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The Impact of Linker Length on P450 Fusion Constructs: Activity, Stability and Coupling
Author(s) -
Hoffmann Sara M.,
Weissenborn Martin J.,
Gricman Łukasz,
Notonier Sandra,
Pleiss Jürgen,
Hauer Bernhard
Publication year - 2016
Publication title -
chemcatchem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.497
H-Index - 106
eISSN - 1867-3899
pISSN - 1867-3880
DOI - 10.1002/cctc.201501397
Subject(s) - linker , fusion , amino acid , chemistry , coupling (piping) , fusion protein , combinatorial chemistry , domain (mathematical analysis) , stereochemistry , biochemistry , materials science , computer science , recombinant dna , gene , mathematics , mathematical analysis , philosophy , linguistics , metallurgy , operating system
Three different reductases have been fused to CYP153 monooxygenase from Marinobacter aquaeolei . The most promising candidate has been analysed in terms of its linker part, which connects the reductase with the haem domain through sequence alignment of the corresponding reductase family CYP116B. To improve the artificial fusion construct, the linker length has been varied, thereby only altering the non‐conserved middle part of the linker. This way seven artificial fusion constructs have been engineered, which varied in linker length between 11 and 32 amino acids (“natural” is 16). These variations showed a substantial impact on the fusion construct. The best mutant, extended by two amino acids, showed an improved activity (67 %), higher stability (67 % more active haem domain after 2 h) and a coupling efficiency of 94 % (55 % higher than before). Presented in this paper is an approach to find and optimise artificial fusion constructs for P450 monooxygenases.