Substrate binding tunes the reactivity of hispidin 3-hydroxylase, a flavoprotein monooxygenase involved in fungal bioluminescence

Abstract

Fungal bioluminescence was recently shown to depend on a unique oxygen-dependent system of several enzymes. However, the identities of the enzymes did not reveal the full biochemical details of this process, as the enzymes do not bear resemblance to those of other luminescence systems, and thus the properties of the enzymes involved in this fascinating process are still unknown. Here, we describe the characterization of the penultimate enzyme in the pathway, hispidin 3-hydroxylase, from the luminescent fungus Mycena chlorophos (McH3H), which catalyzes the conversion of hispidin to 3-hydroxyhispidin. 3-Hydroxyhispidin acts as a luciferin substrate in luminescent fungi. McH3H was heterologously expressed in Escherichia coli and purified by affinity chromatography with a yield of 100 mg/liter. McH3H was found to be a single component monomeric NAD(P)H-dependent FAD-containing monooxygenase having a preference for NADPH. Through site-directed mutagenesis, based on a modeled structure, mutant enzymes were created that are more efficient with NADH. Except for identifying the residues that tune cofactor specificity, these engineered variants may also help in developing new hispidin-based bioluminescence applications. We confirmed that addition of hispidin to McH3H led to the formation of 3-hydroxyhispidin as sole aromatic product. Rapid kinetic analysis revealed that reduction of the flavin cofactor by NADPH is boosted by hispidin binding by nearly 100-fold. Similar to other class A flavoprotein hydroxylases, McH3H did not form a stable hydroperoxyflavin intermediate. These data suggest a mechanism by which the hydroxylase is tuned for converting hispidin into the fungal luciferin. Fungal bioluminescence was recently shown to depend on a unique oxygen-dependent system of several enzymes. However, the identities of the enzymes did not reveal the full biochemical details of this process, as the enzymes do not bear resemblance to those of other luminescence systems, and thus the properties of the enzymes involved in this fascinating process are still unknown. Here, we describe the characterization of the penultimate enzyme in the pathway, hispidin 3-hydroxylase, from the luminescent fungus Mycena chlorophos (McH3H), which catalyzes the conversion of hispidin to 3-hydroxyhispidin. 3-Hydroxyhispidin acts as a luciferin substrate in luminescent fungi. McH3H was heterologously expressed in Escherichia coli and purified by affinity chromatography with a yield of 100 mg/liter. McH3H was found to be a single component monomeric NAD(P)H-dependent FAD-containing monooxygenase having a preference for NADPH. Through site-directed mutagenesis, based on a modeled structure, mutant enzymes were created that are more efficient with NADH. Except for identifying the residues that tune cofactor specificity, these engineered variants may also help in developing new hispidin-based bioluminescence applications. We confirmed that addition of hispidin to McH3H led to the formation of 3-hydroxyhispidin as sole aromatic product. Rapid kinetic analysis revealed that reduction of the flavin cofactor by NADPH is boosted by hispidin binding by nearly 100-fold. Similar to other class A flavoprotein hydroxylases, McH3H did not form a stable hydroperoxyflavin intermediate. These data suggest a mechanism by which the hydroxylase is tuned for converting hispidin into the fungal luciferin. Bioluminescence is a natural phenomenon in which living organisms emit visible light. Such a phenomenon has been observed in a large variety of organisms: Fireflies, jellyfish, bacteria, and fungi. Bioluminescence is often based on a specific precursor molecule, a luciferin substrate, that is converted by a luciferase with concomitant light generation. Although fungal bioluminescence has been observed since ancient times (1Harvey E.N. Shining fish, flesh, and wood.A History of Luminescence From the Earliest Times Until 1900. American Philosophical Society, Philadelphia, PA1957: 461-50710.5962/bhl.title.14249Google Scholar) and a significant number of luminescent fungi have been described (2Chew A.L.C. Tan Y.S. Desjardin D.E. Musa M.Y. Sabaratnam V. Four new bioluminescent taxa of Mycena sect. Calodontes from Peninsular Malaysia.Mycologia. 2014; 106 (24891424): 976-98810.3852/13-274Crossref PubMed Scopus (15) Google Scholar), the precise molecular basis for fungal luminescence has remained elusive. All reported luminescent fungi generate the same glow within the emission range of 520−530 nm (3Desjardin D.E. Oliveira A.G. Stevani C.V. Fungi bioluminescence revisited.Photochemical and Photobiological Sciences. 2008; 7 (18264584): 170-18210.1039/b713328fCrossref PubMed Scopus (85) Google Scholar) and are likely to share a bioluminescent system (4Oliveira A.G. Desjardin D.E. Perry B.A. Stevani C.V. Evidence that a single bioluminescent system is shared by all known bioluminescent fungal lineages.Photochem. Photobiol. Sci. 2012; 11 (22495263): 848-85210.1039/c2pp25032bCrossref PubMed Scopus (41) Google Scholar). Fungal bioluminescence has attracted the interest of many research groups for a long time (3Desjardin D.E. Oliveira A.G. Stevani C.V. Fungi bioluminescence revisited.Photochemical and Photobiological Sciences. 2008; 7 (18264584): 170-18210.1039/b713328fCrossref PubMed Scopus (85) Google Scholar, 5Stevani C.V. Oliveira A.G. Mendes L.F. Ventura F.F. Waldenmaier H.E. Carvalho R.P. Pereira T.A. Current status of research on fungal bioluminescence: Biochemistry and prospects for ecotoxicological application.Photochem. Photobiol. 2013; 89 (23845086): 1318-132610.1111/php.12135Crossref PubMed Scopus (21) Google Scholar). Airth and Foerster (6Airth R.L. McElroy W.D. Light emission from extracts of luminous fungi.J. Bacteriol. 1959; 77 (13630879): 249-25010.1128/JB.77.2.249-250.1959Crossref PubMed Google Scholar, 7Airth R.L. Foerster G.E. The isolation of catalytic components required for cell-free fungal bioluminescence.Arch. Biochem. Biophys. 1962; 97 (13859816): 567-57310.1016/0003-9861(62)90124-8Crossref PubMed Scopus (46) Google Scholar, 8Airth R.L. Foerster G.E. Enzymes associated with bioluminescence in Panus Stypticus luminescens and Panus Stypticus non-luminescens.J. Bacteriol. 1964; 88 (14234795): 1372-137910.1128/JB.88.5.1372-1379.1964Crossref PubMed Google Scholar) demonstrated the luciferin–luciferase reaction by mixing fungal extracts and NAD(P)H. In their work, they suggested that the bioluminescence reaction in fungi is a two-step process involving a NAD(P)H-dependent soluble enzyme and a membrane-bound luciferase (7Airth R.L. Foerster G.E. The isolation of catalytic components required for cell-free fungal bioluminescence.Arch. Biochem. Biophys. 1962; 97 (13859816): 567-57310.1016/0003-9861(62)90124-8Crossref PubMed Scopus (46) Google Scholar). The soluble enzyme catalyzes the first step, producing luciferin. In the second step, the luciferase catalyzes the oxidation of the fungal luciferin, resulting in light emission. For many decades, no significant progress was made concerning the identification of the fungal luciferin and luciferase system. In 2011, Mori et al. (9Mori K. Kojima S. Maki S. Hirano T. Niwa H. Bioluminescence characteristics of the fruiting body of Mycena chlorophos.Luminescence. 2011; 26 (21370386): 604-61010.1002/bio.1280Crossref PubMed Scopus (21) Google Scholar) reported that bioluminescence in Mycena chlorophos depends on a specific enzymatic reaction, confirming Airth's work. Subsequent work by Teranishi (10Teranishi K. Localization of the bioluminescence system in the pileus of Mycena chlorophos.Luminescence. 2016; 31 (26280456): 594-59910.1002/bio.3001Crossref PubMed Scopus (10) Google Scholar) revealed that part of the bioluminescence system in M. chlorophos is localized at the cell membrane. Moreover, it was found that trans-4-hydroxycinnamic acid and trans-3,4-dihydroxycinnnamic acid could increase the light intensity in the living gills of M. chlorophos (11Teranishi K. Trans-p-Hydroxycinnamic acid as a bioluminescence-activating component in the pileus of the luminous fungus Mycena chlorophos.Tetrahedron. 2016; 72: 726-73310.1016/j.tet.2015.12.027Crossref Scopus (11) Google Scholar, 12Teranishi K. Second bioluminescence-activating component in the luminous fungus Mycena chlorophos.Luminescence. 2017; 32 (27271205): 182-18910.1002/bio.3165Crossref PubMed Scopus (6) Google Scholar), and flavins were likely to be the light emitters in bioluminescence (13Teranishi K. Identification of possible light emitters in the gills of a bioluminescent fungus Mycena chlorophos.Luminescence. 2016; 31 (27021064): 1407-141310.1002/bio.3129Crossref PubMed Scopus (4) Google Scholar). Over the last few years, details on the molecular basis of fungal bioluminescence have emerged. Purtov et al. (14Purtov K.V. Petushkov V.N. Baranov M.S. Mineev K.S. Rodionova N.S. Kaskova Z.M. Tsarkova A.S. Petunin A.I. Bondar V.S. Rodicheva E.K. Medvedeva S.E. Oba Y. Oba Y. Arseniev A.S. Lukyanov S. et al.The chemical basis of fungal bioluminescence.Angew. Chem. Int. Ed. Engl. 2015; 54 (26094784): 8124-812810.1002/anie.201501779Crossref PubMed Scopus (68) Google Scholar) showed that hispidin is converted to 3-hydroxyhispidin in the presence of molecular oxygen, NAD(P)H, and a hispidin 3-hydroxylase. Kaskova et al. (15Kaskova Z.M. Dörr F.A. Petushkov V.N. Purtov K.V. Tsarkova A.S. Rodionova N.S. Mineev K.S. Guglya E.B. Kotlobay A. Baleeva N.S. Baranov M.S. Arseniev A.S. Gitelson J.I. Lukyanov S. Suzuki Y. et al.Mechanism and color modulation of fungal bioluminescence.Sci. Adv. 2017; 3 (28508049): e160284710.1126/sciadv.1602847Crossref PubMed Scopus (38) Google Scholar) clearly illustrated the fungal bioluminescence mechanism in which a luciferase oxidizes 3-hydroxyhispidin into a high-energy intermediate which decays with concomitant light emission. The discovery of the fungal luciferin (3-hydroxyhispidin) biosynthesis and recycling pathway was a breakthrough reported by Kotlobay et al. (16Kotlobay A.A. Sarkisyan K.S. Mokrushina Y.A. Marcet-Houben M. Serebrovskaya E.O. Markina N.M. Somermeyer L.G. Gorokhovatsky A.Y. Vvedensky A. Purtov K.V. Petushkov V.N. Rodionova N.S. Chepurnyh T.V. Fakhranurova L.I. Guglya E.B. et al.Genetically encodable bioluminescent system from fungi.Proc. Natl. Acad. Sci. U. S. A. 2018; 115 (30478037): 12728-1273210.1073/pnas.1803615115Crossref PubMed Scopus (51) Google Scholar). In their study, the entire cycle was elucidated which involves a hispidin-synthase, a hispidin 3-hydroxylase (H3H), a luciferase, and a caffeylpyruvate hydrolase. The cluster encompassing the respective genes was found to be conserved in other luminescent fungi. This suggests that all luminescent fungi share the same luciferin/luciferase system. The enzymes and chemistry involved do not show any resemblance with other hitherto known bioluminescence systems (Scheme 1). Recently, a plant was equipped with the fungal genes responsible for luminescence. Upon insertion of the four mentioned genes from the bioluminescent mushroom Neonothopanus nambi into the DNA of tobacco plants, luminous plants were created (17Mitiouchkina T. Mishin A.S. Somermeyer L.G. Markina N.M. Chepurnyh T.V. Guglya E.B. Karataeva T.A. Palkina K.A. Shakhova Fakhranurova L.I. Tsarkova A.S. et with PubMed Scopus Google Scholar). The of the fungal system in tobacco plants that it be with the plant substrate The luminescence was also found to be with the luminescence that the fungal enzyme system has Here, we on the and biochemical characterization of hispidin 3-hydroxylase from Mycena chlorophos of new in the by of the American of and Sciences. Scholar). This enzyme was found to be a monomeric FAD-containing which catalyzes the of hispidin to form 3-hydroxyhispidin (Scheme 1). The the first into the properties of a fungal enzyme and has in enzyme variants with cofactor The system and elucidated catalytic a basis for into the molecular of fungal Although the genes responsible for luminescence in the fungus M. chlorophos have been the respective enzymes have not been in the enzymatic properties of a fungal we to the hispidin hydroxylase from M. A with the McH3H confirmed that this enzyme is a of many fungal that to be in of the A in the of for which the has been elucidated confirmed with class A flavoprotein N.M. a class of PubMed Scopus Google Scholar, S. Biochem. Biophys. 2014; PubMed Scopus Google Scholar). These are single component that a cofactor and depend on NADPH as for The was found with hydroxylase from Pereira M.S. mechanism for the conversion of into by the monooxygenase PubMed Scopus (10) Google Scholar). A of from and hydroxylase from the class A flavoprotein revealed of several and the a is conserved which part of the that the of the cofactor of the of the in acid PubMed Scopus Google Scholar). The of McH3H also other conserved that a binding of the cofactor Identification of a conserved in flavoprotein with a in Sci. PubMed Scopus Google Scholar). the from in the encompassing residues The part in the of has been shown to tune the NADPH of of PubMed Scopus (46) Google Scholar) and may suggest that McH3H has a S. of hydroxylase and flavoprotein 2018; PubMed Scopus (6) Google Scholar). McH3H was as in coli 100 of could be purified affinity on the acid the molecular of is and that of McH3H is to the in a with the of the Upon of the McH3H was with the McH3H revealed molecular of that in the enzyme is The purified color which is in with the that all of a class A flavoprotein monooxygenase that a The of McH3H revealed flavoprotein with at nm and nm The was and that the enzyme is in the form as the by the at nm and the at nm is with in a of of the flavin cofactor 1). The of the cofactor as was confirmed with which in formation of and other are of several class A as and and were no significant on the of McH3H was observed in the presence of 100 of McH3H with were was found that hispidin in the visible and that the of hispidin is by This the of the in which the of is by the at nm of we observed that hispidin to in on the and we that is the to as it showed on the of hispidin at all biochemical of McH3H were The for the of hispidin with This is hispidin several groups with on we for the enzyme by a as McH3H molecular for the of hispidin (Scheme 1). we could that McH3H is on 100 hispidin and a of was the the enzyme a with the and The of the enzyme at was by the the A. a flavin system for and PubMed Scopus Google Scholar) This revealed that McH3H is stable at with of on these we to as for all The was also to the of substrate binding on the of the the was by from in the of hispidin to with hispidin This analysis also of the of For for the of McH3H in 3-hydroxyhispidin we to the product. The enzyme was to hispidin which the reaction was and by was found that McH3H the full of hispidin into 3-hydroxyhispidin other were A range of other and were also as possible However, analysis showed that of the showed any This suggests that McH3H is specific for A substrate is for class A flavoprotein that the (3-hydroxyhispidin) by of McH3H is the substrate for the fungal luciferase light a luminescence reaction was This revealed that visible light be observed in a mixing and coli of any of the components light This that McH3H is a for identifying hispidin as substrate, McH3H was found to show with NADPH as This is for class A flavoprotein they are specific for was a preference for of the we The kinetic were at Although McH3H for NADPH and in the presence of hispidin A and the for NADPH was for The of the enzyme for hispidin was found to be with a of and of the In the of McH3H also as a NADPH this was and These show that a substrate, also acts as A flavoprotein are known to of the which not to resulting in formation A. substrate, many in 2018; PubMed Scopus Google Scholar). The McH3H was by the formation and revealed NADPH was and in the of NADH. the of we a analysis of McH3H to kinetic The reaction cycle of a and A. be not to be the of Biochem. Sci. 31 PubMed Scopus Google Scholar, Google Scholar, S. V. of Biochem. PubMed Scopus Google Scholar). the was by in the of McH3H a preference for NADPH the respective were NADPH. The of the and of the were by the reduction of the at NADPH and the reaction at The reduction of the is boosted hispidin is reduction from to A and binding of substrate efficient reduction of the flavin cofactor by NADPH. The of NADPH for the and were and A and the affinity for hispidin was by the hispidin a of NADPH This the that the hydroxylase has a affinity aromatic substrate The of the flavin cofactor was also mixing the enzyme with NADPH with NADPH and hispidin This showed that the enzyme in the NADPH is This with the of flavin reduction in the of hispidin is a and significant reduction of the flavin is This suggests that the of reduction is not This is also the of reduction The second of the reaction cycle was by mixing the enzyme with the we the mixing the McH3H with in the and presence of In the of substrate, McH3H with a intermediate The intermediate has at nm The reaction led to full of the cofactor in single kinetic hispidin in the a and full was observed The kinetic and these were in the presence of McH3H is not efficient in the flavin intermediate. and the addition of did not help to the This is in with the observed of of the is not phenomenon for class A flavoprotein The of the This is for class A flavoprotein has been observed in M. of a intermediate in the reaction of a flavoprotein 2008; PubMed Scopus Google Scholar, A. the basis of in PubMed Scopus Google Scholar) and suggests a binding of molecular it with the The preference of McH3H for NADPH was as it with other flavoprotein for NADH. In of a of McH3H we a K. S. M. K. and in Four that in 77 PubMed Scopus Google Scholar) and a with the by the of with the and by the work from et al. of of PubMed Scopus (46) Google Scholar, Google Scholar), we in a which in McH3H from to Although the in a of the of the cofactor in the modeled structure, be clearly from the in 32 to on this and on on we residues and as for Upon analysis of with the of a flavoprotein hydroxylase from we to generate at these all for the for NADPH and the were not 1). The mutant was which is more efficient with NADH. This reduction of the for the for NADPH is also by of in the a this mutant McH3H cofactor preference with the the in a which is the 1). the cofactor depends on and be by single The created McH3H may be of interest the hydroxylase as of the of with of McH3H and single in a new In the study, we that hispidin 3-hydroxylase from M. chlorophos be expressed in coli as a soluble monomeric FAD-containing The enzyme could be purified by affinity chromatography and properties were analysis confirmed that McH3H a of hispidin to generate 3-hydroxyhispidin as a single aromatic product. other aromatic for McH3H could be that the hydroxylase is substrate The substrate of McH3H may be to specific in hispidin-based luciferin biosynthesis in fungi as part of the fungal bioluminescence process (16Kotlobay A.A. Sarkisyan K.S. Mokrushina Y.A. Marcet-Houben M. Serebrovskaya E.O. Markina N.M. Somermeyer L.G. Gorokhovatsky A.Y. Vvedensky A. Purtov K.V. Petushkov V.N. Rodionova N.S. Chepurnyh T.V. Fakhranurova L.I. Guglya E.B. et al.Genetically encodable bioluminescent system from fungi.Proc. Natl. Acad. Sci. U. S. A. 2018; 115 (30478037): 12728-1273210.1073/pnas.1803615115Crossref PubMed Scopus (51) Google Scholar). McH3H and with a preference for NADPH. These characteristics are in with of class A flavoprotein In McH3H significant with a FAD-containing hydroxylase and mechanism of PubMed Scopus Google Scholar, and in the of Biochem. Biophys. PubMed Scopus Google Scholar). enzymes also kinetic binding a reduction of the In the of McH3H a significant In the not all is for as significant is In that the enzyme not the as observed with other class A flavoprotein the show that of the of NADPH is to a of A revealed that the hispidin in luminescent fungi identities in the from to in This that hispidin 3-hydroxylase in luminescent fungi may have a preference for NADPH. However, McH3H also to with as and A. K. M. A. of at the of hydroxylase from Biochem. PubMed Scopus Google Scholar, and properties of from Google Scholar, H. of a Chem. PubMed Google Scholar, S. and characterization of from Biophys. 2012; PubMed Scopus Google Scholar). acid analysis that the these enzymes is conserved on several of McH3H were that revealed that the of McH3H could be The are in with the more that the of NADPH to with residues the of to with residues N.S. A. of the of a by PubMed Scopus Google Scholar). Similar with other flavoprotein have been reported S. M. is the of in the monooxygenase Biophys. 2014; PubMed Scopus Google Scholar, N.M. of NADPH in Biochem. PubMed Scopus Google Scholar). McH3H with were Such variants of McH3H may as for of the hydroxylase in developing bioluminescence Except for of the tuned may be for the presence of In the work, we that McH3H is a soluble monomeric FAD-containing hydroxylase that catalyzes the of hispidin to form 3-hydroxyhispidin. Rapid kinetic analysis revealed that McH3H has a affinity for The kinetic data that formation of the the reduction process of by NADPH to of reduction of McH3H is by binding of the reaction and no hispidin is have been that tune the This in with a preference for as The to is for the of the enzyme as a as NADPH is enzyme variants for the of may the of fungal luminescent systems in as engineered luminescent plants (17Mitiouchkina T. Mishin A.S. Somermeyer L.G. Markina N.M. Chepurnyh T.V. Guglya E.B. Karataeva T.A. Palkina K.A. Shakhova Fakhranurova L.I. Tsarkova A.S. et with PubMed Scopus Google Scholar). NADPH and were from was from was from and the enzyme were from coli was as for and All other were from was a M. A of hispidin from Scopus Google Scholar). The coli and from M. chlorophos were by DNA The and genes were into and by the These with for This has and The has and a The reaction and The for and for for was to for and to for to the enzymes. of the reaction was to coli to do the on with were and in with The were and for to the of the The coli the was at for in was was were at and at the for in the were in at and were by and at at the for The was first the cell-free was to the was to from the with was to The enzymes with 100 The McH3H was by the Enzymes were and at The of purified McH3H was by a of at nm The was by of enzyme and with the known A. and and in and in Google Scholar). the of the flavin was that involves of into as described A. and and in and in Google Scholar). The molecular of McH3H was based on the acid by the at S. The of in be from their acid PubMed Scopus Google Scholar, for of of from cell in a with PubMed Scopus Google Scholar). the was into coli were expressed in by of at for were at and at the for in the The was by at and at the aromatic reaction of McH3H by analysis a conversion of hispidin was The reaction 100 in reaction was The reaction was in a at for the reaction was by of and the was with The was and The was by The reaction was the same reaction for analysis on in The reaction was in a at for a was with and the were at for The was by The reaction was the same reaction for analysis on 100 in The reaction was in a at for a was for with The was and by reaction was the same reaction The of the enzyme was at by at purified in a 100 NADPH and The reaction was by the was on system were from the of the reaction the on the of the enzyme was at by the the A. a flavin system for and PubMed Scopus Google Scholar). The purified in at intensity of flavin was the were from to with step, a time of at The of the first of the observed flavin was as the McH3H was by the This was in reaction hispidin and NADPH and the reaction was by was to the reaction the reaction was still in the The addition of increase in as could and The were in at by system The were as A is the and is in the The of McH3H and were in at by of hispidin were in The of in the reaction was The reaction and the reaction was by the was on system were from the of the reaction by the of the substrate at a of the data were with the the to the kinetic The and of McH3H were the of a equipped with a All were in were in by mixing of at The of and were the described S. A. of a monooxygenase from PubMed Scopus Google Scholar). The at nm and nm were to to the observed All data were the and was from the McH3H by McH3H was for analysis on The enzyme McH3H was to a with A and were as for the of the molecular The acid of McH3H was to on the by the A was based on the of Pereira M.S. mechanism for the conversion of into by the monooxygenase PubMed Scopus (10) Google Scholar). Upon of the with the of and and the in was in the McH3H in variants of the The site-directed was by the as a The are in and the are the The reaction and which reaction and The was to the for of for for and for and a at for The were with at to the which the reaction were into coli All data are within the Y. the for a with hispidin 3-hydroxylase from Mycena chlorophos luciferase from Mycena chlorophos from hydroxylase from

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