Daniel Hafki, Jonas Alda, Daniel Pietrus, Susanne Brakmann
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引用次数: 0
Abstract
Biochemical reaction networks adapt to environmental conditions by sensing chemical or physical stimuli and using tightly controlled mechanisms. While most signals come from molecules, many cells can also sense and respond to light. Among the biomolecular structures that enable light sensing, we selected a light-oxygen-voltage (LOV) domain in a previous study that tested the engineering of novel regulatory mechanisms into a nucleic acid polymerase. In this follow-up study, we studied the activities of previously selected variants in kinetic detail, and we generated additional LOV-polymerase fusion variants based on further insertion criteria. Our results provide mechanistic insights into how LOV domain insertion influences polymerase activity in a light-responsive manner: All active and photoresponsive enzyme variants studied by us to date were partially inhibited (i.e., "turned off") after irradiation with blue light at 470 nm, which can be explained by specific obstructions of the polymerase entry or exit structures (substrate entry channels or product exit channels, or both). Although the effects observed are moderate, we anticipate further engineering strategies that could be used to improve the extent of switchability and possibly to develop a "turn-on mode" insertion.
期刊介绍:
ChemBioChem (Impact Factor 2018: 2.641) publishes important breakthroughs across all areas at the interface of chemistry and biology, including the fields of chemical biology, bioorganic chemistry, bioinorganic chemistry, synthetic biology, biocatalysis, bionanotechnology, and biomaterials. It is published on behalf of Chemistry Europe, an association of 16 European chemical societies, and supported by the Asian Chemical Editorial Society (ACES).