Engineering of a bacterial tyrosinase for improved catalytic efficiency towards D-tyrosine using random and site directed mutagenesis approaches
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2013
Authors
Molloy, SusanNikodinović-Runić, Jasmina
Martin, Leona B.
Hartmann, Hermann
Solano, Francisco
Decker, Heinz
O'Connor, Kevin E.
Article (Published version)
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The tyrosinase gene from Ralstonia solanacearum (GenBank NP518458) was subjected to random mutagenesis resulting in tyrosinase variants (RVC10 and RV145) with up to 3.2-fold improvement in kcat, 5.2-fold lower Km and 16-fold improvement in catalytic efficiency for D-tyrosine. Based on RVC10 and RV145 mutated sequences, single mutation variants were generated with all variants showing increased kcat for D-tyrosine compared to the wild type (WT). All single mutation variants based on RV145 had a higher kcat and Km value compared to the RV145 and thus the combination of four mutations in RV145 was antagonistic for turnover, but synergistic for affinity of the enzyme for D-tyrosine. Single mutation variant 145_V153A exhibited the highest (6.9-fold) improvement in kcat and a 2.4-fold increase in Km compared to the WT. Two single mutation variants, C10_N322S and C10_T183I reduced the Km up to 2.6-fold for D-tyrosine but one variant 145_V153A increased the Km 2.4-fold compared to the WT. Homo...logy based modeling of R. solanacearum tyrosinase showed that mutation V153A disrupts the van der Waals interactions with an -helix providing one of the conserved histidine residues of the active site. The kcat and Km values for L-tyrosine decreased for RV145 and RVC10 compared to the WT. RV145 exhibited a 2.1-fold high catalytic efficiency compared to the WT which is a 7.6-fold lower improvement compared to D-tyrosine. RV145 exhibited a threefold higher monophenolase:diphenolase activity ratio for D-tyrosine:D-DOPA and a 1.4-fold higher L-tyrosine:L-DOPA activity ratio compared to the WT. Biotechnol. Bioeng. 2013; 110: 1849-1857.
Keywords:
tyrosinase / D-tyrosine / random mutagenesis / site specific mutagenesis / enzyme catalysis / homology modelingSource:
Biotechnology and Bioengineering, 2013, 110, 7, 1849-1857Publisher:
- Wiley-Blackwell, Hoboken
Note:
- Peer-reviewed manuscript: http://cherry.chem.bg.ac.rs/handle/123456789/3491
- Supplementary material: http://cherry.chem.bg.ac.rs/handle/123456789/3492
DOI: 10.1002/bit.24859
ISSN: 0006-3592
PubMed: 23381872
WoS: 000319525200006
Scopus: 2-s2.0-84878415861
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Inovacioni centar / Innovation CentreTY - JOUR AU - Molloy, Susan AU - Nikodinović-Runić, Jasmina AU - Martin, Leona B. AU - Hartmann, Hermann AU - Solano, Francisco AU - Decker, Heinz AU - O'Connor, Kevin E. PY - 2013 UR - https://cherry.chem.bg.ac.rs/handle/123456789/1359 AB - The tyrosinase gene from Ralstonia solanacearum (GenBank NP518458) was subjected to random mutagenesis resulting in tyrosinase variants (RVC10 and RV145) with up to 3.2-fold improvement in kcat, 5.2-fold lower Km and 16-fold improvement in catalytic efficiency for D-tyrosine. Based on RVC10 and RV145 mutated sequences, single mutation variants were generated with all variants showing increased kcat for D-tyrosine compared to the wild type (WT). All single mutation variants based on RV145 had a higher kcat and Km value compared to the RV145 and thus the combination of four mutations in RV145 was antagonistic for turnover, but synergistic for affinity of the enzyme for D-tyrosine. Single mutation variant 145_V153A exhibited the highest (6.9-fold) improvement in kcat and a 2.4-fold increase in Km compared to the WT. Two single mutation variants, C10_N322S and C10_T183I reduced the Km up to 2.6-fold for D-tyrosine but one variant 145_V153A increased the Km 2.4-fold compared to the WT. Homology based modeling of R. solanacearum tyrosinase showed that mutation V153A disrupts the van der Waals interactions with an -helix providing one of the conserved histidine residues of the active site. The kcat and Km values for L-tyrosine decreased for RV145 and RVC10 compared to the WT. RV145 exhibited a 2.1-fold high catalytic efficiency compared to the WT which is a 7.6-fold lower improvement compared to D-tyrosine. RV145 exhibited a threefold higher monophenolase:diphenolase activity ratio for D-tyrosine:D-DOPA and a 1.4-fold higher L-tyrosine:L-DOPA activity ratio compared to the WT. Biotechnol. Bioeng. 2013; 110: 1849-1857. PB - Wiley-Blackwell, Hoboken T2 - Biotechnology and Bioengineering T1 - Engineering of a bacterial tyrosinase for improved catalytic efficiency towards D-tyrosine using random and site directed mutagenesis approaches VL - 110 IS - 7 SP - 1849 EP - 1857 DO - 10.1002/bit.24859 ER -
@article{ author = "Molloy, Susan and Nikodinović-Runić, Jasmina and Martin, Leona B. and Hartmann, Hermann and Solano, Francisco and Decker, Heinz and O'Connor, Kevin E.", year = "2013", abstract = "The tyrosinase gene from Ralstonia solanacearum (GenBank NP518458) was subjected to random mutagenesis resulting in tyrosinase variants (RVC10 and RV145) with up to 3.2-fold improvement in kcat, 5.2-fold lower Km and 16-fold improvement in catalytic efficiency for D-tyrosine. Based on RVC10 and RV145 mutated sequences, single mutation variants were generated with all variants showing increased kcat for D-tyrosine compared to the wild type (WT). All single mutation variants based on RV145 had a higher kcat and Km value compared to the RV145 and thus the combination of four mutations in RV145 was antagonistic for turnover, but synergistic for affinity of the enzyme for D-tyrosine. Single mutation variant 145_V153A exhibited the highest (6.9-fold) improvement in kcat and a 2.4-fold increase in Km compared to the WT. Two single mutation variants, C10_N322S and C10_T183I reduced the Km up to 2.6-fold for D-tyrosine but one variant 145_V153A increased the Km 2.4-fold compared to the WT. Homology based modeling of R. solanacearum tyrosinase showed that mutation V153A disrupts the van der Waals interactions with an -helix providing one of the conserved histidine residues of the active site. The kcat and Km values for L-tyrosine decreased for RV145 and RVC10 compared to the WT. RV145 exhibited a 2.1-fold high catalytic efficiency compared to the WT which is a 7.6-fold lower improvement compared to D-tyrosine. RV145 exhibited a threefold higher monophenolase:diphenolase activity ratio for D-tyrosine:D-DOPA and a 1.4-fold higher L-tyrosine:L-DOPA activity ratio compared to the WT. Biotechnol. Bioeng. 2013; 110: 1849-1857.", publisher = "Wiley-Blackwell, Hoboken", journal = "Biotechnology and Bioengineering", title = "Engineering of a bacterial tyrosinase for improved catalytic efficiency towards D-tyrosine using random and site directed mutagenesis approaches", volume = "110", number = "7", pages = "1849-1857", doi = "10.1002/bit.24859" }
Molloy, S., Nikodinović-Runić, J., Martin, L. B., Hartmann, H., Solano, F., Decker, H.,& O'Connor, K. E.. (2013). Engineering of a bacterial tyrosinase for improved catalytic efficiency towards D-tyrosine using random and site directed mutagenesis approaches. in Biotechnology and Bioengineering Wiley-Blackwell, Hoboken., 110(7), 1849-1857. https://doi.org/10.1002/bit.24859
Molloy S, Nikodinović-Runić J, Martin LB, Hartmann H, Solano F, Decker H, O'Connor KE. Engineering of a bacterial tyrosinase for improved catalytic efficiency towards D-tyrosine using random and site directed mutagenesis approaches. in Biotechnology and Bioengineering. 2013;110(7):1849-1857. doi:10.1002/bit.24859 .
Molloy, Susan, Nikodinović-Runić, Jasmina, Martin, Leona B., Hartmann, Hermann, Solano, Francisco, Decker, Heinz, O'Connor, Kevin E., "Engineering of a bacterial tyrosinase for improved catalytic efficiency towards D-tyrosine using random and site directed mutagenesis approaches" in Biotechnology and Bioengineering, 110, no. 7 (2013):1849-1857, https://doi.org/10.1002/bit.24859 . .