Crystal Structures of Pinoresinol-Lariciresinol and Phenylcoumaran Benzylic Ether Reductases and Their Relationship to Isoflavone Reductases

Abstract

Despite the importance of plant lignans and isoflavonoids in human health protection (e.g. for both treatment and prevention of onset of various cancers) as well as in plant biology (e.g. in defense functions and in heartwood development), systematic studies on the enzymes involved in their biosynthesis have only recently begun. In this investigation, three NADPH-dependent aromatic alcohol reductases were comprehensively studied, namely pinoresinol-lariciresinol reductase (PLR), phenylcoumaran benzylic ether reductase (PCBER), and isoflavone reductase (IFR), which are involved in central steps to the various important bioactive lignans and isoflavonoids. Of particular interest was in determining how differing regio- and enantiospecificities are achieved with the different enzymes, despite each apparently going through similar enone intermediates. Initially, the three-dimensional x-ray crystal structures of both PLR_Tp1 and PCBER_Pt1 were solved and refined to 2.5 and 2.2 Å resolutions, respectively. Not only do they share high gene sequence similarity, but their structures are similar, having a continuous α/β NADPH-binding domain and a smaller substrate-binding domain. IFR (whose crystal structure is not yet obtained) was also compared (modeled) with PLR and PCBER and was deduced to have the same overall basic structure. The basis for the distinct enantio-specific and regio-specific reactions of PCBER, PLR, and IFR, as well as the reaction mechanism and participating residues involved (as identified by site-directed mutagenesis), are discussed. Despite the importance of plant lignans and isoflavonoids in human health protection (e.g. for both treatment and prevention of onset of various cancers) as well as in plant biology (e.g. in defense functions and in heartwood development), systematic studies on the enzymes involved in their biosynthesis have only recently begun. In this investigation, three NADPH-dependent aromatic alcohol reductases were comprehensively studied, namely pinoresinol-lariciresinol reductase (PLR), phenylcoumaran benzylic ether reductase (PCBER), and isoflavone reductase (IFR), which are involved in central steps to the various important bioactive lignans and isoflavonoids. Of particular interest was in determining how differing regio- and enantiospecificities are achieved with the different enzymes, despite each apparently going through similar enone intermediates. Initially, the three-dimensional x-ray crystal structures of both PLR_Tp1 and PCBER_Pt1 were solved and refined to 2.5 and 2.2 Å resolutions, respectively. Not only do they share high gene sequence similarity, but their structures are similar, having a continuous α/β NADPH-binding domain and a smaller substrate-binding domain. IFR (whose crystal structure is not yet obtained) was also compared (modeled) with PLR and PCBER and was deduced to have the same overall basic structure. The basis for the distinct enantio-specific and regio-specific reactions of PCBER, PLR, and IFR, as well as the reaction mechanism and participating residues involved (as identified by site-directed mutagenesis), are discussed. Vascular plants produce a wide variety of plant defense and health-protecting metabolites, such as the (oligomeric) lignans and isoflavonoids, with both classes of compounds being offshoots of the phenylpropanoid pathway (1.Lewis N.G. Davin L.B. Barton Sir, D.H.R Nakanishi K. Meth-Cohn O. Comprehensive Natural Products Chemistry. Vol. 1. Elsevier, London1999: 639-712Google Scholar, 2.Dixon R.A. Barton Sir, D.H.R Nakanishi K. Meth-Cohn O. Comprehensive Natural Products Chemistry. Vol. 1. Elsevier, London1999: 774-823Google Scholar). The lignans are typically monolignol-derived dimers and are often found as either 8–5′, 8–8′, or 8–O–4′ linked moieties that, depending upon the species, may be either optically active or racemic (1.Lewis N.G. Davin L.B. Barton Sir, D.H.R Nakanishi K. Meth-Cohn O. Comprehensive Natural Products Chemistry. Vol. 1. Elsevier, London1999: 639-712Google Scholar). It is only recently that the biosynthetic pathways to the lignans (3.Davin L.B. Wang H.-B. Crowell A.L. Bedgar D.L. Martin D.M. Sarkanen S. Lewis N.G. Science. 1997; 275: 362-366Crossref PubMed Scopus (577) Google Scholar, 4.Gang D.R. Kasahara H. Xia Z.-Q. Vander Mijnsbrugge K. Bauw G. Boerjan W. Van Montagu M. Davin L.B. Lewis N.G. J. Biol. Chem. 1999; 274: 7516-7527Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 5.Xia Z.-Q. Costa M.A. Pélissier H.C. Davin L.B. Lewis N.G. J. Biol. Chem. 2001; 276: 12614-12628Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) and isoflavonoids (2.Dixon R.A. Barton Sir, D.H.R Nakanishi K. Meth-Cohn O. Comprehensive Natural Products Chemistry. Vol. 1. Elsevier, London1999: 774-823Google Scholar) have been investigated, which in so doing resulted in the isolation and characterization of various lignan reductases, namely pinoresinol-lariciresinol reductases (PLRs) 1The abbreviations used are: PLRpinoresinol-lariciresinol reductaseIFRisoflavone reductaseIFRHisoflavone reductase homologPCBERphenylcoumaran benzylic ether reductaseMALDI-TOFmatrix-assisted laser desorption ionization time-of-flightMES2-(N-morpholino)ethanesulfonic acid. (6.Dinkova-Kostova A.T. Gang D.R. Davin L.B. Bedgar D.L. Chu A. Lewis N.G. J. Biol. Chem. 1996; 271: 29473-29482Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 7.Fujita M. Gang D.R. Davin L.B. Lewis N.G. J. Biol. Chem. 1999; 274: 618-627Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) and phenylcoumaran benzylic ether reductases (PCBERs) (4.Gang D.R. Kasahara H. Xia Z.-Q. Vander Mijnsbrugge K. Bauw G. Boerjan W. Van Montagu M. Davin L.B. Lewis N.G. J. Biol. Chem. 1999; 274: 7516-7527Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar), as well as isoflavone reductases (IFRs) (8.Paiva N.L. Edwards R. Sun Y. Hrazdina G. Dixon R.A. Plant Mol. Biol. 1991; 17: 653-667Crossref PubMed Scopus (145) Google Scholar, 9.Paiva N.L. Sun Y. Dixon R.A. VanEtten H.D. Hrazdina G. Arch. Biochem. Biophys. 1994; 312: 501-510Crossref PubMed Scopus (51) Google Scholar) and their homologs. pinoresinol-lariciresinol reductase isoflavone reductase isoflavone reductase homolog phenylcoumaran benzylic ether reductase matrix-assisted laser desorption ionization time-of-flight 2-(N-morpholino)ethanesulfonic acid. The downstream metabolic products of these enzymatic steps afford, depending upon the species, various substances with important pharmacological and/or plant defense roles. For example, PLRs, which catalyze reduction of the 8–8′ linked lignan pinoresinol (Scheme 1, structure1) (6.Dinkova-Kostova A.T. Gang D.R. Davin L.B. Bedgar D.L. Chu A. Lewis N.G. J. Biol. Chem. 1996; 271: 29473-29482Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 10.Chu A. Dinkova A. Davin L.B. Bedgar D.L. Lewis N.G. J. Biol. Chem. 1993; 268: 27026-27033Abstract Full Text PDF PubMed Google Scholar), generate the precursors of cancer-preventative natural products, such as secoisolariciresinol diglucoside (structure2) derivatives in flaxseed (Linum usitatissimum) (11.Ford J.D. Huang K.-S. Wang H.-B. Davin L.B. Lewis N.G. J. Nat. Prod. 2001; 64: 1388-1397Crossref PubMed Scopus (148) Google Scholar, 12.Teoh K.H. Ford J.D. Kim M.-R. Davin L.B. Lewis N.G. Cunnane S.C. Thompson L.U. Flaxseed in Human Nutrition. 2nd Ed. American Oil Chemist's Society Press, Champaign, IL2003: 41-62Google Scholar) and matairesinol (structure3)/secoisolariciresinol (structure4) in a wide variety of plant species (5.Xia Z.-Q. Costa M.A. Pélissier H.C. Davin L.B. Lewis N.G. J. Biol. Chem. 2001; 276: 12614-12628Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 11.Ford J.D. Huang K.-S. Wang H.-B. Davin L.B. Lewis N.G. J. Nat. Prod. 2001; 64: 1388-1397Crossref PubMed Scopus (148) Google Scholar, 13.Xia Z.-Q. Costa M.A. Proctor J. Davin L.B. Lewis N.G. Phytochemistry. 2000; 55: 537-549Crossref PubMed Scopus (86) Google Scholar). Such lignans are considered to be metabolized in mammals into the cancer-preventing “mammalian” lignans, enterodiol (structure5) and enterolactone (structure6), with the latter metabolites being generated in the gut by the action of gastrointestinal flora such as H. M. Scholar, M. J. PubMed Scopus Google Scholar). PLR is also important enzymatic in the of the in species Z.-Q. Costa M.A. Proctor J. Davin L.B. Lewis N.G. Phytochemistry. 2000; 55: 537-549Crossref PubMed Scopus (86) Google Scholar), as well as to the derivatives and used in treatment Phytochemistry. 2000; PubMed Scopus Google Scholar). metabolites of PLR substances such as that the and of heartwood such as in D.R. M. Davin L.B. Lewis N.G. Lewis N.G. Sarkanen S. and Vol. Scholar). similar in plant defense are also apparently through the action of that catalyze in linked lignans, with alcohol (4.Gang D.R. Kasahara H. Xia Z.-Q. Vander Mijnsbrugge K. Bauw G. Boerjan W. Van Montagu M. Davin L.B. Lewis N.G. J. Biol. Chem. 1999; 274: 7516-7527Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). the isoflavone reductases are involved in the biosynthesis of various such as in (8.Paiva N.L. Edwards R. Sun Y. Hrazdina G. Dixon R.A. Plant Mol. Biol. 1991; 17: 653-667Crossref PubMed Scopus (145) Google Scholar), as well as in a of in W. A. Scholar). and have also been in health protection with a similar to the lignans H. K. S. Scholar), and a of of plant lignans and to a of and K. H. and Scholar). It is considered that lignans and isoflavonoids these of or of their M. M. Y. Biol. Chem. Scopus Google Scholar), PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar), H. K. G. J. Biochem. Mol. Biol. 1993; PubMed Scopus Google Scholar), M. H. G. R. S. A. 1993; PubMed Scopus Google Scholar), and H. Y. J. K. K. J. Biochem. Mol. Biol. PubMed Scopus Google Scholar, L.U. 1996; PubMed Scopus Google Scholar) both and species have been well In the species, is to catalyze the NADPH-dependent of into and the not as a (6.Dinkova-Kostova A.T. Gang D.R. Davin L.B. Bedgar D.L. Chu A. Lewis N.G. J. Biol. Chem. 1996; 271: 29473-29482Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 10.Chu A. Dinkova A. Davin L.B. Bedgar D.L. Lewis N.G. J. Biol. Chem. 1993; 268: 27026-27033Abstract Full Text PDF PubMed Google Scholar). The gene been a of (6.Dinkova-Kostova A.T. Gang D.R. Davin L.B. Bedgar D.L. Chu A. Lewis N.G. J. Biol. Chem. 1996; 271: 29473-29482Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). For each PLR only the is of a and was used as both with of A. Dinkova A. Davin L.B. Bedgar D.L. Lewis N.G. J. Biol. Chem. 1993; 268: 27026-27033Abstract Full Text PDF PubMed Google Scholar). were with the PLR for of and and of and for and (6.Dinkova-Kostova A.T. Gang D.R. Davin L.B. Bedgar D.L. Chu A. Lewis N.G. J. Biol. Chem. 1996; 271: 29473-29482Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). the is having and the NADPH-binding to the (6.Dinkova-Kostova A.T. Gang D.R. Davin L.B. Bedgar D.L. Chu A. Lewis N.G. J. Biol. Chem. 1996; 271: 29473-29482Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). in distinct PLR were and PLR_Tp1 and distinct with the and of pinoresinol and to and and 1, and M. Gang D.R. Davin L.B. Lewis N.G. J. Biol. Chem. 1999; 274: 618-627Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). plants PLR are apparently PCBER is also apparently plant and been in both and in this of in the NADPH-dependent reduction of various phenylcoumaran lignans to the alcohol and alcohol into alcohol and alcohol (4.Gang D.R. Kasahara H. Xia Z.-Q. Vander Mijnsbrugge K. Bauw G. Boerjan W. Van Montagu M. Davin L.B. Lewis N.G. J. Biol. Chem. 1999; 274: 7516-7527Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). in this such enzymatic both the and of the are and the is the also PCBER as a reductase (4.Gang D.R. Kasahara H. Xia Z.-Q. Vander Mijnsbrugge K. Bauw G. Boerjan W. Van Montagu M. Davin L.B. Lewis N.G. J. Biol. Chem. 1999; 274: 7516-7527Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). PCBER high sequence to the NADPH-dependent PLRs, the and their PCBER is typically similar and to and similar and to (4.Gang D.R. Kasahara H. Xia Z.-Q. Vander Mijnsbrugge K. Bauw G. Boerjan W. Van Montagu M. Davin L.B. Lewis N.G. J. Biol. Chem. 1999; 274: 7516-7527Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). not only do they share high gene sequence similarity, but as they share similar with their products in plant as well as apparently having of action (4.Gang D.R. 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Chem. 1999; 274: 7516-7527Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar) only both and For this PCBER was to be the of PLR and IFR the and the enzymes catalyze the (4.Gang D.R. Kasahara H. Xia Z.-Q. Vander Mijnsbrugge K. Bauw G. Boerjan W. Van Montagu M. Davin L.B. Lewis N.G. J. Biol. Chem. 1999; 274: 7516-7527Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). a both and have been identified as in various and A. Kasahara H. Davin L.B. Lewis N.G. S. J. Biochem. 2001; 268: PubMed Scopus Google the on the that these Despite the importance of the lignans and isoflavonoids in plant in human and in that as their systematic studies on the enzymes involved have not been is only recently have enzymes, and been identified in their biosynthetic and only are the for were or x-ray crystal structures for the enzymes in 8–8′ and linked lignan biosynthetic In this have the x-ray of both PLR_Tp1 and site-directed to the of the and reaction to to the various the x-ray crystal structures of both PLR_Tp1 and PCBER_Pt1 were solved and refined to 2.5 and 2.2 Å resolutions, the structures of which studies to be the and reactions of and was into the S. R. Google Scholar) as (4.Gang D.R. Kasahara H. Xia Z.-Q. Vander Mijnsbrugge K. Bauw G. Boerjan W. Van Montagu M. Davin L.B. Lewis N.G. J. Biol. Chem. 1999; 274: 7516-7527Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar), and PLR_Tp1 was into the same in a similar of PLR_Tp1 and of both PLR_Tp1 and PCBER_Pt1 in was by of to a the were to for for PLR_Tp1 and for with The for each were by which the were to The PLR_Tp1 and PCBER_Pt1 were each in and and for with the The the and were to with the and in by a and to as by Gang (4.Gang D.R. Kasahara H. Xia Z.-Q. Vander Mijnsbrugge K. Bauw G. Boerjan W. Van Montagu M. Davin L.B. Lewis N.G. J. Biol. Chem. 1999; 274: 7516-7527Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). The of of PCBER_Pt1 were as (4.Gang D.R. Kasahara H. Xia Z.-Q. Vander Mijnsbrugge K. Bauw G. Boerjan W. Van Montagu M. Davin L.B. Lewis N.G. J. Biol. Chem. 1999; 274: 7516-7527Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar), PLR_Tp1 was as the as by were of (4.Gang D.R. Kasahara H. Xia Z.-Q. Vander Mijnsbrugge K. Bauw G. Boerjan W. Van Montagu M. Davin L.B. Lewis N.G. J. Biol. Chem. 1999; 274: 7516-7527Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar), and in and The was into that were to a in a of PLR_Tp1 not to the and the into was to a in a of PLR_Tp1 not to the of the The PLR_Tp1 was (4.Gang D.R. Kasahara H. Xia Z.-Q. Vander Mijnsbrugge K. Bauw G. Boerjan W. Van Montagu M. Davin L.B. Lewis N.G. J. Biol. Chem. 1999; 274: 7516-7527Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar) and a in A. The was into that were in a of the with PLR_Tp1 was a which was the for PLR_Tp1 were and to a of was by by with and was used for both and were and in and PLR_Tp1 and were used in of the were the and used in the were with the Biophys. J. Full Text PDF PubMed Scopus Google Scholar). were in with PLR_Tp1 of and and of and The were for The were with a of and a of with a and were by the for Ed. Scholar, Biophys. J. 1997; Full Text PDF PubMed Scopus Google Scholar) a of and were the is the the is the is the is the to H. for and Google is the is the to be the the of and and as and and is the were to in and with of a of as with a was used with the was and the were of and were used for of PLR_Tp1 and the of the PCBER_Pt1 was with a and the was to of PCBER_Pt1 were by the with in being a and and with of were to Å a x-ray with and The PCBER_Pt1 crystal to the with in The of 2.2 Å was the a of of PLR_Tp1 were also by the with of in being a and and with of were The PLR_Tp1 crystal to the with in The of 2.5 Å was the a of and structure of PCBER_Pt1 was solved by and and the structure of PLR_Tp1 was solved by the of derivatives were by in and for 2.5 Å and Å were the and the respectively. and and were with and J. Biol. 1999; 55: PubMed Scopus Google Scholar, Biol. 2000; PubMed Scopus Google Scholar). The of structures and the of of the been on the by the of and of in the were and used for the Å The for and the for the were of and The residues are and the was not the of The was refined with 2.2 Å A.T. J. M. Biol. PubMed Scopus Google Scholar). The for of PCBER_Pt1 and is for the The the was and 2.2 Å The structure of PLR_Tp1 was solved by the PCBER_Pt1 and the J. A. 1994; Scopus Google Scholar). The of the was to Å and of of and were to of the residues to the in the PCBER_Pt1 the to residues was not the of The for the for and is The the were and 2.5 Å The of PLR_Tp1 are Å for and for and of PCBER_Pt1 are Å and respectively. The of both PLR_Tp1 and PCBER_Pt1 structures have been in the of the and generate the for the the Scholar) was on the and J. Chem. Scopus Google Scholar) the M.A. S. O. J. M. R. S. J. K. K. J. G. A. R. Martin M.A. A. M. W. M. Ed. Scholar). the of the and were through the of with and the on which is on by a by A.T. J. M. Biol. 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A. R. S. Plant 1996; PubMed Scopus Google and of the a of with G. 1997; PubMed Scopus Google Scholar), the of both PLR_Tp1 and PCBER_Pt1 of the to be in a similar as of in both PCBER_Pt1 and and PLR_Tp1 and that be to the and that are the substrate-binding of these in PCBER_Pt1 in of a in In the the and also is in the crystal structures of both PLR_Tp1 and the residues both PLR_Tp1 and The three of or high are residues and in which is also the for PLR_Tp1 a for respectively. the IFR M. high sequence with both PLR_Tp1 and PCBER_Pt1 a of was also for a with both crystal and were the A.T. J. M. Biol. PubMed Scopus Google Scholar), the of PCBER_Pt1 by A.T. J. M. Biol. PubMed Scopus Google Scholar) with The of the not the and the to be the for the in IFR in reductases, and have in the with both 1996; PubMed Scopus Google Scholar, R. A. S. A. PubMed Scopus Google Scholar) and human reductase S. A. R. O. K. M. Nat. 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Biol. 2001; PubMed Scopus Google Scholar). of the PLR_Tp1 and and are similar to in are also these and enzymes in their of In and the is by a to the of for and for and by a to the of the as for the S. A. R. O. K. M. Nat. Biol. 2001; PubMed Scopus Google Scholar). are PLRs, and The of which is also PLRs, and the of the as in S. O. G. A. J. Mol. Biol. 1997; 268: PubMed Scopus Google Scholar, R. A. S. A. PubMed Scopus Google Scholar). of and and to of the is aromatic alcohol was to studies of the three aromatic to both and as well as to site-directed as that is also to of and In this was the in of high sequence similarity, which the of the crystal structures were in the by the three-dimensional and of the of with of the enzymes of the substrate-binding of and were and the of the were refined as In each the structure of the is of a and and is in the substrate-binding are also to the in such a as to the for alcohol for and for to the of In this the in a with to the aromatic of each of the this being by and by with residues the active as the are for of the is Å the of the to be the and the are in the for the well to the reaction in this active are residues and in and which be to as the for are also and residues in the active of PCBER_Pt1 and a the active of these are not the different a enzymatic of PLR, IFR, and PCBER to be through and reduction of the enone intermediates. the was considered a for site-directed were such that was to the with site-directed as The so both the and the were and (6.Dinkova-Kostova A.T. Gang D.R. Davin L.B. Bedgar D.L. Chu A. Lewis N.G. J. Biol. Chem. 1996; 271: 29473-29482Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 7.Fujita M. Gang D.R. Davin L.B. Lewis N.G. J. Biol. Chem. 1999; 274: 618-627Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). It was found that the not into either and in to that the of the to of the PLR_Tp1 to pinoresinol to and secoisolariciresinol and that the is involved in and PLR and IFR catalyze PCBER only PCBER was to be the of PLR and IFR, PCBER the and the enzymes the (4.Gang D.R. Kasahara H. Xia Z.-Q. Vander Mijnsbrugge K. Bauw G. Boerjan W. Van Montagu M. Davin L.B. Lewis N.G. J. Biol. Chem. 1999; 274: 7516-7527Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). The of the crystal structures of PLR_Tp1 and PCBER_Pt1 to studies of the and regio-specific enzymatic In this PLR_Tp1 the M. Gang D.R. Davin L.B. Lewis N.G. J. Biol. Chem. 1999; 274: 618-627Abstract Full Text Full Text PDF PubMed Scopus (76) Google on the the (8.Paiva N.L. Edwards R. Sun Y. Hrazdina G. Dixon R.A. Plant Mol. Biol. 1991; 17: 653-667Crossref PubMed Scopus (145) Google Scholar). the of sequence and PLR_Tp1 and the overall and structures of and be to be the only are in the and which have to a different on this the structure of was deduced the crystal structures of PLR_Tp1 and PCBER_Pt1 by the same as for the of In this the structures of PLR_Tp1 and in the of the which in their distinct In the of and but in are and and as well as the which of the of pinoresinol the three of PCBER_Pt1 are and which with similar of to and to the in the substrate-binding of the the of which for both of and the a and by the of these three residues and to and In the only is with both the and the of the the residues in PLR_Tp1 in PCBER_Pt1 and in the a of structures the substrate-binding in PCBER_Pt1 and in and in PLR_Tp1 in and in and and In also either to the of the substrate-binding as for PCBER_Pt1 or to as for with the structures of the lignan aromatic alcohol PLR_Tp1 and which a of aromatic alcohol were the and to 2.5 and 2.2 Å resolutions, and a was by with PLR_Tp1 and through a and of three reductases, the basis for and was and the of the is in a with the in of the of the was achieved a the and residues on the In the substrate-binding of and the of the was Å the of the for It was deduced that a (e.g. for was for and in with this site-directed a PLR_Tp1 was of which as and which the for the by the so that the and to PLR_Tp1 and respectively. the the of PCBER_Pt1 be by the of the a and In in the of and with and are and be