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Engineering of a bacterial tyrosinase for improved catalytic efficiency towards D-tyrosine using random and site directed mutagenesis approaches
dc.creator | Molloy, Susan | |
dc.creator | Nikodinović-Runić, Jasmina | |
dc.creator | Martin, Leona B. | |
dc.creator | Hartmann, Hermann | |
dc.creator | Solano, Francisco | |
dc.creator | Decker, Heinz | |
dc.creator | O'Connor, Kevin E. | |
dc.date.accessioned | 2019-10-03T16:26:46Z | |
dc.date.available | 2014-01-22 | |
dc.date.issued | 2013 | |
dc.identifier.issn | 0006-3592 | |
dc.identifier.uri | https://cherry.chem.bg.ac.rs/handle/123456789/3491 | |
dc.description.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. | en |
dc.publisher | Wiley-Blackwell, Hoboken | |
dc.rights | embargoedAccess | |
dc.rights.uri | https://creativecommons.org/licenses/by-nc/4.0/ | |
dc.source | Biotechnology and Bioengineering | |
dc.subject | tyrosinase | en |
dc.subject | D-tyrosine | en |
dc.subject | random mutagenesis | en |
dc.subject | site specific mutagenesis | en |
dc.subject | enzyme catalysis | en |
dc.subject | homology modeling | en |
dc.title | Engineering of a bacterial tyrosinase for improved catalytic efficiency towards D-tyrosine using random and site directed mutagenesis approaches | en |
dc.type | article | |
dc.rights.license | BY-NC | |
dcterms.abstract | Мартин, Леона Б.; Никодиновић-Рунић, Јасмина; Моллоy, Сусан; Солано, Францисцо; Децкер, Хеинз; О'Цоннор, Кевин Е.; Хартманн, Херманн; | |
dc.citation.volume | 110 | |
dc.citation.issue | 7 | |
dc.citation.spage | 1849 | |
dc.citation.epage | 1857 | |
dc.identifier.wos | 000319525200006 | |
dc.identifier.doi | 10.1002/bit.24859 | |
dc.citation.other | 110(7): 1849-1857 | |
dc.citation.rank | M21 | |
dc.identifier.pmid | 23381872 | |
dc.description.other | This is the peer-reviewed version of the following article: Molloy, S.; Nikodinović-Runić, J.; Martin, L. B.; Hartmann, H.; Solano, F.; Decker, H.; O’Connor, K. E. Engineering of a Bacterial Tyrosinase for Improved Catalytic Efficiency towards D-Tyrosine Using Random and Site Directed Mutagenesis Approaches. Biotechnology and Bioengineering 2013, 110 (7), 1849–1857. [https://doi.org/10.1002/bit.24859] | |
dc.description.other | Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3492] | |
dc.type.version | acceptedVersion | |
dc.identifier.scopus | 2-s2.0-84878415861 | |
dc.identifier.fulltext | https://cherry.chem.bg.ac.rs/bitstream/id/15257/Engineering_of_a_acc_2013.pdf |