Light Regulation of Enzyme Allostery through Photo-responsive Unnatural Amino Acids

Andrea C Kneuttinger; Kristina Straub; Philipp Bittner; Nadja A Simeth; Astrid Bruckmann; Florian Busch; Chitra Rajendran; Enrico Hupfeld; Vicki H Wysocki; Dominik Horinek; Burkhard König; Rainer Merkl; Reinhard Sterner

doi:10.1016/j.chembiol.2019.08.006

Light Regulation of Enzyme Allostery through Photo-responsive Unnatural Amino Acids

Cell Chem Biol. 2019 Nov 21;26(11):1501-1514.e9. doi: 10.1016/j.chembiol.2019.08.006. Epub 2019 Sep 5.

Affiliations

¹ Institute of Biophysics and Physical Biochemistry, University of Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany.
² Institute of Organic Chemistry, University of Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany.
³ Institute of Organic Chemistry, University of Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany; Centre for Systems Chemistry, Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, the Netherlands.
⁴ Institute of Biochemistry, Genetics and Microbiology, University of Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany.
⁵ Department of Chemistry and Biochemistry and Resource for Native Mass Spectrometry Guided Structural Biology, The Ohio State University, Columbus, OH 43210, USA.
⁶ Institute of Physical and Theoretical Chemistry, University of Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany.
⁷ Institute of Biophysics and Physical Biochemistry, University of Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany. Electronic address: [email protected].

Abstract

Imidazole glycerol phosphate synthase (ImGPS) is an allosteric bienzyme complex in which substrate binding to the synthase subunit HisF stimulates the glutaminase subunit HisH. To control this stimulation with light, we have incorporated the photo-responsive unnatural amino acids phenylalanine-4'-azobenzene (AzoF), o-nitropiperonyl-O-tyrosine (NPY), and methyl-o-nitropiperonyllysine (mNPK) at strategic positions of HisF. The light-mediated isomerization of AzoF at position 55 (fS55AzoF^E ↔ fS55AzoF^Z) resulted in a reversible 10-fold regulation of HisH activity. The light-mediated decaging of NPY at position 39 (fY39NPY → fY39) and of mNPK at position 99 (fK99mNPK → fK99) led to a 4- to 6-fold increase of HisH activity. Molecular dynamics simulations explained how the unnatural amino acids interfere with the allosteric machinery of ImGPS and revealed additional aspects of HisH stimulation in wild-type ImGPS. Our findings show that unnatural amino acids can be used as a powerful tool for the spatiotemporal control of a central metabolic enzyme complex by light.

Keywords: allostery; enzyme catalysis; photo-control; protein design; synthetic biology; unnatural amino acids.

Publication types

Research Support, N.I.H., Extramural

MeSH terms

Allosteric Regulation
Allosteric Site
Amino Acids / chemical synthesis
Amino Acids / chemistry*
Amino Acids / metabolism
Aminohydrolases / chemistry
Aminohydrolases / metabolism*
Glutamine / chemistry
Glutamine / metabolism
Isomerism
Kinetics
Light*
Molecular Dynamics Simulation
Protein Subunits / chemistry
Protein Subunits / metabolism

Substances

Amino Acids
Protein Subunits
Glutamine
imidazole glycerol phosphate synthase
Aminohydrolases

Grants and funding

P41 GM128577/GM/NIGMS NIH HHS/United States