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• W2105201265 abstract "Cytochrome P450s are heme-containing proteins that catalyze the oxidative metabolism of many physiological endogenous compounds. Because of their unique oxygen chemistry and their key role in drug and xenobiotic metabolism, particular attention has been devoted in elucidating their mechanism of substrate recognition. In this work, we analyzed the three-dimensional structures of a monomeric cytochrome P450 from Saccharopolyspora erythraea, commonly called EryK, and the binding kinetics to its physiological ligand, erythromycin D. Three different structures of EryK were obtained: two ligand-free forms and one in complex with its substrate. Analysis of the substrate-bound structure revealed the key structural determinants involved in substrate recognition and selectivity. Interestingly, the ligand-free structures of EryK suggested that the protein may explore an open and a closed conformation in the absence of substrate. In an effort to validate this hypothesis and to investigate the energetics between such alternative conformations, we performed stopped-flow absorbance experiments. Data demonstrated that EryK binds erythromycin D via a mechanism involving at least two steps. Contrary to previously characterized cytochrome P450s, analysis of double jump mixing experiments confirmed that this complex scenario arises from a pre-existing equilibrium between the open and closed subpopulations of EryK, rather than from an induced-fit type mechanism. Cytochrome P450s are heme-containing proteins that catalyze the oxidative metabolism of many physiological endogenous compounds. Because of their unique oxygen chemistry and their key role in drug and xenobiotic metabolism, particular attention has been devoted in elucidating their mechanism of substrate recognition. In this work, we analyzed the three-dimensional structures of a monomeric cytochrome P450 from Saccharopolyspora erythraea, commonly called EryK, and the binding kinetics to its physiological ligand, erythromycin D. Three different structures of EryK were obtained: two ligand-free forms and one in complex with its substrate. Analysis of the substrate-bound structure revealed the key structural determinants involved in substrate recognition and selectivity. Interestingly, the ligand-free structures of EryK suggested that the protein may explore an open and a closed conformation in the absence of substrate. In an effort to validate this hypothesis and to investigate the energetics between such alternative conformations, we performed stopped-flow absorbance experiments. Data demonstrated that EryK binds erythromycin D via a mechanism involving at least two steps. Contrary to previously characterized cytochrome P450s, analysis of double jump mixing experiments confirmed that this complex scenario arises from a pre-existing equilibrium between the open and closed subpopulations of EryK, rather than from an induced-fit type mechanism. The cytochrome P450 (P450) family consists of heme-containing enzymes with extremely diverse functions, distributed in virtually all organisms, from bacteria to human (1Wreck-Reichhart D. Feyereisen R. Genome Biol. 2000; 1: 3003.1-3003.9Google Scholar). They catalyze the oxidation of non-reactive C-H bonds and are involved in numerous metabolic reactions of many physiological endogenous compounds (i.e. antibiotics, lipids, and steroids), as well as xenobiotic molecules (2Davydov D.R. Fernando H. Baas B.J. Sligar S.G. Halpert J.R. Biochemistry. 2005; 44: 13902-13913Crossref PubMed Scopus (83) Google Scholar, 3Lamb D.C. Waterman M.R. Kelly S.L. Guengerich F.P. Curr. Opin. Biotechnol. 2007; 18: 504-512Crossref PubMed Scopus (109) Google Scholar, 4Pylypenko O. Schlichting I. Annu. Rev. Biochem. 2004; 73: 991-1018Crossref PubMed Scopus (75) Google Scholar). Because of their unique oxygen chemistry and key role in drug metabolism, multidisciplinary research has been focused on understanding the basis of their mechanism of substrate selectivity, both regarding binding and hydroxylation (5Isin E.M. Guengerich F.P. Anal. Bioanal. Chem. 2008; 392: 1019-1030Crossref PubMed Scopus (95) Google Scholar, 6Poulos T.L. Johnson E.F. Cytochrome P450s: Structure Mechanism and Biochemistry.in: Ortiz de Montellano P.R. 3rd Ed. Kluwer Academic/Plenum Publishers, New York2005: 377-530Google Scholar). Despite a highly conserved fold, P450s display a wide range of substrate specificities (7Denisov I.G. Makris T.M. Sligar S.G. Schlichting I. Chem. Rev. 2005; 105: 2253-2277Crossref PubMed Scopus (1606) Google Scholar). Some cytochrome P450s show broad substrate and hydroxylation specificity and generally act as xenobiotic metabolizing enzymes, reacting with over 75% of pharmaceutical products on the market (8Guengerich F.P. Chem. Res. Toxicol. 2008; 21: 70-83Crossref PubMed Scopus (1196) Google Scholar, 9Wienkers L.C. Heath T.G. Nat. Rev. Drug Discov. 2005; 4: 825-833Crossref PubMed Scopus (723) Google Scholar, 10Williams J.A. Hyland R. Jones B.C. Smith D.A. Hurst S. Goosen T.C. Peterkin V. Koup J.R. Ball S.E. Drug Metab. Dispos. 2004; 32: 1201-1208Crossref PubMed Scopus (791) Google Scholar). On the other hand, other P450s, mostly involved in biosynthetic pathways, perform highly regio- and stereospecific hydroxylations. The P450 from Saccharopolyspora erythraea CYP113A1, commonly called EryK, catalyzes one of the final steps of erythromycin A biosynthesis (Fig. 1). The initial steps of this biosynthetic pathway are performed by a polyketide synthase that carries out the condensation and stereospecific reduction of propionate with six molecules of methylmalonyl-CoA, producing the macrolactone 6-deoxyerythronolide B (11Khosla C. Tang Y. Chen A.Y. Schnarr N.A. Cane D.E. Annu. Rev. Biochem. 2007; 76: 195-221Crossref PubMed Scopus (218) Google Scholar). EryK acts as a hydroxylase on the C-12 of the macrolactone ring of the metabolic intermediate erythromycin D (ErD). 5The abbreviations used are: ErDerythromycin DErAerythromycin AErBerythromycin BSRSsubstrate recognition sitesBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolPDBProtein Data Bank. This catalytic step follows the initial C-6 hydroxylation, performed by the P450 EryF, and the insertion of two deoxysugar units on the fifth and third positions of 6-deoxyerythronolide B, performed by glycosyl transferases EryBV and EryCIII, respectively. The biosynthetic pathway is then completed by a methyl transferase, EryG, which transfers a methyl moiety on the mycarosyl sugar (12Lambalot R.H. Cane D.E. Aparicio J.J. Katz L. Biochemistry. 1995; 34: 1858-1866Crossref PubMed Scopus (75) Google Scholar, 13Stassi D. Donadio S. Staver M.J. Katz L. J. Bacteriol. 1993; 175: 182-189Crossref PubMed Google Scholar). EryG may also competitively act on ErD yielding a shunt metabolite, erythromycin B (ErB), which is an unsuitable substrate for further conversion by EryK to erythromycin A (14Chen Y. Deng W. Wu J. Qian J. Chu J. Zhuang Y. Zhang S. Liu W. Appl. Env. Microbiol. 2008; 74: 1820-1828Crossref PubMed Scopus (66) Google Scholar). This gives rise to significant shunt product accumulation during commercial fermentation of erythromycin A. erythromycin D erythromycin A erythromycin B substrate recognition sites 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol Protein Data Bank. Here we report a structural and biophysical characterization of recombinant EryK. We obtained three different crystal structures, two in the ligand-free form and one bound to ErD. On the basis of these structures we propose a mechanism for substrate recognition and regioselectivity and we highlight the conformational changes involved in substrate binding to EryK. Furthermore, we show that the ligand-free structure of EryK may be shifted from an open to a closed state by increasing ionic strength conditions. In an effort to support these observations, we present an extensive characterization of the binding kinetics of EryK to ErD, using both single and double mixing time-resolved stopped-flow experiments. The significance of our results, in the context of previous characterization of the structure and function of P450s, will be also discussed. Recombinant His-tagged EryK was overexpressed in the BL21 STARTM (DE3) Escherichia coli strain (Invitrogen) and purified as described previously (15Savino C. Sciara G. Miele A.E. Kendrew S.G. Vallone B. Protein Pept. Lett. 2008; 15: 1138-1141Crossref PubMed Scopus (7) Google Scholar). Purified EryK was concentrated and stored at 193 K. To assess its oligomerization state, EryK was subjected to ultracentrifugation in a Beckman Optima XL-1 instrument equipped with absorbance optics. Protein samples, measured at a concentration of 15 μm in the presence of 50 mm Tris·HCl, pH 7.5, at 298 K, were found to be monomeric (supplemental Fig. S1). Three crystal forms of EryK were obtained in different crystallization conditions: with His tag (His6-EryK) in low salt conditions, without tag (EryK) in high salt, and in complex with its substrate erythromycin D (ErD-EryK) (Table 1). All crystallization protocols were carried out at 294 K by vapor diffusion and ErD was introduced by co-crystallization. Diffraction data were collected at 100 K at the ESRF synchrotron (Grenoble, France) beamlines ID14-3 using a marCCD detector, and ID14-2 using a ADSC Q4R detector. Data were integrated and scaled using the HKL suite (16Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar), except for His6-EryK data, which were processed with MOSFLM (17Leslie A.G.W. Joint CCP4/ESF-EACBM Newsl. Prot. Crystallogr. 1992; 26: 27-33Google Scholar) and scaled with SCALA in the CCP4 suite (18Collaborative Computational Project, N.4Acta Crystallogr. D Biol. Crystallogr. 1994; 50: 7690-7763Google Scholar). A summary is shown in Table 1.TABLE 1Data collections and refinements statisticsProtein name, PDB IDOpen EryK, PDB 2WIOClosed EryK, PDB 2JJNErD-EryK, PDB 2JJOCrystallization conditions25% PEG 3350, 0.2 m NaCl,0.1 m Tris-HCl, pH 8.02.0 m (NH4)2SO4,0.1 m BisTris, pH 6.525% PEG 3350, 0.2 m CH3COONH4,0.1 m Tris-HCl, pH 8.5Data collectionSpace groupP212121P21P21Cell dimensions (Å)a = 37.93, b = 57.34,c = 179.56a = 53.46, b = 68.00, c = 57.41,β = 101.05°a = 57.84, b = 36.53, c = 96.00,β = 93.92°Resolution (Å)2.00 (2.01-1.91)1.60 (1.66-1.60)2.00 (2.15-2.00)Completeness (%)82.2 (85.9)99.2 (97.9)91.5 (80.4)Redundancy4.8 (4.7)3.1 (2.6)4.0 (3.1)RmergeaRmerge = ΣiΣj|II,j − 〈Ij〉|/ΣiΣjIi,j, where i runs over multiple observations of the same intensity, and j runs over all crystallographically unique intensities. (%)19.3 (43.8)3.9 (18.9)5.9 (24.5)I/σ(I)10.9 (3.1)19.9 (19.1)13.0 (10.9)No. reflections22,45953,46125,564B Wilson (Å2)19.716.119.5RefinementResolution (Å)89.9-2.0056.3-1.6095.8-2.00No. of atomsProtein/amino acid range3096/19-4113269/17-4113096/19-411Heme/ligand434343/49Water/sulfate309546/25335Rcryst/RfreebRwork = Σ‖Fobs| −|Fcalc‖/Σ|Fobs|, where |Fobs| > 0. Rfree is based on 5% of the data randomly selected and is not used in the refinement. (%)18.3/23.216.1/19.816.9/23.6Mean B-factors (Å2)Protein25.121.022.9Heme16.811.813.0Substrate15.7Water/ion33.332.0/31.126.5Root mean square deviationsBond (Å)0.0140.0160.013Angles (°)1.5431.6651.624Ramachandran (%)Most favored/allowed/generous/disallowed89.3/10.7/0/091.2/18.2/0.3/0.390.8/8.0/0.9/0.3a Rmerge = ΣiΣj|II,j − 〈Ij〉|/ΣiΣjIi,j, where i runs over multiple observations of the same intensity, and j runs over all crystallographically unique intensities.b Rwork = Σ‖Fobs| −|Fcalc‖/Σ|Fobs|, where |Fobs| > 0. Rfree is based on 5% of the data randomly selected and is not used in the refinement. Open table in a new tab In all cases molecular replacement was carried out to determine the initial crystallographic phases using the program MolRep (19Vagin A. Teplyakov A. Acta Crystallogr. D Biol. Crystallogr. 1998; 54: 400-402Crossref PubMed Scopus (41) Google Scholar) from the CCP4 suite (18Collaborative Computational Project, N.4Acta Crystallogr. D Biol. Crystallogr. 1994; 50: 7690-7763Google Scholar). The ligand-free form of His6-EryK was determined using EryF coordinates (20Cupp-Vickery J.R. Poulos T.L. Nat. Struct. Biol. 1995; 2: 144-153Crossref PubMed Scopus (408) Google Scholar) (33.9% sequence identity, supplemental Fig. S2) as a search model (PDB code 1OXA). EryK was similarly determined by molecular replacement with His6-EryK coordinates as a search model. EryK coordinates were used to solve the structure of ErD-EryK. Initial atomic models were subjected to iterative rounds of building and refinement. Refinement was carried out with Refmac5 (21Pannu N.S. Murshudov G.N. Dodson E.J. Read R.J. Acta Crystallogr. D Biol. Crystallogr. 1998; 54: 1285-1294Crossref PubMed Scopus (197) Google Scholar) in CCP4, followed by model building and manual readjustment performed in QUANTA (Accelrys Inc.). Solvent molecules were added into the Fo − Fc density map, contoured at 3σ, with the X-SOLVATE tool of QUANTA. Secondary structure assignment was performed using the Kabsch and Sander algorithm and geometrical quality of final models was assessed using PROCHECK (22Laskowski R.A. Moss D.S. Thornton J.M. J. Mol. Biol. 1993; 231: 1049-1067Crossref PubMed Scopus (1087) Google Scholar). The first 14 to 18 N-terminal residues, depending on the crystal form, are missing in all models due to insufficient electron density in this region. Final statistics are shown in Table 1. All figures were produced with PyMOL. Root mean square deviation and sequence identity among the structures of EryK, PikC, and EryF were determined using CE (23Shindyalov I.N. Bourne P.E. Protein Eng. 1998; 11: 739-747Crossref PubMed Scopus (1702) Google Scholar). Sequence numbering refers to EryK (SwissProt accession A4FNo_SACEN; NCBI accession YP_001102980); secondary structure elements are named following P450 convention. Because catalysis would require the presence of external reducing agents and EryK was purified in its oxidized form, addition of ErD results in monitoring only the binding reaction (12Lambalot R.H. Cane D.E. Aparicio J.J. Katz L. Biochemistry. 1995; 34: 1858-1866Crossref PubMed Scopus (75) Google Scholar). Thus, all the experiments reported in this work refer to the binding reaction of EryK to ErD, in the absence of catalysis. Binding affinities of ErD to EryK in different ionic strength conditions were determined (at 298 K) by titrating 2 μm enzyme with the ligand, ErD, in a total volume of 800 μl of 50 mm Tris·HCl buffer, pH 7.5, with and without 2 m NaCl. Ligand concentration covered a suitable range from 0 to 300 μm. UV visible spectra (190–820 nm) were recorded after each addition and the absorbance intensities at 420 and 390 nm were plotted against the logarithm of the added ligand concentrations (data not shown). From each of the substrate-bound spectra were subtracted the appropriate blank. The dissociation constant (KD) was estimated using the Kaleidagraph software package. A nonlinear regression analysis was applied using hyperbolic equation ΔAUobs = ΔAUmax [L]/(KD + [L]), where ΔAUobs is the absorbance difference, ΔAUmax is the maximum absorbance difference extrapolated to infinite ligand concentration, and [L] is the ligand analytical concentration. Data were also globally fitted with the program Prism (Graphpad). The time evolution of the EryK-ErD interaction was followed by monitoring the change in absorbance at 390 nm on a stopped-flow apparatus. Measurements were performed on a SX18-MV stopped-flow instrument (Applied Photophysics, Leatherhead, UK) using both symmetric and asymmetric (1:10) mixing. In the symmetric mixing mode, EryK, at a final constant concentration of 7.5 μm, was mixed with ErD diluted to the desired final concentration, varying in a range from 9 to 150 μm. The traces were recorded in 50 mm Tris·HCl, pH 7.5, at 288 K, both in the absence and presence of 2 m NaCl. In the asymmetric mixing mode, varying the NaCl concentration in a range from 0 to 3.6 m, the kinetic traces of the interaction between EryK (final constant concentration of 5.5 μm) and ErD (final constant concentration of 100 μm) were recorded at 288 K in 50 mm Tris·HCl, pH 7.5. Kinetic traces were extracted from the acquired spectra (recorded as average of at least three experiments) and analyzed using the Kaleidagraph software package. To characterize the pre-equilibrium stage of the open and closed conformations of EryK, we performed double mixing measurements carried out in 50 mm Tris·HCl, pH 7.5, by using an Applied Photophysics SX18-MV stopped-flow instrument at 288 K. EryK (13.5 μm) was incubated in 2 m NaCl for different delay times and then symmetrically mixed with ErD at a final concentration of 100 μm, in 50 mm Tris·HCl, pH 7.5, with 2 m NaCl. The observed amplitudes, reflecting the fraction of molecules populating the open and closed conformations, were plotted as a function of delay times and fitted to a single exponential decay. Classically, complex kinetics for a monomeric protein, as EryK, undergoing a ligand-induced conformational change, is described by the Scheme 1. A binding event progressing through pathways 1 and 2 is representative of an induced fit model, whereby ligand binding is postulated to induce a conformational change (24Koshland Jr., D.E. Némethy G. Filmer D. Biochemistry. 1966; 5: 365-385Crossref PubMed Scopus (2208) Google Scholar), which may be transmitted to neighboring residues as shown in Scheme 2. Under such conditions, observations of biphasic kinetics arise from the accumulation of the EryK-ErD reaction intermediate. When and if the fast phase is λ1 ≫ λ2, the fast phase λ1 is due to the initial encounter between the EryK and ErD molecules, with an observed rate constant shown in Equation 1. λ1=k1[ErD]+K1(Eq. 1) Furthermore, the slow phase λ2 is then expected to increase hyperbolically with increasing ErD concentration as shown in Equation 2. λ2=k-2+k2[ErD]KD'+[ErD](Eq. 2) where KD'=k-1k1(Eq. 3) In this case, λ2 is expected to equal k−2 at [ErD] = 0 and (k−2 + k2) at [ErD] → ∞.SCHEME 2View Large Image Figure ViewerDownload Hi-res image Download (PPT) Alternatively, a binding event progressing through pathways 3 and 4 assumes that two alternative conformations are in pre-equilibrium in the absence of the ligand, formally similar to a concerted or conformational selection model (25Monod J. Wyman J. Changeux J.P. J. Mol. Biol. 1965; 12: 88-118Crossref PubMed Scopus (6184) Google Scholar). Accordingly, complex kinetics may arise from a heterogeneous conformational ensemble of EryK, which may populate, in the absence of ligand, experimentally detectable populations of both EryK and EryK*. Under such conditions, and if k−2 ≫ (k1 + k−1), the fast phase would arise from an initial population of monomers in the EryK* conformation and would occur with a rate constant as described in Equation 4. λ1=k2[ErD]+k-2(Eq. 4) On the other hand, the slow phase λ2 is expected to decrease hyperbolically with increasing ErD concentration as shown in Equation 5. λ2=k1+k-1KD'KD'+[ErD](Eq. 5) where KD'=k-2k2(Eq. 6) In this case, λ2 is expected to approach (k−1 + k1) at [ErD] = 0 and k1 at [ErD] → ∞. EryK displays the typical topology of previously characterized P450s (Fig. 2). This enzyme shows the highest sequence identity with EryF (33.9%), which is involved in the same biosynthetic pathway (see supplemental Fig. S2). The conserved protein core is formed by a four-helix bundle, three parallel helices (D, L, and I) and an antiparallel one (E). The prosthetic heme group is located between the distal helix I and the proximal helix L and it is bound to Cys353, the highly conserved fifth ligand of the heme iron. Despite a conserved overall structure, P450s display a wide range of substrate specificities (1Wreck-Reichhart D. Feyereisen R. Genome Biol. 2000; 1: 3003.1-3003.9Google Scholar). At a structural level, P450 substrate recognition and binding is assured by six so called “substrate recognition sites” (SRS) that line the active site (26Gotoh O. J. Biol. Chem. 1992; 267: 83-90Abstract Full Text PDF PubMed Google Scholar). Although the definition of SRS becomes obsolete once the structure has been determined and precise protein-substrate contacts have been revealed, we will use in this section this nomenclature to define the amino acid topology because in EryK the elements that contact the substrate correspond to these SRS. These regions are: SRS1, formed either by the BC loop (as in EryK) or the B′ helix (as in EryF); SRS2 formed by the C-terminal part of helix F; SRS3 formed by the N-terminal part of helix G; SRS4 formed by the central part of the distal helix I; SRS5 formed by the loop at the C-terminal end of helix K; SRS6 formed by the β-hairpin β4 (supplemental Fig. S2). Some SRSs may rearrange upon substrate binding, formally following an induced fit scenario (4Pylypenko O. Schlichting I. Annu. Rev. Biochem. 2004; 73: 991-1018Crossref PubMed Scopus (75) Google Scholar). The superposition of the crystal structures of the two ligand-free forms of EryK (Fig. 2C), obtained at low and high salt, reveals that there are two different conformations for this protein in the unbound state, involving, among others, a movement of the BC loop of about 11 Å. Furthermore, helix G kinks at the level of Pro192, causing its N-terminal part to shift about 10 Å toward the BC loop. Interestingly, Pro192 is not conserved in homologous P450. The movement of helix G also pulls the FG loop and helix F, causing the latter to unwind almost completely (Figs. 2C and 3A, and supplemental Movie 1). This reorganization, observed in the EryK structure at high ionic strength, closes the access channel for the active site of the molecule. Although the last two C-terminal turns of helix F keep parallel to the moving part of helix G, its N-terminal part (Asp158–Asp164) adopts a coil conformation upon active site closing. As shown in detail in Fig. 3C, the coil conformation of helix F causes the loss of a H-bond between His243 and Ser166 and contact between Trp165 and Pro192 that connect helix I to helix G through helix F in an open ligand-free EryK structure obtained at low ionic strength. Moreover, Trp165 rotates and docks in a new position pointing toward helix I and interacts with His243 (supplemental Movie 2). In summary, these rearrangements result in a marked reduction of the exposure of the heme distal site that identifies two different conformations to which we will refer to as “open” and “closed” depending on the position of helices F and G and the BC loop. Importantly, an open/closed transition has been previously observed in other P450s, as in the case of the Bacillus megaterium flavocytochrome P450 BM3 (CYP102) (27Li H. Poulos T.L. Acta Crystallogr. D Biol. Crystallogr. 1995; 51: 21-32Crossref PubMed Scopus (108) Google Scholar, 28Ravichandran K.G. Boddupalli S.S. Hasermann C.A. Peterson J.A. Deisenhofer J. Science. 1993; 261: 731-736Crossref PubMed Scopus (912) Google Scholar) and PikC (CYP107L1), the Streptomyces venezuelae P450 involved in C-12 hydroxylation in the pikromycin biosynthetic pathway (29Sherman D.H. Li S. Yermalitskaya L.V. Kim Y. Smith J.A. Waterman M.R. Podust L.M. J. Biol. Chem. 2006; 281: 26289-26297Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). We co-crystallized EryK in a complex with ErD. The well defined electron density of the substrate was unambiguously positioned in the active site (Fig. 3B). The cavity hosting the substrate involves essentially two regions: a fixed one, formed by the loop at the helix K C-terminal, the β-hairpins β4 and β1 (Asp46–Gly49), and a mobile one, formed by the BC loop and the F and G helices. A central feature of ErD recognition involves anchoring of the two sugar substituents, a desosamine on C-5 and a mycarosyl group on C-3 of the macrolactone ring (supplemental Table S1 and Fig. 3B). Desosamine interacts mainly with the loop at the helix K C-terminal, by two H-bonds with Gln290 and Gln292; moreover, Ile392 in the β-hairpin β4 ensures the sugar clamping by van der Waals contacts. It is worth emphasizing that this is the only amino acid close to the forbidden conformation in the Ramachandran plot for the refined ErD-EryK structure, suggesting a critical structural role (30Cheng G. Qian B. Samudrala R. Baker D. Nucleic Acids Res. 2005; 33: 5861-5867Crossref PubMed Scopus (68) Google Scholar, 31Heringa J. Argos P. Proteins. 1999; 37: 44-55Crossref PubMed Scopus (32) Google Scholar). All these interactions would be absent for a ligand lacking the desosamine sugar as in the case of the biosynthetic intermediate MycEB (see Fig. 1). Mycarose interacts with the mobile part of the channel establishing four crossed H-bonds with His88 and Glu89 on BC loop (Fig. 3B). Its position is also restricted by van der Waals interactions with the FG loop, which closes down on top of it. Substrate binding can therefore be described as landing on the fixed region followed by closure of two “arms” consisting of the BC loop and the F and G helices. The selectivity for ErD against ErB is clearly explained by this structure. Indeed, methylation of 3′-OH would interfere with the complete closure of the arms to embrace the substrate, caused by clashes with Met86, His88, and Glu89 in the BC loop. The macrolactone ring of ErD establishes hydrophobic interactions with residues on helix I, helix K on the C-terminal, and β-hairpin β4 (Table S1). Ile244 and Thr245 on helix I lie in the active site, and the amino acids in these topological positions are believed to be involved in catalysis, playing a role in stabilization and proton delivery to the iron-bound dioxygen species (32Meunier B. de Visser S.P. Shaik S. Chem. Rev. 2004; 104: 3947-3980Crossref PubMed Scopus (1871) Google Scholar). Altogether packing the macrolactone ring against the heme and SRS4 is very tight, explaining why limited hydroxylation at C-12 was observed when bulky substituents were introduced in C-13 (33Pacey M.S. Dirlam J.P. Geldart R.W. Leadlay P.F. McArthur H.A. McCormick E.L. Monday R.A. O'Connell T.N. Staunton J. Winchester T.J. J. Antibiot. 1998; 51: 1029-1034Crossref PubMed Scopus (56) Google Scholar, 34Ruan X. Pereda A. Stassi D.L. Zeidner D. Summers R.G. Jackson M. Shivakumar A. Kakavas S. Staver M.J. Donadio S. Katz L. J. Bacteriol. 1997; 179: 6416-6425Crossref PubMed Scopus (122) Google Scholar) and also why there is no flexibility allowed for substrate hydroxylation sites, as observed for PikC (29Sherman D.H. Li S. Yermalitskaya L.V. Kim Y. Smith J.A. Waterman M.R. Podust L.M. J. Biol. Chem. 2006; 281: 26289-26297Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), where empty pockets in the binding site are present. In ErD-EryK, the target of hydroxylation C-12 of the macrolactone ring is positioned 5.3 Å from the heme iron. Similar distances between the hydroxylation site and the iron have been reported for other P450s, such as EryF (Fe-C6 4.8 Å) and P450cam (Fe-C5 4.2 Å) (35Poulos T.L. Finzel B.C. Howard A.J. J. Mol. Biol. 1987; 195: 687-700Crossref PubMed Scopus (1296) Google Scholar). The water molecule bound to the oxidized heme iron in the ligand-free structure of EryK disappears and a new one (Wat102) is found at 2.7 Å from the metal, which is H-bonded to the substrate 11-OH group. This water occupies a position that almost coincides with the O-2 atom of dioxygen bound to P450cam. Two more water molecules are found in the vicinity of Thr245, Wat4 and Wat38, which in P450cam appear upon dioxygen binding (36Schlichting I. Berendzen J. Chu K. Stock A.M. Maves S.A. Benson D.E. Sweet R.M. Ringe D. Petsko G.A. Sligar S.G. Science. 2000; 287: 1615-1622Crossref PubMed Scopus (1219) Google Scholar). Moreover, Wat27 is also positioned as in P450cam, hydrogen bonded to conserved Asp362. Therefore, taking into account the carbonyl atom of Gly242, in ErD-EryK we find a water molecule chain that leads from the active site to the bulk. This network corresponds closely to that observed in the oxygen-bound form of P450cam, with Wat102 mimicking the bound dioxygen (supplemental Fig. S3). From this work we cannot determine the mechanism of EryK catalysis, but given the conservation of water molecules in the active site and Thr241, we may infer that the general catalytic mechanism proposed for bacterial P450 is conserved. Structural rearrangements due to substrate binding occur in the most dynamic regions of EryK (F and G helices and BC loop). Movements of these elements are not hindered from crystal contacts in open, closed, and closed-bound EryK. The ErD-EryK structure resembles the closed ligand-free form (Fig. 2, B and D), but new interactions are found that work as a molecular device that switches upon substrate binding. The movement of the BC loop in ErD-EryK is less pronounced than in the closed ligand-free form, because interactions with the mycarose hinder its full transition. Moreover, helices F and G move less on top of the substrate channel (9 Å displacement instead of 11 Å); helix G kinks at the level of Pro192 and, together with the FG loop, adopts a conformation similar to the closed ligand-free structure (Cα differences “ligand-free closed”/ErD-EryK ∼ 1.5 Å). Helix F in the ErD-EryK structure follows the movement of helix G, being lifted about one turn (3.7 Å) and tilted by 13°. This helix is not unwound on its N-terminal side, whereas in the ligand-free closed structure it loses the α helix geometry almost completely (Figs. 2B and 3A, and supplemental Movie 3). Therefore, in the open and substrate-bound structures, helix F is present and held by H-bonds wit" @default.
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