Structural studies of codeinone reductase reveal novel insights into aldo-keto reductase function in benzylisoquinoline alkaloid biosynthesis

Abstract

Benzylisoquinoline alkaloids (BIAs) are a class of specialized metabolites with a diverse range of chemical structures and physiological effects. Codeine and morphine are two closely related BIAs with particularly useful analgesic properties. The aldo-keto reductase (AKR) codeinone reductase (COR) catalyzes the final and penultimate steps in the biosynthesis of codeine and morphine, respectively, in opium poppy (Papaver somniferum). However, the structural determinants that mediate substrate recognition and catalysis are not well defined. Here, we describe the crystal structure of apo-COR determined to a resolution of 2.4 Å by molecular replacement using chalcone reductase as a search model. Structural comparisons of COR to closely related plant AKRs and more distantly related homologues reveal a novel conformation in the β1α1 loop adjacent to the BIA-binding pocket. The proximity of this loop to several highly conserved active-site residues and the expected location of the nicotinamide ring of the NADP(H) cofactor suggest a model for BIA recognition that implies roles for several key residues. Using site-directed mutagenesis, we show that substitutions at Met-28 and His-120 of COR lead to changes in AKR activity for the major and minor substrates codeinone and neopinone, respectively. Our findings provide a framework for understanding the molecular basis of substrate recognition in COR and the closely related 1,2-dehydroreticuline reductase responsible for the second half of a stereochemical inversion that initiates the morphine biosynthesis pathway. Benzylisoquinoline alkaloids (BIAs) are a class of specialized metabolites with a diverse range of chemical structures and physiological effects. Codeine and morphine are two closely related BIAs with particularly useful analgesic properties. The aldo-keto reductase (AKR) codeinone reductase (COR) catalyzes the final and penultimate steps in the biosynthesis of codeine and morphine, respectively, in opium poppy (Papaver somniferum). However, the structural determinants that mediate substrate recognition and catalysis are not well defined. Here, we describe the crystal structure of apo-COR determined to a resolution of 2.4 Å by molecular replacement using chalcone reductase as a search model. Structural comparisons of COR to closely related plant AKRs and more distantly related homologues reveal a novel conformation in the β1α1 loop adjacent to the BIA-binding pocket. The proximity of this loop to several highly conserved active-site residues and the expected location of the nicotinamide ring of the NADP(H) cofactor suggest a model for BIA recognition that implies roles for several key residues. Using site-directed mutagenesis, we show that substitutions at Met-28 and His-120 of COR lead to changes in AKR activity for the major and minor substrates codeinone and neopinone, respectively. Our findings provide a framework for understanding the molecular basis of substrate recognition in COR and the closely related 1,2-dehydroreticuline reductase responsible for the second half of a stereochemical inversion that initiates the morphine biosynthesis pathway. Opiates are essential and currently irreplaceable medicines for the management of severe pain associated with severe burns, postoperative recovery, cancer treatment, and palliative care (1World Health Organization Model List of Essential Medicines, 21st List. World Health Organization, Geneva2019Google Scholar). Globally, the licit demand for eight billion defined daily doses per year (459 tons of morphine equivalents) is almost entirely supplied by the agricultural production of opium poppy plants in Turkey, Tasmania, and Eastern Europe (2International Narcotics Control Board Narcotic drugs - technical report. United Nations Location, Vienna2019Google Scholar). While several opiate pharmaceuticals are isolated directly from the plant (e.g., morphine and codeine), others are derived from the structurally related, nonmedicinal alkaloid thebaine to yield a suite of semisynthetic opiates with refined pharmacological properties (e.g., oxycodone, hydrocodone, and buprenorphine (2International Narcotics Control Board Narcotic drugs - technical report. United Nations Location, Vienna2019Google Scholar)). Driven by the large capital investment required to establish pharmaceutical manufacturing capacity, coupled with the challenges of sustaining agricultural productivity in an increasingly unpredictable climate and securing global supply chains in a frequently unstable geopolitical environment, recent attention has focused on the potential biosynthesis of medicinal opiates in engineered microorganisms. Heterologous production systems also provide new opportunities to introduce novel enzyme biocatalysts and to direct the flux of metabolites toward specific products through protein and genome engineering, thus offering the possibility of creating novel compounds generally inaccessible through traditional plant breeding or postextraction chemical modification. Key to the success of this emerging synthetic biology strategy is a deep understanding of the genes, enzymes, and pathways that have evolved in opium poppy over tens of millions of years (3Li Y. Winzer T. He Z. Graham I.A. Over 100 million years of enzyme evolution underpinning the production of morphine in the Papaveraceae family of flowering plants.Plant Commun. 2020; 1: 100029Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). In opium poppy, the first committed step in morphine biosynthesis is the stereochemical inversion of (S)-reticuline to (R)-reticuline by reticuline epimerase (REPI) (4Guo L. Winzer T. Yang X. Li Y. Ning Z. He Z. Teodor R. Lu Y. Bowser T.A. Graham I.A. Ye K. The opium poppy genome and morphinan production.Science. 2018; 362: 343-347Crossref PubMed Scopus (118) Google Scholar, 5Farrow S.C. Hagel J.M. Beaudoin G. a W. Burns D.C. Facchini P.J. Stereochemical inversion of (S)-reticuline by a cytochrome P450 fusion in opium poppy.Nat. Chem. Biol. 2015; 11: 728-732Crossref PubMed Scopus (84) Google Scholar). Next, a carbon–carbon phenol-coupling establishes the promorphinan scaffold in salutaridine (CYP719B1; salutaridine synthase; SalSyn), and synthase; to the morphinan structure of medicinal opiates Facchini P.J. Benzylisoquinoline alkaloid biosynthesis in opium Scopus Google Scholar, Facchini The alkaloid Scopus Google Scholar). the major to morphine through an of the a codeinone is to codeine by codeinone reductase of the In an minor from and by is to morphine in two steps in with the major of to codeine is to opiate biosynthesis and has as a in in engineered K. for and semisynthetic Chem. Biol. PubMed Scopus Google Scholar). physiological in the of codeinone and establishes an the two at R. The of to codeinone in morphine biosynthesis Scopus Google Scholar). COR is to the in codeinone and to yield codeine or respectively, COR the with codeine as the substrate L. Facchini P.J. reductase with and in opium 2018; Scopus Google Scholar). a the of COR in of engineered to the of at the of codeine or K. for and semisynthetic Chem. Biol. PubMed Scopus Google Scholar, L. Facchini P.J. reductase with and in opium 2018; Scopus Google Scholar, K. biosynthesis of in 2015; PubMed Scopus Google Scholar). the of and COR the of to a that in opium poppy plants X. Hagel J.M. L. R. Facchini P.J. is in codeine and morphine biosynthesis in opium poppy.Nat. Chem. Biol. PubMed Scopus Google Scholar). the of to thus the of for to by the of to the production of opiates in engineered of COR and the of opiate this enzyme a COR with a more activity a of on the structural responsible for substrate recognition in COR has the success of X. Hagel J.M. L. R. Facchini P.J. is in codeine and morphine biosynthesis in opium poppy.Nat. Chem. Biol. PubMed Scopus Google Scholar). COR to the large and AKR is by the and activity J.M. The aldo-keto reductase (AKR) Biol. PubMed Scopus Google Scholar). AKRs are in and have to a The aldo-keto Biol. 2015; PubMed Scopus Google Scholar). In AKRs to a range of of and specialized The range of plant metabolites AKR in biosynthesis R. in plants by of a PubMed Scopus Google (e.g., and alkaloids of the and in aldo-keto as in diverse plant and 2015; PubMed Scopus (84) Google Scholar)). of BIA chalcone reductase in biosynthesis R. of a novel reductase with chalcone in the biosynthesis of Scopus Google and alkaloid biosynthesis evolved in the and PubMed Scopus Google are closely related to COR and structure has for Structural of chalcone reductase and for Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). While several describe the of as a for the of the and have to an BIA-binding Structural of chalcone reductase and for Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The COR structure novel the substrate of BIA-binding AKR enzymes, has the of COR with a substrate that the production of opiates over The crystal structure of the to as using molecular replacement and refined to a resolution of 2.4 Å the to the severe in the the of that a the Å the of the the to the of the the of to a resolution of 2.4 Å resolution the and and 2.4 Å resolution in the and as a the in the and of COR with are in the and as chains The residues not have to with residues in chains residues in chains and in in chains and residues in residues in chains and residues in and for and of from in a new The structure of COR a and to the AKR The aldo-keto Biol. 2015; PubMed Scopus Google in eight a by eight to the The of the is with an and the of the is by two and large major roles in substrate The aldo-keto Biol. 2015; PubMed Scopus Google Scholar, J.M. of the reductase PubMed Scopus Google and are and by and to the loop to substrate and cofactor β1α1 and also to substrate and The β1α1 loop to the substrate and cofactor the loop of the substrate and the two key residues two residues and two residues and of the in and codeine to in the at a location expected for this to a of The crystal structure in this thus an with a for the cofactor to that for AKRs to or The for codeine or codeinone also to and in conformation with the closely related in the The of COR of with the of a of in of the in the The L. structure by of Biol. PubMed Scopus Google to structures in the with the structural to The the to as a enzyme from Å by chalcone reductase from Å is with of structures from the AKR show that the is more distantly related of the family as AKR Å show structural to COR of of of structural in AKRs are in the that major roles in substrate recognition J.M. of the reductase PubMed Scopus Google Scholar). The structural of COR is the of the β1α1 loop from the cofactor is particularly with structures of and The conformation of this loop to on of the more The conformation by the β1α1 loop the cofactor and to the for the alkaloid through the and of and the of show that Met-28 is to is also in the AKR to in BIA the of the β1α1 loop from the cofactor not of the residues in NADP(H) The loop in and is not the of the two residues and at the of the residues of loop in COR not the to this of the structure that residues a conformation that is to that in and from the in to the loop of COR and is residues and that of is residues COR loop over and the of the the key The residues in the loop from the the of the loop to the are to is in and show that residues are residues are in of the apo-COR crystal structure and a or on the substrate pocket. Structural comparisons of and show that or two residues of the the pocket. In residues are and However, the conformation by the β1α1 loop in COR from the pocket. to the cofactor and Structural of residues and expected this residues to cofactor the of at the the highly conserved at the of the loop the pocket. conserved at the structure the to the and a the of the and of the pocket. the loop in to the the loop in COR to the The and loop of COR that are to and to a is particularly in is residues the of structural the in COR and residues in and that a conserved in substrate at the of the that is not to COR in of the in the for this crystal the of the as crystal to at of that are the structure of the the structure of apo-COR that the highly conserved cofactor in AKRs is also in COR and structural of the AKR suggest a highly conserved of The aldo-keto Biol. 2015; PubMed Scopus Google Scholar, J.M. of the reductase PubMed Scopus Google Scholar). The an by and is expected to in an with the nicotinamide in the of the and the more on the Structural of chalcone reductase and for Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J.M. of the reductase PubMed Scopus Google Scholar). The loop and the β1α1 loop in Structural of chalcone reductase and for Biol. Chem. Full Text Full Text PDF PubMed Scopus Google is not in of over the NADP(H) cofactor as in the β1α1 loop is toward the as in more of the in COR is the of in a that is by and residues in of the AKR The of this closely with the nicotinamide ring in to for J.M. of the reductase PubMed Scopus Google Scholar). of also with the of NADP(H) in the The on the nicotinamide ring is expected to with the chains of and are to the and the chains of and The also with and in COR to a in that an with the that is not in COR Structural of chalcone reductase and for Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In COR and the is also by several with the and with the and with the In the apo-COR a in the with from the adjacent that this not in the NADP(H) of The and are The substrates in the plant for the by COR are codeinone and in and in engineered COR also and the and L. 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Graham I.A. biosynthesis in opium poppy a fusion 2015; PubMed Scopus Google Scholar). residues to codeine are and as well as and are of the of codeine toward a conformation to that of The of of with The of this crystal structure of apo-COR with The a with two on of the is of the of the that the in and is in a is not conserved in COR and to for the The of of the and the of COR in a in the plant suggest that the the of and COR in an environment, and protein at to of or at in to to the and of as in K. model for understanding and evolution of plant Scopus Google Scholar). The of the adjacent to the is also to the potential roles of residues in the of The apo-COR crystal of and AKR closely and to a of the of key residues with to and substrate In and COR protein two and two the of in that the lead to protein with the of in the COR crystal structure the of a minor at the expected molecular of and COR using to the major and and to the minor reductase activity as L. Facchini P.J. reductase with and in opium 2018; Scopus Google Scholar). the and not for in the protein half the of the activity in the and respectively, with that of residues enzyme the activity to protein of the and in a of activity and of activity and residues in substrate and recognition for on the of the plant AKRs and substitutions at the of the substrate to COR activity and The residues that by the not COR activity and are in loop the of the and or The of residues the substrate that are with the of the residues for substrates in for and residues of the and the ring in in a the activity of in activity not that of the The that BIA-binding activity more activity that the of the substitutions on the substrate for in is to the structurally the on the of the substrate for in from and more The The first the that not COR activity and of of residues the substrate and The second the that not COR of residues the substrate and the two COR activity more activity more The are the that and COR of the and of a the also using not enzyme activity as the for the enzyme or the more COR a reductase activity In the of in the are to in the to codeine at from COR also to in of However, and to a more to of of the two the of codeine as COR to The to COR in the and of codeine of of to the COR structure the closely related AKR structure with to the biosynthesis enzyme chalcone reductase model using the structure Structural of chalcone reductase and for Biol. Chem. Full Text Full Text PDF PubMed Scopus Google as a of the in COR responsible for BIA COR also of residues and through and of COR in L. Facchini P.J. reductase with and in opium 2018; Scopus Google Scholar). the in that in of the apo-COR in a location expected on the structure to the in location of the β1α1 loop toward the BIA-binding the of Met-28 and the BIA-binding pocket. the of β1α1 loop conformation in related and is that the β1α1 loop several to an However, of Met-28 that the conformation of the β1α1 loop in apo-COR has for the first the of residues the structure of the BIA-binding not on from with structures of the β1α1 the chains of the β1α1 Met-28 loop a on to and lead to changes in the activity of in residues and on that residues in a of loop the of the not for substrate and The of loop to BIA substrate recognition and catalysis are to the in the loop and of from the L. Facchini P.J. reductase with and in opium 2018; Scopus Google that changes to the of the loop that is conserved COR in and in loop is almost in BIA that enzyme and not to L. Facchini P.J. reductase with and in opium 2018; Scopus Google as to the on activity is the of or a of Our show particularly changes in activity from specific substitutions at two The in and the a and in the substitutions in and by the as an of enzyme specific activity and the as a of an activity by (e.g., by with The of the substitutions that the of the of the BIA substrate more the or the the more the the catalysis of the of codeine to a the of The COR loop is to Met-28 and to directly the BIA However, that the in of codeine and production L. Facchini P.J. reductase with and in opium 2018; Scopus Google Scholar). Our structure an of this through an in the at are expected to the of the of the and of the pocket. also with is also of the pocket. is by show a of the β1α1 loop to directly with the BIA Our structure also for the first and His-120 in and for with and of His-120 with residues on COR COR the with and also the conformation to and we that the of range for In the on COR the not with and to with the BIA The of from the of His-120 with a that of the COR and with is not the of the of the BIA-binding to the of codeine and The substrates for the by and through a in COR activity (e.g., of the from thebaine by to the to the for the production of codeine using engineered COR has to to to in the L. Facchini P.J. reductase with and in opium 2018; Scopus Google the of the and a of an K. for and semisynthetic Chem. Biol. PubMed Scopus Google Scholar, L. of morphinan alkaloids in 2015; PubMed Scopus Google Scholar). The of of in a to in the to the enzyme has to the of and codeinone X. Hagel J.M. L. R. Facchini P.J. is in codeine and morphine biosynthesis in opium poppy.Nat. Chem. Biol. PubMed Scopus Google Scholar). The of as an to the of and as well as and also a molecular to and in engineered X. Hagel J.M. L. R. Facchini P.J. is in codeine and morphine biosynthesis in opium poppy.Nat. Chem. Biol. PubMed Scopus Google Scholar). with flux from the COR to of this in the to the of codeinone over neopinone, that the in codeinone and with the of the in reveal that the enzyme as codeine as is of a highly from crystal structures of COR in with codeinone and to and and also a for codeine over the production in in a to L. Facchini P.J. reductase with and in opium 2018; Scopus Google Scholar). the COR are from in the in the provide a of the in as engineered and thus suggest that COR is a to in with the of The of the COR crystal structure for the first the of more for 1,2-dehydroreticuline reductase the AKR in BIA is the AKR of the reticuline epimerase (REPI) fusion in a cytochrome P450 first (S)-reticuline to and catalyzes a of the in 1,2-dehydroreticuline to (R)-reticuline S.C. Hagel J.M. Beaudoin G. a W. Burns D.C. Facchini P.J. Stereochemical inversion of (S)-reticuline by a cytochrome P450 fusion in opium poppy.Nat. Chem. Biol. 2015; 11: 728-732Crossref PubMed Scopus (84) Google Scholar, T. Teodor Li Y. R. Bowser T.A. 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D.C. and structure for and PubMed Scopus Google Scholar). model of COR with and codeinone by the structure of the the structure of the COR to the of and structural of residues in the AKR J.M. The aldo-keto reductase (AKR) Biol. PubMed Scopus Google Scholar, The aldo-keto Biol. 2015; PubMed Scopus Google is expected to in Using and reductase for the of residues and to with the conformation of The residues in loop from the COR structure using the G. and to protein loop Google range of of the β1α1 loop also using to show that a in for a of the to the of alkaloid The COR substrates codeine and codeinone the using T.A. and a model of for Chem. PubMed Scopus Google and W. T. R. for Chem. PubMed Scopus Google Scholar). The of the to Å from the of the nicotinamide ring of and Å from the of The model with protein structure using using COR as a using the L. 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