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• W2972077284 abstract "•Allostery in the HisH-HisF bienzyme complex was regulated by light•Photo-sensitive unnatural amino acids in HisF were used to control HisH activity•HisH activity depends on the conformational organization of the catalytic H178 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 (fS55AzoFE ↔ fS55AzoFZ) 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. 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 (fS55AzoFE ↔ fS55AzoFZ) 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. In the last decade, regulation of enzyme activity by light has received increasing attention in the field of synthetic biology. Various approaches have been presented, which range from the binding of light-responsive ligands at the active site to the fusion of an enzyme with a light-oxygen-voltage sensing domain (Szymański et al., 2013Szymański W. Beierle J.M. Kistemaker H.A.V. Velema W.A. Feringa B.L. Reversible photocontrol of biological systems by the incorporation of molecular photoswitches.Chem. Rev. 2013; 113: 6114-6178Crossref PubMed Scopus (819) Google Scholar, Baker and Deiters, 2014Baker A.S. Deiters A. Optical control of protein function through unnatural amino acid mutagenesis and other optogenetic approaches.ACS Chem. Biol. 2014; 9: 1398-1407Crossref PubMed Scopus (69) Google Scholar, Hüll et al., 2018Hüll K. Morstein J. Trauner D. In vivo photopharmacology.Chem. Rev. 2018; 118: 10710-10747Crossref PubMed Scopus (400) Google Scholar, Losi et al., 2018Losi A. Gardner K.H. Möglich A. Blue-light receptors for optogenetics.Chem. Rev. 2018; 118: 10659-10709Crossref PubMed Scopus (124) Google Scholar, Lachmann et al., 2019Lachmann D. Lahmy R. König B. Fulgimides as light-activated tools in biological investigations.Eur. J. Org. Chem. 2019; https://doi.org/10.1002/ejoc.201900219Crossref Scopus (19) Google Scholar, Schmermund et al., 2019Schmermund L. Jurkaš V. Özgen F.F. Barone G.D. Büchsenschütz H.C. Winkler C.K. Schmidt S. Kourist R. Kroutil W. Photo-biocatalysis: biotransformations in the presence of light.ACS Catal. 2019; 9: 4115-4144Crossref Scopus (145) Google Scholar). Less consideration has been dedicated to the photo-control of allosteric interactions, which are crucial regulatory features of enzymes in practically all metabolic pathways (Kastritis and Gavin, 2018Kastritis P.L. Gavin A.-C. Enzymatic complexes across scales.Essays Biochem. 2018; 62: 501-514Crossref PubMed Scopus (25) Google Scholar). Allostery in enzyme complexes describes the binding of a ligand to one subunit by which the activity of another, associated subunit is influenced (Makhlynets et al., 2015Makhlynets O.V. Raymond E.A. Korendovych I.V. Design of allosterically regulated protein catalysts.Biochemistry. 2015; 54: 1444-1456Crossref PubMed Scopus (46) Google Scholar). Thus, light regulation of allostery becomes most interesting for the temporal control of synthetic processes in industrial biocatalysis, such as in enzyme cascades that generally mimic metabolic pathways (Schmidt-Dannert and Lopez-Gallego, 2016Schmidt-Dannert C. Lopez-Gallego F. A roadmap for biocatalysis – functional and spatial orchestration of enzyme cascades.Microb. Biotechnol. 2016; 9: 601-609Crossref PubMed Scopus (93) Google Scholar). We have recently described photo-control of allostery within the bienzyme complex imidazole glycerol phosphate synthase (ImGPS) from Thermotoga maritima (Figure 1A) (Kneuttinger et al., 2018Kneuttinger A.C. Winter M. Simeth N.A. Heyn K. Merkl R. König B. Sterner R. Artificial light regulation of an allosteric bienzyme complex by a photosensitive ligand.ChemBioChem. 2018; 19: 1750-1757Crossref Scopus (15) Google Scholar). ImGPS consists of the glutaminase subunit HisH, which lacks measurable activity as a monomer, and the cyclase subunit HisF, which poorly activates HisH by complexation (List et al., 2012List F. Vega M.C. Razeto A. Häger M.C. Sterner R. Wilmanns M. Catalysis uncoupling in a glutamine amidotransferase bienzyme by unblocking the glutaminase active site.Chem. Biol. 2012; 19: 1589-1599Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Although the active sites of the two subunits are 25 Å apart (Douangamath et al., 2002Douangamath A. Walker M. Beismann-Driemeyer S. Vega-Fernandez M.C. Sterner R. Wilmanns M. Structural evidence for ammonia tunneling across the (βα)8 barrel of the imidazole glycerol phosphate synthase bienzyme complex.Structure. 2002; 10: 185-193Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), significant allosteric stimulation of HisH is initiated by binding of the HisF substrate N'-[(5′-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (PrFAR) (Lisi et al., 2017Lisi G.P. East K.W. Batista V.S. Loria J.P. Altering the allosteric pathway in IGPS suppresses millisecond motions and catalytic activity.Proc. Natl. Acad. Sci. U S A. 2017; 114: E3414-E3423Crossref PubMed Scopus (42) Google Scholar) or its analog N'-[(5′-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (ProFAR) (List et al., 2012List F. Vega M.C. Razeto A. Häger M.C. Sterner R. Wilmanns M. Catalysis uncoupling in a glutamine amidotransferase bienzyme by unblocking the glutaminase active site.Chem. Biol. 2012; 19: 1589-1599Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Several computational studies have described an allosteric network that connects the active sites of HisF and HisH and might transmit the stimulation signal (Rivalta et al., 2012Rivalta I. Sultan M.M. Lee N.-S. Manley G.A. Loria J.P. Batista V.S. Allosteric pathways in imidazole glycerol phosphate synthase.Proc. Natl. Acad. Sci. U S A. 2012; 109: E1428-E1436Crossref PubMed Scopus (151) Google Scholar, VanWart et al., 2012VanWart A.T. Eargle J. Luthey-Schulten Z. Amaro R.E. Exploring residue component contributions to dynamical network models of allostery.J. Chem. Theor. Comput. 2012; 8: 2949-2961Crossref PubMed Scopus (127) Google Scholar, Lisi et al., 2016Lisi G.P. Manley G.A. Hendrickson H. Rivalta I. Batista V.S. Loria J.P. Dissecting dynamic allosteric pathways using chemically related small-molecule activators.Structure. 2016; 24: 1155-1166Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, Negre et al., 2018Negre C.F.A. Morzan U.N. Hendrickson H.P. Pal R. Lisi G.P. Loria J.P. Rivalta I. Ho J. Batista V.S. Eigenvector centrality for characterization of protein allosteric pathways.Proc. Natl. Acad. Sci. U S A. 2018; 115: E12201-E12208Crossref PubMed Scopus (95) Google Scholar). At the stimulation endpoint, chemical activation of glutamine catalysis in HisH has been postulated to occur through a backbone flip in the substrate binding site that leads to the stabilization of the oxyanion reaction intermediate (Chaudhuri et al., 2001Chaudhuri B.N. Lange S.C. Myers R.S. Chittur S.V. Davisson V.J. Smith J.L. Crystal structure of imidazole glycerol phosphate synthase: a tunnel through a (β/α)8 barrel joins two active sites.Structure. 2001; 9: 987-997Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, Chaudhuri et al., 2003Chaudhuri B.N. Lange S.C. Myers R.S. Davisson V.J. Smith J.L. Toward understanding the mechanism of the complex cyclization reaction catalyzed by imidazole glycerolphosphate synthase: crystal structures of a ternary complex and the free enzyme.Biochemistry. 2003; 42: 7003-7012Crossref PubMed Scopus (59) Google Scholar, Lipchock and Loria, 2010Lipchock J.M. Loria J.P. Nanometer propagation of millisecond motions in V-type allostery.Structure. 2010; 18: 1596-1607Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, Rivalta et al., 2012Rivalta I. Sultan M.M. Lee N.-S. Manley G.A. Loria J.P. Batista V.S. Allosteric pathways in imidazole glycerol phosphate synthase.Proc. Natl. Acad. Sci. U S A. 2012; 109: E1428-E1436Crossref PubMed Scopus (151) Google Scholar). Activated HisH then hydrolyzes glutamine to glutamate and ammonia, which subsequently travels through an intermolecular channel to the active site of HisF (Douangamath et al., 2002Douangamath A. Walker M. Beismann-Driemeyer S. Vega-Fernandez M.C. Sterner R. Wilmanns M. Structural evidence for ammonia tunneling across the (βα)8 barrel of the imidazole glycerol phosphate synthase bienzyme complex.Structure. 2002; 10: 185-193Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Ammonia there reacts with PrFAR to produce imidazole glycerol phosphate (ImGP) and 5-aminoimidazol-4-carboxamidribotide (AICAR), which are used for histidine and de novo purine biosynthesis, respectively. By associating a photo-responsive ligand to the active site of HisF, we inhibited proper binding of PrFAR and were, hence, able to photo-control the allosterically stimulated activity of HisH by a factor of 2 (Kneuttinger et al., 2018Kneuttinger A.C. Winter M. Simeth N.A. Heyn K. Merkl R. König B. Sterner R. Artificial light regulation of an allosteric bienzyme complex by a photosensitive ligand.ChemBioChem. 2018; 19: 1750-1757Crossref Scopus (15) Google Scholar). We have now established a more efficient and versatile light regulation of allostery within the ImGPS complex using translational incorporation of unnatural amino acids (UAAs). By means of orthogonal tRNACUA/aminoacyl-tRNA-synthetase pairs, UAAs can be site-specifically incorporated into proteins, providing new opportunities for synthetic biology (Liu and Schultz, 2010Liu C.C. Schultz P.G. Adding new chemistries to the genetic code.Annu. Rev. Biochem. 2010; 79: 413-444Crossref PubMed Scopus (1275) Google Scholar). Although light-responsive UAAs have been implemented by targeting positions that are directly associated with the active site of an enzyme either as a substrate (Lemke et al., 2007Lemke E.A. Summerer D. Geierstanger B.H. Brittain S.M. Schultz P.G. Control of protein phosphorylation with a genetically encoded photocaged amino acid.Nat. Chem. Biol. 2007; 3: 769-772Crossref PubMed Scopus (173) Google Scholar), a catalytic residue (Luo et al., 2017Luo J. Torres-Kolbus J. Liu J. Deiters A. Genetic encoding of photocaged tyrosines with improved light-activation properties for the optical control of protease function.ChemBioChem. 2017; 18: 1442-1447Crossref PubMed Scopus (31) Google Scholar), or a residue close to substrate binding (Luo et al., 2017Luo J. Torres-Kolbus J. Liu J. Deiters A. Genetic encoding of photocaged tyrosines with improved light-activation properties for the optical control of protease function.ChemBioChem. 2017; 18: 1442-1447Crossref PubMed Scopus (31) Google Scholar, Schlesinger et al., 2018Schlesinger O. Dandela R. Bhagat A. Adepu R. Meijler M.M. Xia L. Alfonta L. Photo-switchable microbial fuel-cells.Biotechnol. Bioeng. 2018; 115: 1355-1360Crossref PubMed Scopus (7) Google Scholar, Wang et al., 2019Wang J. Liu Y. Liu Y. Zheng S. Wang X. Zhao J. Yang F. Zhang G. Wang C. Chen P.R. Time-resolved protein activation by proximal decaging in living systems.Nature. 2019; 569: 509-513Crossref PubMed Scopus (95) Google Scholar), light-responsive UAAs have hitherto only rarely been used to control allostery (Luo et al., 2018Luo J. Samanta S. Convertino M. Dokholyan N.V. Deiters A. Reversible and tunable photoswitching of protein function through genetic encoding of azobenzene amino acids in mammalian cells.ChemBioChem. 2018; 19: 2178-2185Crossref PubMed Scopus (30) Google Scholar). The most commonly used light-responsive UAAs bear a natural amino acid as a scaffold caged with a photolabile protecting group (Figure 1B) (Curley and Lawrence, 1999Curley K. Lawrence D.S. Light-activated proteins.Curr. Opin. Chem. Biol. 1999; 3: 84-88Crossref PubMed Scopus (69) Google Scholar, Courtney and Deiters, 2018Courtney T. Deiters A. Recent advances in the optical control of protein function through genetic code expansion.Curr. Opin. Chem. Biol. 2018; 46: 99-107Crossref PubMed Scopus (69) Google Scholar, Bardhan and Deiters, 2019Bardhan A. Deiters A. Development of photolabile protecting groups and their application to the optochemical control of cell signaling.Curr. Opin. Struct. Biol. 2019; 57: 164-175Crossref PubMed Scopus (58) Google Scholar). Irradiation with UV light induces a decaging reaction, setting free the natural amino acid (Klán et al., 2013Klán P. Šolomek T. Bochet C.G. Blanc A. Givens R. Rubina M. Popik V. Kostikov A. Wirz J. Photoremovable protecting groups in chemistry and biology: reaction mechanisms and efficacy.Chem. Rev. 2013; 113: 119-191Crossref PubMed Scopus (1129) Google Scholar). One of the first caged UAA that has been incorporated into proteins was o-nitrobenzyl-O-tyrosine (NBY) (Deiters et al., 2006Deiters A. Groff D. Ryu Y. Xie J. Schultz P.G. A genetically encoded photocaged tyrosine.Angew. Chem. Int. Ed. 2006; 45: 2728-2731Crossref PubMed Scopus (150) Google Scholar). Disadvantages of this UAA, however, include slow and incomplete decaging, which could be improved by the addition of a methylenedioxy moiety to form o-nitropiperonyl-O-tyrosine (NPY) and by an additional methyl substituent (Berroy et al., 2001Berroy P. Viriot M.L. Carré M.C. Photolabile group for 5′-OH protection of nucleosides: synthesis and photodeprotection rate.Sens. Actuators B. 2001; 74: 186-189Crossref Scopus (21) Google Scholar, Luo et al., 2017Luo J. Torres-Kolbus J. Liu J. Deiters A. Genetic encoding of photocaged tyrosines with improved light-activation properties for the optical control of protease function.ChemBioChem. 2017; 18: 1442-1447Crossref PubMed Scopus (31) Google Scholar). Selective incorporation has also been accomplished for other UAAs using, for example, serine (Lemke et al., 2007Lemke E.A. Summerer D. Geierstanger B.H. Brittain S.M. Schultz P.G. Control of protein phosphorylation with a genetically encoded photocaged amino acid.Nat. Chem. Biol. 2007; 3: 769-772Crossref PubMed Scopus (173) Google Scholar), cysteine (Uprety et al., 2014Uprety R. Luo J. Liu J. Naro Y. Samanta S. Deiters A. Genetic encoding of caged cysteine and caged homocysteine in bacterial and mammalian cells.ChemBioChem. 2014; 15: 1793-1799Crossref PubMed Scopus (43) Google Scholar), or lysine (methyl-o-nitropiperonyllysine [mNPK]) (Gautier et al., 2010Gautier A. Nguyen D.P. Lusic H. An W. Deiters A. Chin J.W. Genetically encoded photocontrol of protein localization in mammalian cells.J. Am. Chem. Soc. 2010; 132: 4086-4088Crossref PubMed Scopus (195) Google Scholar) as a scaffold. A general drawback of caged UAAs from the perspective of light regulation is the irreversibility of the decaging reaction. For reversible photo-control, the azobenzene moiety has been successfully integrated into many biologically relevant molecules (Morstein et al., 2019Morstein J. Hill R.Z. Novak A.J.E. Feng S. Norman D.D. Donthamsetti P.C. Frank J.A. Harayama T. Williams B.M. Parrill A.L. et al.Optical control of sphingosine-1-phosphate formation and function.Nat. Chem. Biol. 2019; 15: 623-631Crossref PubMed Scopus (42) Google Scholar, Schmermund et al., 2019Schmermund L. Jurkaš V. Özgen F.F. Barone G.D. Büchsenschütz H.C. Winkler C.K. Schmidt S. Kourist R. Kroutil W. Photo-biocatalysis: biotransformations in the presence of light.ACS Catal. 2019; 9: 4115-4144Crossref Scopus (145) Google Scholar) and specifically incorporated into proteins as a derivative of phenylalanine (AzoF) (Bose et al., 2006Bose M. Groff D. Xie J. Brustad E. Schultz P.G. The incorporation of a photoisomerizable amino acid into proteins in E. coli.J. Am. Chem. Soc. 2006; 128: 388-389Crossref PubMed Scopus (143) Google Scholar). Irradiation with UV light converts its E isomer into a Z isomer, which is thermodynamically less stable and reverts upon irradiation with light above 400 nm (Zimmerman et al., 1958Zimmerman G. Chow L.-Y. Paik U.-J. The photochemical isomerization of azobenzene.J. Am. Chem. Soc. 1958; 80: 3528-3531Crossref Scopus (472) Google Scholar). The switching occurs rapidly, with high quantum yields and little photobleaching (Szymański et al., 2013Szymański W. Beierle J.M. Kistemaker H.A.V. Velema W.A. Feringa B.L. Reversible photocontrol of biological systems by the incorporation of molecular photoswitches.Chem. Rev. 2013; 113: 6114-6178Crossref PubMed Scopus (819) Google Scholar). Our strategy to photo-control allosteric stimulation of HisH in ImGPS via the incorporation of UAAs in HisF (AzoF, NBY, NPY, and mNPK) is schematically outlined in Figure 1C. With the intention to disturb activation of HisH by HisF, we chose positions close to three known sites that are part of the previously described allosteric network (Rivalta et al., 2012Rivalta I. Sultan M.M. Lee N.-S. Manley G.A. Loria J.P. Batista V.S. Allosteric pathways in imidazole glycerol phosphate synthase.Proc. Natl. Acad. Sci. U S A. 2012; 109: E1428-E1436Crossref PubMed Scopus (151) Google Scholar, VanWart et al., 2012VanWart A.T. Eargle J. Luthey-Schulten Z. Amaro R.E. Exploring residue component contributions to dynamical network models of allostery.J. Chem. Theor. Comput. 2012; 8: 2949-2961Crossref PubMed Scopus (127) Google Scholar, Lisi et al., 2016Lisi G.P. Manley G.A. Hendrickson H. Rivalta I. Batista V.S. Loria J.P. Dissecting dynamic allosteric pathways using chemically related small-molecule activators.Structure. 2016; 24: 1155-1166Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, Negre et al., 2018Negre C.F.A. Morzan U.N. Hendrickson H.P. Pal R. Lisi G.P. Loria J.P. Rivalta I. Ho J. Batista V.S. Eigenvector centrality for characterization of protein allosteric pathways.Proc. Natl. Acad. Sci. U S A. 2018; 115: E12201-E12208Crossref PubMed Scopus (95) Google Scholar). Furthest away from the HisH active site, the highly flexible loop 1 of HisF is thought to be involved in coupling both catalytic activities of HisF and HisH (Douangamath et al., 2002Douangamath A. Walker M. Beismann-Driemeyer S. Vega-Fernandez M.C. Sterner R. Wilmanns M. Structural evidence for ammonia tunneling across the (βα)8 barrel of the imidazole glycerol phosphate synthase bienzyme complex.Structure. 2002; 10: 185-193Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Mutational studies (Beismann-Driemeyer and Sterner, 2001Beismann-Driemeyer S. Sterner R. Imidazole glycerol phosphate synthase from Thermotoga maritima. Quaternary structure, steady-state kinetics, and reaction mechanism of the bienzyme complex.J. Biol. Chem. 2001; 276: 20387-20396Crossref PubMed Scopus (81) Google Scholar) and NMR experiments (Lisi et al., 2016Lisi G.P. Manley G.A. Hendrickson H. Rivalta I. Batista V.S. Loria J.P. Dissecting dynamic allosteric pathways using chemically related small-molecule activators.Structure. 2016; 24: 1155-1166Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) have supported the significance of loop 1 and suggested that its dynamics (Lisi et al., 2016Lisi G.P. Manley G.A. Hendrickson H. Rivalta I. Batista V.S. Loria J.P. Dissecting dynamic allosteric pathways using chemically related small-molecule activators.Structure. 2016; 24: 1155-1166Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) and interactions (Rivalta et al., 2012Rivalta I. Sultan M.M. Lee N.-S. Manley G.A. Loria J.P. Batista V.S. Allosteric pathways in imidazole glycerol phosphate synthase.Proc. Natl. Acad. Sci. U S A. 2012; 109: E1428-E1436Crossref PubMed Scopus (151) Google Scholar) with hydrophobic residues in the core of HisF change upon PrFAR binding. The other two sites are associated with PrFAR-stimulated motions at the HisH:HisF interface. First, for ammonia to pass into the intermolecular channel, the gate at the entrance, composed of residues fR5, fE46, fK99, and fE167 (f for HisF), needs to open (Chaudhuri et al., 2001Chaudhuri B.N. Lange S.C. Myers R.S. Chittur S.V. Davisson V.J. Smith J.L. Crystal structure of imidazole glycerol phosphate synthase: a tunnel through a (β/α)8 barrel joins two active sites.Structure. 2001; 9: 987-997Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, Douangamath et al., 2002Douangamath A. Walker M. Beismann-Driemeyer S. Vega-Fernandez M.C. Sterner R. Wilmanns M. Structural evidence for ammonia tunneling across the (βα)8 barrel of the imidazole glycerol phosphate synthase bienzyme complex.Structure. 2002; 10: 185-193Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Similarly, PrFAR promotes a so-called “breathing motion” hinged at the cation-π interaction fR249-hW123 (h for HisH) that opens the interface above the glutamine binding site, possibly to allow for glutamine binding (Amaro et al., 2007Amaro R.E. Sethi A. Myers R.S. Davisson V.J. Luthey-Schulten Z.A. A network of conserved interactions regulates the allosteric signal in a glutamine amidotransferase.Biochemistry. 2007; 46: 2156-2173Crossref PubMed Scopus (71) Google Scholar, Rivalta et al., 2012Rivalta I. Sultan M.M. Lee N.-S. Manley G.A. Loria J.P. Batista V.S. Allosteric pathways in imidazole glycerol phosphate synthase.Proc. Natl. Acad. Sci. U S A. 2012; 109: E1428-E1436Crossref PubMed Scopus (151) Google Scholar). Based on these considerations, we have incorporated the photo-responsive UAAs AzoF, NBY, NPY, and mNPK into positions close to loop 1, the ammonia channel, and the hinge region, and identified three UAA-HisFs that showed the potential to regulate HisH activity by light. We then characterized these proteins with respect to their structure, stability, and function, and optimized the isomerization and decaging processes. In the following, we could directly light regulate HisH activity 10-fold by our AzoF-HisF and 4- to 6-fold by our caged UAA-HisFs. Finally, molecular dynamics (MD) simulations revealed how each UAA affects HisH activity. We applied three criteria to identify suitable positions in HisF for the incorporation of AzoF, NBY, and mNPK. First, the position in HisF should at least be 10 Å away from the HisH active site avoiding direct interactions of the UAA with catalytic residues. Moreover, UAAs should not hamper substrate binding in either the HisF or HisH active sites, nor impair the overall structure of the ImGPS complex. Based on these criteria, we tested ten positions in T. maritima HisF for the incorporation of light-responsive UAAs. The positions include conserved (∗) residues and are localized in or close to the allosteric sites of loop 1 (fK13, ∗fK19, ∗fF23, fS29, fL35, ∗fY39, and ∗fS55), the ammonia gate (∗fR5 and ∗fK99), and the interface hinge (fD74) (Figure 2A). NBY was used to replace tyrosine ∗fY39, phenylalanine ∗fF23, and aspartate fD74 (a phenylalanine in yeast ImGPS), whereas mNPK was used to replace lysines fK13, ∗fK19, and ∗fK99. For the design of AzoF replacements, we first calculated its rotamer library and incorporated AzoF into each of the ten positions in silico. The nine residues where the rotamers minimally overlapped with van der Waals radii of neighboring residues were replaced by AzoF (fR5, fK13, fK19, fF23, fS29, fL35, fY39, fS55, and fK99). Fifteen UAA-HisFs were then expressed in Escherichia coli cells using previously designed tRNACUA/aminoacyl-tRNA-synthetase pairs; these pairs have been shown to scarcely incorporate endogenous, natural amino acids, while each UAA was incorporated with high efficiency (Bose et al., 2006Bose M. Groff D. Xie J. Brustad E. Schultz P.G. The incorporation of a photoisomerizable amino acid into proteins in E. coli.J. Am. Chem. Soc. 2006; 128: 388-389Crossref PubMed Scopus (143) Google Scholar, Deiters et al., 2006Deiters A. Groff D. Ryu Y. Xie J. Schultz P.G. A genetically encoded photocaged tyrosine.Angew. Chem. Int. Ed. 2006; 45: 2728-2731Crossref PubMed Scopus (150) Google Scholar, Gautier et al., 2010Gautier A. Nguyen D.P. Lusic H. An W. Deiters A. Chin J.W. Genetically encoded photocontrol of protein localization in mammalian cells.J. Am. Chem. Soc. 2010; 132: 4086-4088Crossref PubMed Scopus (195) Google Scholar). Purification resulted in yields of 1–87 mg/L expression medium. UAA incorporation was analyzed by tryptic digest coupled to mass spectrometry (MS) analysis and for fK13AzoF with UV-visible spectroscopy (Figure S1). The two variants fK13mNPK and fK19mNPK were discarded after this step, because we could not confirm their identity. For the sake of brevity, we use the term fposUAA to name a HisF protein containing a UAA at position pos, e.g., fS55AzoF designates the HisF protein with AzoF at position S55; likewise ImGPS(fS55AzoF) designates the ImGPS complex containing AzoF at position S55 of HisF. The remaining 13 UAA-HisFs were screened for their ability to activate HisH in the presence of ProFAR in their “as isolated” states (caged or E), after irradiation with UV light at a wavelength of 365 nm (decaged or Z) and, for AzoF-HisFs, after additional irradiation with visible light at a wavelength of 420 nm (E) (Figure 2B). Suitable candidates for in-depth characterization were selected according to the following criteria: (1) At least 20% wild-type (WT) HisH activity had to be retained in ImGPS complexes containing the irradiated caged UAA-HisFs or the more active isomer of AzoF; (2) HisH activity was altered at least 1.5-fold upon irradiation (light regulation factor [LRF]). HisH activities in ImGPS(fK19AzoF), ImGPS(fF23AzoF), ImGPS(fY39AzoF), ImGPS(fF23NBY), and ImGPS(fY39NBY) did not reach 20% WT activity, though irradiation of fY39NBY should in principle restore the WT situation. Native MS suggests that considerable quantities (20%–60%) of reduced NBY existed in both fF23NBY and fY39NBY, rationalizing missing decaging. Moreover, also the remaining intact NBY-HisF proteins could not be decaged (Figure S2). However, during the course of these studies and as an alternative to NBY, the UAA NPY was introduced and shown to possess higher decaging efficiencies (Luo et al., 2017Luo J. Torres-Kolbus J. Liu J. Deiters A. Genetic encoding of photocaged tyrosines with improved light-activation properties for the optical control of protease function.ChemBioChem. 2017; 18: 1442-1447Crossref PubMed Scopus (31) Google Scholar). Indeed, HisH activities in ImGPS(fF23NPY) and ImGPS(fY39NPY) were >20% WT after irradiation (Figure 2B). In the second selection step, LRFs of HisH in ImGPS(fF23NPY), ImGPS(fY39NPY), and ImGPS containing the remaining eight UAA-HisFs were measured and the results were displayed in a histogram (Figure 2C). Only four UAA-HisFs led to LRFs >1.5, of which fF23NPY was the only caged UAA-HisF whose decaging did not restore WT-HisH activity. This result was rationalized by a control experiment with fF23Y, which showed that a tyrosine residue at position 23 leads to a drastic reduction to ∼30% of WT-HisH activity. We therefore excluded fF23NPY from our further studies and finally selected fS55AzoF, fY39NPY, and fK99mNPK (Figure 2D) as most promising UAA-HisFs for light regulation of ImGPS allostery. Before analyzing light regulation in detail, we characterized the non-irradiated UAA-HisFs fS55AzoF, fY39NPY, and fK99mNPK with respect to structure, stability, and function. Circular dichroism spectroscopy and analytical size-exclusion chromatography demonstrated that the three UAA-HisFs are properly folded monomers and form stoichiometric complexes with WT-HisH (Figures 3A–3C ). Crystal structure analysis of fY39NPY (PDB: 6rtz) and fS55AzoF (PDB: 6ru0) in complex with WT-HisH showed that only the tyrosine and phenylalanine moieties could be resolved (Figures S3A–S3B), confirming the high flexibility of the UAA side chains, as observed in MD simulations (Figures S3C–S3F). Nevertheless, in fY39NPY, an additional electron density cloud indicated the approximate position of the o-nitropiperonyl moiety. This moiety shoved aside fH228, causing a destabilization of helix α8′ in HisF (Figure S3G). As a consequence, certain residues of this helix took on unallowed conformations in the Ramachandran plot (Lovell et al., 2003Lovell S.C. Davis I.W. Arendall W.B. Bakker P.I.W. de Word J.M. Prisant M.G. Richardson J.S. Richardson D.C. Structure validation by Cα geometry: Φ, Ψ and Cβ deviation.Proteins. 2003; 50: 437-450Crossre" @default.
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• W2972077284 date "2019-11-01" @default.
• W2972077284 modified "2023-10-10" @default.
• W2972077284 title "Light Regulation of Enzyme Allostery through Photo-responsive Unnatural Amino Acids" @default.
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