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

2003; Elsevier BV; Volume: 278; Issue: 50 Linguagem: Inglês

10.1074/jbc.m308493200

ISSN

1083-351X

Autores

Tongpil Min, Hiroyuki Kasahara, Diana L. Bedgar, BuHyun Youn, Paulraj K. Lawrence, David R. Gang, Steven C. Halls, HaJeung Park, Jacqueline L. Hilsenbeck, Laurence Davin, Norman Lewis, ChulHee Kang,

Tópico(s)

Phytoestrogen effects and research

Resumo

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 Clostridia sp (14.Setchell K.D.R. Lawson A.M. Borriello S.P. Adlercreutz H. Axelson M. Falk Symp. 1982; 31: 93-97Google Scholar, 15.Borriello S.P. Setchell K.D.R. Axelson M. Lawson A.M. J. Appl. Bacteriol. 1985; 58: 37-43Crossref PubMed Scopus (348) Google Scholar). PLR is also an important enzymatic step in the formation of the antiviral agent podophyllotoxin (structure7) in Podophyllum species (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), as well as to the semi-synthetic chemotherapeutic derivatives etoposide (structure8), etopophos (structure9), and teniposide (structure10), widely used in cancer treatment (16.Canel C. Moraes R.M. Dayan F.E. Ferreira D. Phytochemistry. 2000; 54: 115-120Crossref PubMed Scopus (370) Google Scholar). Other metabolites of physiological significance derived from PLR catalysis involve substances such as plicatic acid (structure11) that help ensure the durability and structural integrity of heartwood tissue, such as in Western red cedar (Thuja plicata) (17.Gang D.R. Fujita M. Davin L.B. Lewis N.G. Lewis N.G. Sarkanen S. Lignin and Lignan Biosynthesis. Vol. 697. ACS Symposium Series, Washington, D.C.1998: 389-421Google Scholar). Somewhat similar roles in plant defense are also apparently effectuated through the action of PCBERs that catalyze comparable reductive processes in 8–5′ linked lignans, e.g. with dehydrodiconiferyl alcohol (structure12) (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). Additionally, the isoflavone reductases are involved in the biosynthesis of various phytoalexins, such as (-)-medicarpin (structure13) in alfalfa (Medicago sativa) (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 generating a number of heartwood-protective substances, e.g. hildecarpin (structure14) in Tephrosia hildebrandtii (18.Lwande W. Hassanali A. Njoroge P.W. Bentley M.D. Delle Monache F. Jondiko J.I. Insect Sci. Applic. 1985; 6: 537-541Google Scholar). Particular isoflavones, e.g. daidzein (structure15) and genistein (structure16), have also been implicated in health protection with a somewhat similar role to the lignans (19.Adlercreutz H. Hökerstedt K. Bannwart C. Hämäläinen E. Fotsis T. Bloigu S. Prog. Cancer Res. Ther. 1988; 35: 409-412Google Scholar), and a striking body of evidence now exists linking dietary ingestion of plant lignans and isoflavones to a significantly reduced risk incidence of breast, prostate, and colon cancers (20.Griffiths K. Adlercreutz H. Boyle P. Denis L. Nicholson R.I. Morton M.S. Nutrition and Cancer. Isis Medical Media, Oxford, UK1996Google Scholar). It is now considered that dietary lignans and isoflavonoids impart these protective effects because of one or more of their antioxidant (21.Osawa T. Nagata M. Namiki M. Fukuda Y. Agric. Biol. Chem. 1985; 49: 3351-3352Crossref Scopus (1) Google Scholar), weak estrogenic/antiestrogenic (22.Martin P.M. Korwitz K.B. Ryan D.S. McGuire W.L. Endocrinology. 1978; 103: 1860-1867Crossref PubMed Scopus (550) Google Scholar, 23.Waters A.P. Knowler J.T. J. Reprod. Fert. 1982; 66: 379-381Crossref PubMed Scopus (60) Google Scholar), anti-aromatase (24.Adlercreutz H. Bannwart C. Wähälä K. Mäkelä T. Brunow G. Hase T. Arosemena P.J. Kellis Jr., J.T. Vickery L.E. J. Steroid Biochem. Mol. Biol. 1993; 44: 147-153Crossref PubMed Scopus (498) Google Scholar), antiangiogenic (25.Fotsis T. Pepper M. Adlercreutz H. Fleischmann G. Hase T. Montesano R. Schweigerer L. Proc. Natl. Acad. Sci., U. S. A. 1993; 90: 2690-2694Crossref PubMed Scopus (731) Google Scholar), and anticarcinogenic/antitumor (26.Adlercreutz H. Mousavi Y. Clark J. Höckerstedt K. Hämäläinen E. Wähälä K. Mäkelä T. Hase T. J. Steroid Biochem. Mol. Biol. 1992; 41: 331-337Crossref PubMed Scopus (401) Google Scholar, 27.Thompson L.U. Seidl M.M. Rickard S.E. Orcheson L.J. Fong H.H.S. Nutr. Cancer. 1996; 26: 159-165Crossref PubMed Scopus (230) Google Scholar) properties. PLRs from both Forsythia and Thuja species have been well studied. In the former species, it is an ∼35-kDa protein able to catalyze the sequential NADPH-dependent enantiospecific reductions of (+)-pinoresinol (structure1a) into (+)-lariciresinol (structure17a) and (-)-secoisolariciresinol (structure4b), respectively (Fig. 1A); the (-)-antipode (structure1b) does not serve as a substrate (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 PLR_Fi1 has been cloned; it encodes a protein of 34.9 kDa (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 reductive step, only the 4-pro-R hydride is abstracted, indicative of a class A reductase, and when [7,7′-2H2]-pinoresinol (1) was used as substrate, both reductions occurred with an inversion of configuration at C-7/C-7′ (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). Various kinetic parameters were determined with the purified PLR giving values for Km of 27 ± 1.5 and 121 ± 6.0 μm and Vmax of 16.2 ± 0.4 and 25.2 ± 0.7 μmol h-1 mg-1 protein for (+)-pinoresinol (structure1a) and (+)-lariciresinol (structure17a), respectively (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). Moreover, the protein is cytosolic, having no secretory system targeting sequence, and has the NADPH-binding region localized closed to the N terminus (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). By comparison, in T. plicata, two distinct PLR genes were cloned (PLR_Tp1 and PLR_Tp2) whose corresponding recombinant proteins, PLR_Tp1 and PLR_Tp2, catalyzed distinct enantiospecific reductions with the (-)- and (+)- antipodes of pinoresinol (structures1b and 1a, respectively) to ultimately afford (+)- and (-)-secoisolariciresinol (structures4a and 4b; Fig. 1, A and B) (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). (In all vascular plants examined thus far PLR homologs are apparently ubiquitous.) PCBER is also apparently an ubiquitous vascular plant enzyme and has been studied in both Pinus taeda and Populus trichocarpa. This 33.6-kDa protein is, in this case, capable of catalyzing in vitro the regiospecific NADPH-dependent 7–O–4′ reduction of various defense-related phenylcoumaran lignans to afford the corresponding diphenols, e.g. dehydrodiconiferyl alcohol (structure12) and dihydrodehydrodiconiferyl alcohol (structure18) into isodihydrodehydrodiconiferyl alcohol (structure19) and tetrahydrodehydrodiconiferyl alcohol (structure20), respectively (Fig. 1C) (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). Although in this case such conversions proceed at relatively low enzymatic rates, both the (+)- and (-)-enantiomeric forms of the substrates are utilized, and the 4-pro-R hydride is abstracted from the NADPH cofactor, thereby also classifying PCBER as a class 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 shows high sequence homology to the NADPH-dependent PLRs, the IFRs, and their corresponding homologs (IFRHs); PCBER is typically ∼66% similar and ∼45% identical to PLRs and ∼65% similar and ∼50% identical to IFRs (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). Yet not only do they share high gene sequence similarity, but as described above they share similar physiological roles with their products (i.e. in plant defense) as well as apparently having comparable catalytic modes of action (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). However, the PLRs (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 IFRs (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) known so far catalyze enantiospecific conversions, whereas PCBER (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) only shows regiospecific discrimination, reducing both (+)- and (-)-enantiomeric forms. For this reason, PCBER was proposed to be the likely progenitor of PLR and IFR because it catalyzes the more general conversion, and the other two enzymes catalyze the more specific conversions (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). Furthermore, from a physiological perspective, both PCBERs and IFRs have been identified as minor allergens in various fruits and vegetables (28.Karamloo F. Wangorsch A. Kasahara H. Davin L.B. Lewis N.G. Vieths S. Eur. J. Biochem. 2001; 268: 5310-5320Crossref PubMed Scopus (107) Google Scholar); the regions on the proteins that confer these allergenic responses are, however, unknown. Despite the importance of the lignans and isoflavonoids in plant biology, in human health, and in industries that use heartwoods as their main resource, systematic studies on the enzymes involved have not been done. This is because only fairly recently have several proteins, enzymes, and genes been identified in their biosynthetic pathways, and hence only now are the proteins available for structural analyses. Accordingly, there were no NMR or x-ray crystal structures available for the enzymes in 8–8′ and 8–5′ linked lignan biosynthetic pathways. In this work, we have completed the x-ray crystallographic analysis of both PLR_Tp1 and PCBER_Pt1, including using site-directed mutagenesis to probe the stereochemistry of the chiral center and reaction mechanism, to begin to address the various structural issues involved. As described below, the threedimensional x-ray crystal structures of both PLR_Tp1 and PCBER_Pt1 were solved and refined to 2.5 and 2.2 Å resolutions, respectively, the structures of which now allow incisive comparative studies to be made between the enantiospecific and regiospecific reactions of PCBER_Pt1, PLR_Tp1, and IFR. Plasmid Construction—PCBER_Pt1 was cloned into the overexpression plasmid pSBETa (29.Schenk P.M. Baumann S. Mattes R. Steinbiss H.-H. BioTechniques. 1995; 19: 196-200PubMed Google Scholar) as previously described (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 cloned into the same vector in a similar manner. Purification of PLR_Tp1 and PCBER_Pt1—Expression of both PLR_Tp1 and PCBER_Pt1 in Escherichia coli (B834-DE3) was individually induced by addition of isopropyl-β-d-thiogalactopyranoside to a 1 mm final concentration at mid-log phase (A600 = 0.5–0.7). Following induction the two cell lines were allowed to grow for 21 h at 23 °C for PLR_Tp1 and 10 h at 37 °C for PCBER_Pt1, respectively, with shaking at 250 rpm. The cells for each were then harvested by centrifugation (3,000 × g), after which the individual pellets were frozen (∼2 h) to facilitate cell lysis. The pellets containing PLR_Tp1 and PCBER_Pt1 were each thawed at room temperature, then individually suspended in Buffer A (20 mm Bis-Tris-propane, pH 8.0, containing 2 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, and 5 mm dithiothreitol), and sonicated (three times for 30 s), with the resulting suspensions centrifuged (20,000 × g). The supernatant from the PLR_Tp1- and PCBER_Pt1-containing cell lines were next subjected to ammonium sulfate precipitation, with the proteins precipitating between 40 and 70% ammonium sulfate reconstituted in buffer A, followed by a desalting step over PD-10 columns (Amersham Biosciences) and then subjected to Affi-Blue gel column chromatography as described by Gang et al. (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 final stages of purification of PCBER_Pt1 were as previously described (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), whereas PLR_Tp1 was purified as follows. Fractions from the Affi-Blue gel column containing PLR_Tp1, as determined by SDS-polyacrylamide gel electrophoresis analysis, were combined (186 mg of protein), concentrated (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 desalted over PD10 columns equilibrated in Buffer B (20 mm Bis-Tris-propane, pH 7.0, containing 2 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, and 5 mm dithiothreitol). The desalted protein solution was next divided into five equal aliquots that were sequentially applied to a POROS 20 HQ column (PerSeptive Biosystem) pre-equilibrated in Buffer B at a flow rate of 7 ml min-1. PLR_Tp1 did not bind to the column, and the combined fraction (95.6 mg), divided into four equal aliquots (23.9 mg × 4), was directly applied to a POROS 20 SP cation exchange column (PerSeptive Biosystem) pre-equilibrated in Buffer B at a flow rate of 10 ml min-1. Again PLR_Tp1 did not bind to the column, whereas most of the remaining E. coli contaminating proteins did. The protein solution containing PLR_Tp1 (67.25 mg) was next concentrated (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 desalted over a PD10 column equilibrated in buffer A. The resulting protein solution was divided into four equal aliquots that were sequentially loaded onto an ADP-agarose (Sigma) column (50 × 4.6 mm) pre-equilibrated in Buffer A at a flow rate of 2 ml min-1. After washing the column with Buffer A (16 ml), PLR_Tp1 was eluted using a linear NaCl gradient (0–2 m, 5 ml), which was then held at the 2 m NaCl concentration for another 5 ml. Fractions containing PLR_Tp1 were combined and concentrated to give a yield of ∼21 mg, whose purity was confirmed by SDS-polyacrylamide gel electrophoresis analysis followed by silver staining. Analytical Ultracentrifugation—A Beckman Optima XL-A analytical ultracentrifuge equipped with absorbance optics and an An60Ti rotor was used for both sedimentation equilibrium and sedimentation velocity experiments. Sedimentation equilibrium experiments were preequilibrated (1 h) at 4 °C and performed in Epon six-channel (1.2 cm path) centerpieces at 6,000, 9,000, and 12,000 rpm monitored at 280 nm. Different PLR_Tp1 concentrations (0.2, 0.6, and 2.0 mg ml-1) were used in potassium phosphate buffer (20 mm, pH 7.5) containing NaCl (100 mm). All of the samples were dialyzed (16 h) against the desired buffer conditions and used in the reference cells. Equilibrium profiles were analyzed with the program WinNONLIN (30.Johnson M.L. Correia J.J. Yphantis D.A. Halvorson H.R. Biophys. J. 1981; 36: 575-588Abstract Full Text PDF PubMed Scopus (778) Google Scholar). Sedimentation velocity experiments were performed at 25 °C in Epon double channel centerpieces (1.2 cm path) with PLR_Tp1 concentrations of 0.2, 0.6, and 2.0 mg ml-1 and at speeds of 25,000, 30,000, and 36,000 rpm. The samples were previously dialyzed against potassium phosphate buffer (20 mm, pH 7.5) containing 100 mm NaCl for 24 h. The sedimentation velocity data were collected at 280 nm with a radial increment of 0.3 mm and a delay between scans of 800 s. Only data with a defined meniscus and plateau were analyzed by the Svedberg 5.01 (31.Philo J.S. Data Analysis Software for XL-A Sedimentation Experiments, 5.01 Ed. Amgen Inc., Thousand Oaks, CA1992Google Scholar, 32.Philo J.S. Biophys. J. 1997; 72: 435-444Abstract Full Text PDF PubMed Scopus (215) Google Scholar) program using a modified Fujita-MacCosham function. Values of s and D were obtained from the relation Mw = sRT/D(1 - &̄nu;ρ), where Mw is the molecular weight (calculated from the sequence), s is the sedimentation coefficient, R is the gas constant, T is the temperature (corrected according to Durchschlag (33.Durchschlag H. Hinz H.-J. Thermodynamic Data for Biochemistry and Biotechnology. Springer-Verlag, New York1986: 45-116Crossref Google Scholar)), D is the diffusion coefficient, &̄nu; is the partial specific volume (calculated to be 0.750 from the amino acid composition using the method of Cohn and Edsall (34.Cohn E.J. Edsall J.T. Proteins, Amino Acids and Peptides as Ions and Dipolar Ions. Rheinhold, New York1943: 370-381Google Scholar)), and ρ is the solvent density. MALDI-TOF Mass Spectrometry—PLR_Tp1 samples were diluted to 10–20 pmol in H2O (0.4 μl) and mixed with an equal volume of a saturated (1:1 H2O:acetonitrile) solution of sinapinic acid as matrix. A Voyager DERP mass spectrometer with a PerSeptive Biosystems Voyager Biospectrometry Work station was used with data analyzed using the Data Explorer 5.1 software package. Laser fluence was about 20% above threshold, and the collected spectra were from 256 scans. Mass standards of cytochrome c (12,360 Da) and trypsinogen (23,980 Da) were used for instrument calibration. Crystallization of PLR_Tp1 and PCBER_Pt1—For crystallization, the buffer of the purified PCBER_Pt1 solution was replaced with a solution containing 20 mm Tris, pH 8.0, 1 mm EDTA, 5 mm dithiothreitol, and the protein concentration was adjusted to 50 mg ml-1. Crystals of PCBER_Pt1 were grown by the vapor diffusion method, with drops ∼4 μl in size being equilibrated against a reservoir containing 30% polyethylene glycol 8000, 0.1 m sodium cacodylate and 0.2 m sodium citrate. Diffraction quality crystals usually appeared after 10 days, and larger crystals with dimensions of ∼0.5 × 0.5 × 0.3 mm were obtained after 2 weeks. Some crystals diffracted up to 2.7 Å using a fine focusing RIGAKU rotating anode x-ray generator with mirror optics at 50 kV and 100 mA. The PCBER_Pt1 crystal belongs to the monoclinic space group P21 with two molecules in an asymmetric unit (a = 66.20 Å, b = 67.94 Å, c = 75.11 Å, β = 115.09°). The native data of 2.2 Å resolution was collected from the Advanced Light Source (ALS, beam line 8.2.2) at a temperature of -170 °C. Crystals of PLR_Tp1 were also grown by the vapor diffusion method, with drops of ∼4 μl in size being equilibrated against a 0.6-ml reservoir containing 20% polyethylene glycol 8000, 0.1 m MES, pH 6.2, 0.1 m NaCl, and 0.1 m calcium acetate. Diffraction quality crystals appeared after 2 weeks, and larger crystals with dimensions of ∼0.4 × 0.3 × 0.3 mm were obtained after 4 weeks. The PLR_Tp1 crystal belongs to the orthorhombic space group P212121 with four molecules in an asymmetric unit (a = 82.574 Å, b = 125.959 Å, c = 128.578 Å). The native data of 2.5 Å resolution was collected from the Stanford Synchrotron Radiation Laboratory (SSRL, beam line 9-2) at a temperature of -170 °C. Structure Solution and Refinement—The structure of PCBER_Pt1 was solved by combining multiwavelength anomalous dispersion (MAD) (platinum) and multiple isomorphous replacement (MIR) (mercury) methods, and the structure of PLR_Tp1 was solved by molecular replacement using the coordinates of PCBER_Pt1. After screening 20 heavy metal compounds, two marginal quality heavy atom derivatives were prepared by soaking crystals in solutions containing potassium tetrachloroplatinum and p-chloronitrophenyl mercury for ∼5 days, 2.5 Å

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