGTACCAGACGCCAAGCAGACC 60kd_R03 CGAGATCAGAGCCACCAATGCC 60kd_R04 GATGATGTTGGTCAGCTTCTTG 60kd_R05 ACCCTCCTTTTTCTCTGCATTTTCGG 60kd_R06 TTCTTTTCTTACACCGTATTTAACTG 60kd_R09 GCGCTGGATCCGTCAGAGGTAATGGC GPI_F11_EcoRI GCGAATTCATCGAAGGTCGTATGGCCCCCGCAAACACTCTTCCC GPI_int1-R TTACCATGACGGGGCCGAGATCAGAGCCACCAATG GPI_int1-F ATTGGTGGCTCTGATCTCGGCCCCGTCATGGTAAC GPI_int2-R GGAGGTATTGGTCGAAGGGAGCAACCAGGCGAGTC GPI_int2-F GACTCGCCTGGTTGCTCCCTTCGACCAATACCTCC GPI_int3-R CGCCAAACAGAATGGAGCCAGTGGTGTACTTGGCG GPI_int3-F CGCCAAGTACACCACTGGCTCCATTCTGTTTGGCG GPI_R11_XhoI GCGCCTCGAGTTAGATCTTGGAGAACGACTTG Table S3. CS2 hydrolysis activity of purified protein of recombinant GST-fused COSase measured by the production of H2S*
CS2** Protein 460 μM 80 mM 28.8 ± 9.2 ppmv GST-fused COSase ND (3.8 ± 1.2 nmol mg-1 min-1)
GST ND ND
*Sampling of the headspace gas was in 10 min after the addition of CS2. The amount of H2S produced represents average value of triplicate experiments. Standard deviation of value is shown at right.
**ND means that concentrations of H2S in the headspace gas were lower than 100 pptv of detection limit. Table S4. Multiple sequence alignment of the active site of COSase, CS2 hydrolase and clade D β-CA family enzymes from Ascomycota , Basidiomycota , Bacteria and Archaea * Phylum/Division Class Organism name** Enzyme name*** Accession number Used sequence ★ ★ ★
Crenarchaeota Thermoprotei Acidianus sp. strain A1-3 CS2 hydrolase AEL19654 Trp 31 - Gly 92 1 W VLT C M D E R V H I E Q S L G I Q - -P D D ------A H I Y R N A G G I VT D D A I R SASLTT N F F G T- --KEI IVVT H T D C G 62 Euryarchaeota Methanobacteria Methanothermobacter thermautotrophicus strain Delta H CA AAB86055 Cys 33 - Gly 96 1 C I IT C M D S R L I D L LE R A L G I G R G D ------AKVIK N A G N I V D D G V I R SAAVAI YAL G - - -V N EI I IV G H T D C G 64 Euryarchaeota Methanobacteria Methanothermobacter thermautotrophicus CA WP_010877190 Cys 33 - Gly 96 1 C I IT C M D S R L I D L LE R A L G I G R G D ------AKVIK N A G N I V D D G V I R SAAVAI YAL G - - -V N EI I IV G H T D C G 64 Acidobacteria Acidobacteria Acidobacteria bacterium 13 2 20CM 2 57 6 HP OLB85114 Ala 32 - Gly 92 1 A V L T C M D T R LS- T R T L G LK- -E G D ------A H I I R N A G G I VT D D T L R SLVVS H H L L G T- - -EEF M V V N H T D C G 61 Acidobacteria Acidobacteria Acidobacteria bacterium 13 2 20CM 57 17 HP OLB39356 Ala 32 - Gly 92 1 A V L T C M D T R LS- T R T L G LK- -E G D ------A H I I R N A G G I VT D D T L R SLVVS H H L L G T- - -EEF M V I N H T D C G 61 Acidobacteria Acidobacteria Bryobacterales bacterium KBS 96 CA WP_020722144 Ala 34 - Gly 95 1 A I L T C M D A R L D PAKFA G LS- -E G D ------A H V I R N A G G R ATE D A I R SLVISYKLL G T- - -KE W FV I H H V D C G 62 Acidobacteria Acidobacteria Edaphobacter aggregans CA WP_035357967 Ala 34 - Gly 95 1 A I L T C M D A R L D PAKFA G LS- -E G D ------A H V I R N A G G R A S D D A I R SLVISYKLL G T- - - R EFFV I H H T N C G 62 Actinobacteria Actinobacteria Actinosynnema mirum DSM 43827 CA ACU34313 Thr 32 - Gly 93 1 T I L T C M D S R I R VFEI F G LK- - Q G E------A H V L R N A G G VVT D D M I R SLALS Q R K L G T- - - R EVLLV H H T N C G 62 Actinobacteria Actinobacteria Amycolatopsis keratiniphila CA WP_016333233 Ala 31 - Gly 92 1 A V L A C M D A R L N V Y G A L G L Q - -E G E------A H V I R N A G G VVTE D E I R SLAIS Q R L L G T- - - R EI ILI H H T D C G 62 Actinobacteria Actinobacteria Dietzia maris DSM 43672 CA KZO58441 Ala 32 - Gly 93 1 A V V A C M D A R L N P Y G L L G LT- -E G D ------A H V I R N A G G VVT D D V L R SLTIS Q R L L G T- --EEIVLI H H T D C G 62 Actinobacteria Actinobacteria Geodermatophilus obscurus DSM 43160 CA ADB77019 Ala 31 - Gly 92 1 A V V A C M D S R M PLFP M L G LE- -V G D ------A H V I R N A G G VI TE D V I R SLTVS Q H V L G T- - - R EI LLV H H T Q C G 62 Actinobacteria Actinobacteria Gordonia bronchialis DSM 43247 CA ACY22488 Ala 31 - Gly 92 1 A V V A C M D A R L D V Y R I L G L D - - D G E------A H V I R N A G G VI T D D E I R SLAIS Q R L L G T---TEI ILI H H T D C G 62 Actinobacteria Actinobacteria Kitasatospora CA WP_014135273 Ala 41 - Gly 102 1 A V V A C M D A R L D LFAAL G LE- -L G D ------A H I I R N A G G VVT D D A I R SLTIS Q R A L G T- - - R TVVL I H H T G C G 62 Actinobacteria Actinobacteria Mycolicibacterium phlei CA WP_003887124 Ala 31 - Gly 92 1 A V V A C M D A R L D V Y R I L G LK- - D G E------A H V I R N A G G VVT D D E I R SLAIS Q R L L G T- - - R EI ILI H H T D C G 62 Actinobacteria Actinobacteria Mycobacterium tuberculosis strain H37Rv CA NP_215800 Ala 31 - Gly 92 1 A I V A C M D A R L D V Y R M L G IK- -E G E------A H V I R N A G C VVT D D V I R SLAIS Q R L L G T- - - R EI ILL H H T D C G 62 Actinobacteria Actinobacteria Mycolicibacterium rhodesiae CA WP_005146653 Ala 31 - Gly 92 1 A V V A C M D A R L D V Y R I L G LK- - D G E------A H V I R N A G G VI T D D E I R SLAIS Q R L L G T---KEI ILI H H T D C G 62 Actinobacteria Actinobacteria Nocardia jiangxiensis CA WP_040830785 Ala 31 - Gly 92 1 A V V A C M D A R L D V Y R I L G L G - -E G E------A H V I R N A G G VVT D D E I R SLAIS Q R L L G T---TEI ILI H H T D C G 62 Actinobacteria Actinobacteria Streptomyces ambofaciens ATCC23877 CA AKZ55233 Ala 57 - Gly 118 1 A V V A C M D A R L D VTAAL G L R - - N G D ------C H V I R N A G G AVT D D V I R SLTIS Q R A L G T- - - R SVI LV H H T A C G 62 Actinobacteria Actinobacteria Streptosporangium roseum CA WP_012893510 Ala 32 - Gly 93 1 A V V T C M D S R I D P L G V F G LK- -P G D ------AKIL R N A G A R V T D D V L R TLVLAVYLL G V- - - N R VLV M P H T D C G 62
Proteobacteria Acidithiobacillia Acidithiobacillus thiooxidans strain G8 CS2 hydrolase AGQ48122 Trp 32 - Gly 95 1 W V C A C M D E R LPV D D A L G I R - - G D R ---- G D A H V F R N A G G L I T D D A I R S A M L T C N F F G T---EEIVI I N H T E C G 64
Proteobacteria Acidithiobacillia Acidithiobacillus thiooxidans strain S1p CS2 hydrolase AGQ48123 Trp 35 - Gly 102 1 W VLA C M D E R LPV D EAL G I H - -V D TPA G G G D A H C F R N A G G I VT D D A I R S A M L T C N F F G T---KEIVIV Q H T Q C G 68 Proteobacteria Betaproteobacteria Thiobacillus thioparus strain THI115 COSase BAL45931 Ala 40 - Gly 101 1 A V V A C M D A R L D V E D L L G L Q - -T G E------A H I I R N A G G V I N E D A I R C LI IS H H L L N T- - - H EI ILV H H T R C G 62 Verrucomicrobia unclassified Methylacidiphilum infernorum CA WP_012464703 Ala 34 - Gly 95 1 A I L T C M D A R L H P G KFA G LK- -E G D ------A H I I R N A G G R A S D D A I R SLI ISYKLL G T- - -KE W FV I H H T D C G 62 Verrucomicrobia Verrucomicrobiae Verrucomicrobiae bacterium strain DG 1235 CA WP_008102208 Ala 34 - Gly 95 1 A I L T C M D A R L D PAKYA G LA- -E G D ------A H V I R N A G G R A S D D A I R SLVIS H KL L G T- - - N E W FV I H H T D C G 62 Ascomycota Dothideomycetes Alternaria alternata strain Z7 CA OWY55259 Thr 34 - Gly 94 1 T I V T C M D A R I D PTAAF G IP- -L G A------A H V I R N A G G - C V K D A F R SIVIS Q Q L L G T- - - R EVI LVK H T G C G 61 Ascomycota Dothideomycetes Parastagonospora nodorum strain SN15 HP XP_001805507 Ala 38 - Gly 98 1 A V L T C M D A R I D PTAAF G IP- -L G A------A H V I R N A G A- SA R D A F R SIVIS Q Q L L G T- - -TEVLVVK H T G C G 61 Ascomycota Dothideomycetes Pyrenophora tritici-repentis strain Pt-1C-BFP CA EDU40754 Thr 34 - Gly 94 1 T I V T C M D A R I D PTAAF G IP- -L G A------A H V I R N A G A- SVK D A F R SIVIS Q Q L L G T- - - R E V M I VK H T C C G 61 Ascomycota Eurotiomycetes Aspergillus fumigatus strain Af293 CA XP_751882 Ala 32 - Gly 92 1 A V V T C M D A R I D VFSVL G LT- -E G D ------A H V I R N A G G - R ASEAL R SLI IS Q R L L G T- - -EEVVVI H H T D C G 61 Ascomycota Eurotiomycetes Aspergillus terreus NIH2624 HP XP_001209372 Ala 32 - Gly 92 1 A I V A C M D A R L D V A R A L G LE- -E G D ------A H V I R N A G G - R V T D A L R SLI IS Q Q L L G T- - - R EI I IV H H T D C G 61 Ascomycota Eurotiomycetes Byssochlamys spectabilis CBS 101075 CA RWQ91563 Ala 32 - Gly 92 1 A V L A C M D A R L D P A R A L G LE- -E G D ------A H V I R N A G G - R V A D A V R SLVIS Q Q L L G T- - - R EIVIV H H T D C G 61 Ascomycota Eurotiomycetes Coccidioides immitis strain RS CD XP_001241380 Leu 34 - Gly 94 1 L V L T C M D A R I D PAAAF G I D - -L G D ------A H V I R N A G A - N S R D A L R SI I IS Q Q L L N T- - -KEILLVK H T G C G 61 Ascomycota Eurotiomycetes Penicillium griseofulvum strain PG3 CA KXG48348 Ala 32 - Gly 92 1 A V V A C M D A R L D P A R V L G LE- -E G D ------A H V I R N A G G - R V S D A L R SI I IS Q Q L L G T- - - R EIVIV H H T N C G 61 Ascomycota Leotiomycetes Botrytis cinerea strain B05.10 HP XP_001561102 Ala 52 - Gly 112 1 A V V T C M D A R I D PSAAF G IA- -L G D ------A H V I R N A G G - SA R D A L R SLVIS Q Q L L G T- - - N EI LLVK H T G C G 61 Ascomycota Leotiomycetes Coleophoma cylindrospora strain BP6252 HP RDW69535 Val 42 - Gly 102 1 V I I S C S D P R VI PE Q F L D M N - -L G E G A------VI R N A G G - R T M D A M R SI LSL D A V G N L- - - G T I VVV H H T D C G 61 Ascomycota Leotiomycetes Meliniomyces bicolor strain E CA XP_024727572 Val 42 - Gly 102 1 V I I S C A D P R I TPE H F L G L N - -F G EAA------II R N G G G - R T V D A L R SI L D L D S L G AT- - - G TVVV I H H T D C G 61 Ascomycota Leotiomycetes Scytalidium lignicola DSM 105466 HP RFU27146 Val 43 - Gly 104 1 V V L S C S D P R L N P Y Q I L G I D Q -TLKAT------M V R N A G G - R A F D A I R TLSVL Q T I G AP- - - G T I VV M H H T D C G 62 Ascomycota Pezizomycetes Ascobolus immersus strain RN42 CA RPA84159 Ala 41 - Gly 101 1 A I V T C M D A R I H PSKAF G IT- -L G D ------A H I I R N A G A- S G V D A L R SLVIS Q Q L L G T- - -EEVFIVK H T D C G 61 Ascomycota Pezizomycetes Ascodesmis nigricans CBS 389.68 CA TGZ79294 Ala 34 - Gly 94 1 A V V T C M D A R L H PAKAL G I N - -L G D ------A H I I R N A G G - SA Q D A L R SLTIS Q Q L L G T- - -EVVVVVK H T D C G 61 Ascomycota Pezizomycetes Morchella conica strain CCBAS932 CA RPB12401 Ala 36 - Ala 96 1 A V V T C M D T N I M PASAL G FK- -L G D ------A H L I R N G G G - SA R EAL R SLVVS Q Q L F G T---KAILIIK H T D C A 61 Ascomycota Saccharomycetes Scheffersomyces stipitis CBS 6054 CD XP_001383682 Ala 32 - Gly 92 1 A V V I C M D A R I D PAKAL G LS- -E G D ------A H V I R N A G G - R A S D A L R SVI IS Q R L L G T- - - R EI VVV H H T D C G 61 Ascomycota Sordariomycetes Beauveria brongniartii strain RCEF 3172 CA OAA43988 Thr 32 - Gly 92 1 T V V T C M D A R I D PAKI L G LT- -E G D ------A H V I R N A G G - R A A D S I R SLI IS Q Q L L G T- - - R EIVLI H H T D C G 61 Ascomycota Sordariomycetes Chaetomium globosum CBS 148.51 HP XP_001225170 Leu 35 - Gly 95 1 L V L T C M D A R I D P A R A F G I D - -L G D ------A H V I R N A G A- SAV D G L R SVVI S Q Q L L G T- - -KEVILVK H T G C G 61 Ascomycota Sordariomycetes Fusarium oxysporum f. sp. pisi strain HDV247 CA EXA35532 Ala 34 - Gly 94 1 A V I A C M D A R L D P A Q V L G IE- -L G S------A H V I R N A G G - R A A D A L R SVI IS Q Q L L G T- - - R EIVIV H H T D C G 61 Ascomycota Sordariomycetes Pochonia chlamydosporia strain 170 CA XP_018139119 Ala 32 - Gly 92 1 A V V A C M D A R L D PAKVL G LS- -E G D ------A H V I R N A G G - R T IEAL R SVVI S Q Q L L G T- - - R EIVIL H H T D C G 61 Ascomycota Sordariomycetes Podospora anserina strain S mat+ UP XP_001911575 Leu 33 - Gly 93 1 L V L T C M D A R I D P A R A F G IE- -L G D ------A H V I R N A G A- SAV D A L R SIVISE Q L L G T- - - N EIVLVK H T G C G 61 Ascomycota Sordariomycetes Purpureocillium lilacinum strain PLFJ-1 CA XP_018178900 Ala 32 - Gly 92 1 A V V A C M D A R L D PAKI L G LE- -E G D ------A H V I R N A G G - R T IEAL R SI I IS Q Q L L G T- - - R EIAIV H H T D C G 61 Ascomycota Sordariomycetes Pyricularia oryzae strain Y34 CA ELQ41354 Leu 34 - Gly 94 1 L V L T C M D A R I D PAAAF G I D - -L G D ------A H V I R N A G A- SA R D G L R SVVI S Q LLL G T- - - R EVLLLK H T G C G 61 Ascomycota Sordariomycetes Sordaria macrospora CA CAT00782 Leu 37 - Gly 97 1 L L L T C M D A R I D P A R A F G IE- -L G D ------A H V I R N A G A-STK D A L R SI I IS Q Q LLAT- - -EAIAIVK H T G C G 61 Ascomycota Sordariomycetes Trichoderma harzianum strain T6776 CA KKP06884 Phe 32 - Gly 92 1 F V L T C M D A R L D PAKVL G LE- -E G H ------A H V Y R N A G G - R AAEAL R SLI IS Q Q A L G T- - -EEVVVI H H T D C G 61 Ascomycota Sordariomycetes Trichoderma reesei strain RUT C-30 CA ETR97590 Phe 32 - Gly 92 1 F V L T C M D A R L D PAKFL G LE- -E G H ------A H V Y R N A G G - R AAEAL R SLI IS Q Q A L G T- - - R EVVV I H H T D C G 61 Ascomycota Sordariomycetes Trichoderma harzianum strain THIF08 COSase LC499780 Phe 32 - Gly 92 1 F V L T C M D A R L D PAKFL G LE- -E G H ------A H V Y R N A G G - R AAEAL R SLI IS Q Q A L G T- - -EEVVVI H H T D C G 61 Basidiomycota Agaricomycetes Lentinula edodes NBRC 111202 CA GAW00580 Leu 32 - Gly 92 1 L I V T C M D A R I D C YKSL G LE- -L G E------A H I V R N A G G - SA R D A F R SILIS Q R L L G T- - - R EI AVF H H T N C G 61 Basidiomycota Agaricomycetes Mycena chlorophos CA GAT43668 Leu 30 - Gly 89 1 L V V T C M D P R I H PYE Q F G LK- -F G E C G ------IV R N G G G - STE N ALPSILIA Q K F G G ---- H H I AVV H H T D C G 60 Basidiomycota Agaricomycetes Sparassis crispa CA XP_027618783 Ala 33 - Gly 93 1 A V V T C M D A R L N V F G H L G IK- -E G E------A H I I R N A G G - AAK D A L R SI I IS Q R L L G T- - - R EI AVF H H T A C G 61 Basidiomycota Dacrymycetes Calocera cornea strain HHB12733 CA KZT55846 Ile 31 - Gly 91 1 I V V T C M D A R I D PAASL G I N - -L G E------A H V I R N A G G - R A T D A L R SILIS Q R L L G T- - -SEIVLY H H T G C G 61 Basidiomycota Exobasidiomycetes Acaromyces ingoldii strain MCA 4198 CA PWN93467 Ala 32 - Gly 92 1 A V L T C M D A R I LPEAAL G IK- -E G Q ------A H V I R N A G G - R APEAL R SVI IS Q Q L L G T- - -EEVVVV Q H T D C G 61 Basidiomycota Exobasidiomycetes Ceraceosorus guamensis strain MCA 4658 CA PWN41936 Thr 32 - Gly 92 1 T V L T C M D A R I D PVAAF G IK- -E G D ------A H V I R N A G A - R A P D A V R SLVIS Q Q L L G T- - - D TILVI G H S D C G 61 Basidiomycota Exobasidiomycetes Tilletiopsis washingtonensis strain MCA 4186 CA PWO00771 Ile 62 - Gly 122 1 I L V T C M D G R I LPEEAF G IK- -A G T V N ------VL R N A G G - R V A G AIPSI LVS Q VAL G S- - - H EI IVV H H T D C G 61 Basidiomycota Hymenomycetes Wolfiporia cocos strain MD-104 SS10 CA PCH39405 Ala 36 - Gly 96 1 A I V T C M D A R L N VFA Q L G LK- -E G D ------A H I I R N A G G - M A R D A I R SI I IS Q R M L D T- - - R EI AVF R H T L C G 61 Basidiomycota Microbotryomycetes Leucosporidium creatinivorum strain 62-1032 CA ORY90289 Ala 53 - Gly 113 1 A I V G C M D A R L D TSAAT G L H - -E G D ------S H H I R N A G G - R AAEAL R SLVIS Q EL L G T- - - R EI IVI H H T D C G 61 Basidiomycota Microbotryomycetes Rhodotorula graminis strain WP1 HP KPV77109 Ala 62 - Gly 122 1 A V L C C M D A R L D P N A M L G LE- -V G D ------A H I I R N G G G - R A A D A L R SL I G S Q EAL Q T- - - R EI IVI H H E D C G 61 Ascomycota Sordariomycetes Sordaria macrospora CA FM878639 Leu 41 - Gly 105 1 L W I G C S D S R C PETTI L G M Q - -P G D ------VFV H R N I A N I VSPT D I N TTAVI EYAVA H LKVK H I VL C G H S A C G 65 *Amino acid sequences used here were as follows: COSase of Trichoderma harzianum strain THIF08; clade D β-CA family enzyme of Ascomycota and Basidiomycota referred to Elleuche and Pöggeler (2010) and listed in the NCBI genome databases; the other clade D β-CA family enzyme referred to Ogawa et al. (2013, 2016). Sequence alignment was carried out on the active site that is homologous to the amino acid sequence of the active site of β-CA of Sordaria macrospora [CAS1 (FM878639), Leu41 to Gly105, Elleuche and Pöggeler, (2010)]. All sequences except for that of S. macrospora belong to the clade D β-CA family. The alignment was constructed using ClustalW with MEGA X. The residues that are identical in all sequences and identical in more than 50% are boxed in red and grey background, respectively. Closed stars indicate the zinc binding residues of β-CA. The accession number of each sequence is shown by NCBI. ** The name of organisms was based on the data from NCBI. The organism name in bold indicates that the in vitro experiment on the organisms exhibited the COSase activity. Acidobacteria bacterium 13 2 20CM 2 57 6 and 13 2 20CM 57 17; sequence data from soil metagenome.
*** The name of enzymes was based on the data from NCBI. CS2 hydrolase, carbon disulfide hydrolase; CA, carbonic anhydrase; COSase, carbonyl sulfide hydrolase; CD, carbonate dehydratase; HP, hypothetical protein; UP, uncharacterized protein.
Fig. S1. Micrograph picture of T. harzianum strain THIF08 culture on PDA medium at 7 days.
1600 160
1200 120
800 80 S (ppmv) S 2 H COS (ppmv) COS 400 40
0 0 0 10 20 30 40 50 60 70 Time (min)
Fig. S2. COS degradation and production of H2S by cell-free extract of T. harzianum strain THIF08. (Closed circle), cell-free extract after heat treatment; (open circle), cell- free extract. The amount of COS and H2S are shown as solid line and dashed line, respectively. Sampling of the headspace gas was started 10 min after the addition of COS.
Fig. S3. Schematic summary of constructed genomic DNA fragments encoding (A) COSase and (B) GPI. (A) To amplify the DNA fragment encoding COSase, oligonucleotide primers for PCR were designed based on the genomic DNA sequence encoding CA (Joint Genome Institute [JGI] Protein ID: 784479) of T. harzianum strain TR274, in the JGI Genome Portal (https://genome.jgi.doe.gov) (Grigoriev et al., 2012; Nordberg et al., 2014). The primer sequences were as follows: 20kd_F01, 20kd_F02, 20kd_R02, and 20kd_R03 (Table S2). The total sequence size determined using primers was 779 bp. Sequence analysis of the amplified genomic DNA fragments revealed the presence of two putative introns (102 bp and 66 bp). To construct an expression plasmid, the putative exon regions (86 bp, 178 bp, and 228 bp) were amplified by using the two- step PCR method, and the fused protein-coding DNA fragment (492 bp) was cloned into
the expression vector pGEX-6P-1 (GE Healthcare Life Sciences) as follows. In the first PCR step, the three exon fragments (86 bp, 178 bp, and 228 bp) were amplified using the following primers: 20kd_F06_EcoRI and 20kd_int1_R, 20kd_int1_F and 20kd_int2_R, and 20kd_int2_F and 20kd_R09_XhoI (Table S2). In the second PCR step, the reaction mixture from the first step was used as a template, with the primers 20kd_F06_EcoRI and 20kd_R09_XhoI. The resultant DNA fragment containing the putative coding sequence of COSase was digested with the restriction enzymes EcoRI and XhoI, and cloned between the EcoRI and XhoI sites of pGEX-6P-1 to construct the expression plasmid pGEX-COS (5479 bp). The nucleotide sequence of the inserted DNA fragment was confirmed by sequencing. (B) For amplification of the genomic DNA encoding GPI, primers were designed based on the whole genome sequence of T. harzianum (DNA Data Bank of Japan [DDBJ] SRA data accession number: SRR976278). The primer sequences were as follows: 334 60kd_F05, 60kd_F06, 60kd_F01, 60kd_F02, 60kd_F04, 60kd_F07, 60kd_R05, 60kd_R06, 60kd_R09, 60kd_R01, 60kd_R02, 60kd_R03, and 60kd_R04 (Table S2). The total sequence size determined using primers was 1928 bp. Sequence analysis revealed the presence of three putative introns (89 bp, 77 bp and 66 bp). To construct the expression plasmid, the putative exon regions (497 bp, 534 bp, 107 bp and 521 bp) were amplified by using the two step-PCR method, and the fused protein-coding DNA fragment was cloned into pGEX-6P-1. In the first PCR step, the four exon fragments (497 bp, 534 bp, 107 bp and 521 bp) were amplified with the following primers: GPI_F11_EcoRI and GPI_int1_R, GPI_int1_F and GPI_int2_R, GPI_int2_F and GPI_int3_R, and GPI_int3_F and GPI_R11_XhoI (Table S2). In the second PCR step, the reaction mixture from the first PCR step was used as a template with the primers GPI_F11_EcoRI and GPI_R11_XhoI. The resultant DNA fragment containing the putative coding sequence of GPI was digested with EcoRI and XhoI, and then cloned between the EcoRI and XhoI sites of pGEX-6P-1 to construct the expression plasmid pGEX-GPI (6,646 bp). The nucleotide sequence of the inserted DNA fragment was confirmed by sequencing.
T. harzianum THIF08 1 TT C T C TT C A T C T C C A A G C C A T A T - C A A G A C A G T C TT C A A G A C A TT G C A TTT C T C TTT C C A T - C A C C TT A TTT A G C A A C A - C A C A C T A T C G A A A A T G A C C G 97 T. harzianum TR274 1 TT C T C TT C A T C T A C A A G C C A T A T - C A A G A C A G T C TT C A A G A C A TT G C A TTT C T C TTTT C A C A C A G C TT A TTT A G C A A C A - C A C A C T A T C G A A A A T G A C C G 98 T. harzianum T6776 1 TT C T C TT C A C C T C C A A G C C A T A T T C A A G A T A G T C TT C A A G A C A TT G C G TTT C T C TTTT C A C A C A G C TT A TTT A G C A A G A G C A C A C T A T C G A A A A T G A C C G 100 *****************************************************************************************
T. harzianum THIF08 98 T C G C C AG C G AG TTT G A AG TT G C C A A C C AG C A A T A T G TT G C T A C A TTT G A C A AGG C C G A T C T G C C T A T G C C T C C C GG C C G gtgcgtttcatc t agct t tca 197 T. harzianum TR274 99 T C G C C AG C G AG TTT G A AG TT G C C A A C C AG C A A T A T G TT G C T A C G TTT G A C A AGG C C G A T C T G C C T A T G C C T C C C GG C C G gtgcgt ttca c ccagct t tca 198 T. harzianum T6776 101 T C G C C AG C G AG TTT G A AG TT G C C A A C C AG C A A T A T G TT G C T A C A TT C G A C A AGG C C G A T C T G C C T A T G C C T C C T AG C C G gtgcgtttcatccagc c t tca 200 *********************************************************************************************
T. harzianum THIF08 198 ttattctctacagcaatccatcttttctacacagcaa c t tgag g attctcatgatttgctaatttgagatgagata c t tag A A AGG TTTTT G T C TT G A C T 297 T. harzianum TR274 199 ttattctctacagcagtccatcttttctacacagcaa t t tgag t attctcatgatttgctaatttgagatgagatatttag A A AGG TTTTT G T C TT G A C C 298 T. harzianum T6776 201 t t t ttctctaca c g a a tccatcttttctaca - - - c g a a t tgag a a t t c c catgatttgctaat a tgagatgagatat t tag A A AGG TTTTT G T T C T G A C T 297 ************************************************************************************
T. harzianum THIF08 298 T G C A T GG A C G C T C G T C T GG A C C C AG C C A A A TT C C TT GG C C T T G A AG AGGG C C A C G C C C A C G T C T A C C G C A A T G C T GG AGG A C G AG C T G C C G AGG C G C T G C 397 T. harzianum TR274 299 T G C A T GG A C G C T C G T C T GG A C C C AG C C A AG TT C C T C GG C C T GG AGG AGGG C C A C G C C C A C G T C T A C C G C A A T G C C GG AGG A C G AG C T G C C G AGG C G C T G C 398 T. harzianum T6776 298 T G C A T GG A C G C T C G T C T GG A C C C AG C C A AGG T A C T C GG C C T GG A AG AGGG C C A C G C T C A C G T C T A C C G C A A T G C C GG AGG A C G AG C T G C C G AGG C G C T G C 397 ********************************************************************************************
T. harzianum THIF08 398 G C T C C TT G A T C A TTT C T C AG C A AG C T C T GGG A A C C G A AG A AG T C G T T G TT A T C C A T C A C gtatgaattgtc t c c c t a-gaagct t tgatgca c atagaat 496 T. harzianum TR274 399 G C T C C TT G A T C A TTT C T C AG C A AG C T C T GGG A A C C G A AG A AG T T G T C G TT A T C C A C C A C gtatgaat tgtcccccca-gaag t t t tgatgcagatagaat 497 T. harzianum T6776 398 G C T C T TT G A T C A TTT C T C AG C A AG C T C T GGG A A C C G A AG A AG T C G T C G TT A T C C A C C A C gtatgaat tg c c c c c c c c c aaagc t a t acctat ga t g gaa t 497 ********************************************************************************
T. harzianum THIF08 497 tggt tagctgacaa g ttattt-ataag A C C G A C T G C GG T A T G C TT C T C A T C C A C G AGG AGG AG TT C C G C A A C A C T G T C A AG A AG A A C A C T GG C G AGG A T G 595 T. harzianum TR274 498 t gg c tagctgacaaattattt-ataag A C C G A C T G C GG C A T G C TT C T C A T T C A C G AGG AGG AG TT C C G C A A C A C T G T C A AG A AG A A C A C T GG AG AGG A T G 596 T. harzianum T6776 498 tggt t g gttgacaaattattt t ataag A C C G A C T G C GG T A T G C TT C T C A T C C A C G A AG AGG AG TT C C G C A A C A C T G T C A A A A AG A A C A C T GG C G AGG A T G 597 ******************************************************************************************
T. harzianum THIF08 596 T C AG C C A C A TT G C A TT C TT G A C C A T C C AGG A C C T G C AG A AG AG C G T C A AG A C T G A T G T C G AG C TTTT G AGG A AG A A T G C T G C G A T C A AG A A T G T G C C T A T 695 T. harzianum TR274 597 T C AG C C A C G TT G C A TT C TT G A C C A T C C A AG A C C T G C AG A AG AG C G T C A AG A C C G A T G T C G AG C TTTT G AGG A AG A A T G C T G C G A T C A AGG A T G T G C C T A T 696 T. harzianum T6776 598 T C AG C C A C G TT G C A TT C TT G A C C A T C A AGG A T C T G C AGG AG AG C G T C A AG A C T G A T G T C G AG C TT C T G AGG A AG A A T G C C G C G A T C A AG A A C G T G C C C A T 697 *****************************************************************************************
T. harzianum THIF08 696 TT C GGG C T A C A T C TTT G A T G T C A AG A C GGG C A AG A TT A A C A AGG TT G A T G TTT A AG C G C A T G T A C G T C T A G A GG C A A A T T A T A T 779 T. harzianum TR274 v1.0 697 TT C GGG C T A C A T C TTT G A T G T C A AG A C GGG C A AG A TT A A C A AGG TT A A T G TTT A AG C A C A T G T A C G T C T A G A GGG A A A A T A T A T 780 T. harzianum T6776 698 TT C GGG C T A C A T C TTT G A T G T C A AG A C GGG C A AG A TT A A C A AGG TT G A T G TTT A AG C G C A T G T A T G T G T A G A ------C A T A T 774 ************************************************************************ Fig.S4 Multiple alignment of partial nucletotide sequences of T. harzianum strain TR274 and T. harzianum strainT6776 corresponding to sequence-determined region of Trichoderma harzianum strain THIF08 in this study. Nucleotides which are identical among three strains are indicated by asterisk. Nucleotide which is different from those of other two strains is shown in red. Nucleotides in putative COSase regions and intron regions are shown in bold upper case letter and lower case letter.
9
COSase 8.5 8.3
8 Blank
7.5 pH Bovine CA
7 6.8
6.5
6 -10 0 10 20 30 40 50 Time (second)
Fig. S5. Determination of the CA activity based on blanks and enzyme solutions. The time course of pH decrease from 8.3 to 7.0 was recorded. TBlank, Treq(CA) and Treq(COSase) were 25.6, 15.3 and 20, respectively.
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