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Substrate Recognition by the Multifunctional Cytochrome P450 MycG in Mycinamicin Hydroxylation and Epoxidation Reactions*

  • Shengying Li
    Affiliations
    Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109

    Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong 266101, China
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  • Drew R. Tietz
    Affiliations
    Department of Chemistry and Rosenstiel Basic Medical Sciences Institute, Brandeis University, Waltham, Massachusetts 02454
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  • Florentine U. Rutaganira
    Affiliations
    Center for Discovery and Innovation in Parasitic Diseases, University of California, San Francisco, California 94158
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  • Petrea M. Kells
    Affiliations
    Center for Discovery and Innovation in Parasitic Diseases, University of California, San Francisco, California 94158
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  • Yojiro Anzai
    Affiliations
    Faculty of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan
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  • Fumio Kato
    Affiliations
    Faculty of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan
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  • Thomas C. Pochapsky
    Affiliations
    Department of Chemistry and Rosenstiel Basic Medical Sciences Institute, Brandeis University, Waltham, Massachusetts 02454
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  • David H. Sherman
    Affiliations
    Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109

    Department of Medicinal Chemistry, Chemistry, and Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan 48109
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  • Larissa M. Podust
    Correspondence
    To whom correspondence should be addressed: Dept. of Pathology, University of California, San Francisco, CA 94158. Tel.: 415-514-1381; Fax: 415-502-8193
    Affiliations
    Center for Discovery and Innovation in Parasitic Diseases, University of California, San Francisco, California 94158

    Department of Pathology, University of California, San Francisco, California 94158
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grants RO1 GM078553 (to D. H. S. and L. M. P.) and GM44191 (to T. C. P.). This work was also supported by the Hans W. Vahlteich Professorship (to D. H. S.).
Open AccessPublished:September 05, 2012DOI:https://doi.org/10.1074/jbc.M112.410340
      The majority of characterized cytochrome P450 enzymes in actinomycete secondary metabolic pathways are strictly substrate-, regio-, and stereo-specific. Examples of multifunctional biosynthetic cytochromes P450 with broader substrate and regio-specificity are growing in number and are of particular interest for biosynthetic and chemoenzymatic applications. MycG is among the first P450 monooxygenases characterized that catalyzes both hydroxylation and epoxidation reactions in the final biosynthetic steps, leading to oxidative tailoring of the 16-membered ring macrolide antibiotic mycinamicin II in the actinomycete Micromonospora griseorubida. The ordering of steps to complete the biosynthetic process involves a complex substrate recognition pattern by the enzyme and interplay between three tailoring modifications as follows: glycosylation, methylation, and oxidation. To understand the catalytic properties of MycG, we structurally characterized the ligand-free enzyme and its complexes with three native metabolites. These include substrates mycinamicin IV and V and their biosynthetic precursor mycinamicin III, which carries the monomethoxy sugar javose instead of the dimethoxylated sugar mycinose. The two methoxy groups of mycinose serve as sensors that mediate initial recognition to discriminate between closely related substrates in the post-polyketide oxidative tailoring of mycinamicin metabolites. Because x-ray structures alone did not explain the mechanisms of macrolide hydroxylation and epoxidation, paramagnetic NMR relaxation measurements were conducted. Molecular modeling based on these data indicates that in solution substrate may penetrate the active site sufficiently to place the abstracted hydrogen atom of mycinamicin IV within 6 Å of the heme iron and ∼4 Å of the oxygen of iron-ligated water.

      Introduction

      Mycinamicins are macrolide antibiotics produced by the actinomycete Micromonospora griseorubida (
      • Satoi S.
      • Muto N.
      • Hayashi M.
      • Fujii T.
      • Otani M.
      Mycinamicins, new macrolide antibiotics. I. Taxonomy, production, isolation, characterization, and properties.
      ). They are composed of a 16-membered macrolactone ring and two sugars, desosamine and mycinose, at the C-5 and C-21 positions respectively (Fig. 1). Wild-type M. griseorubida mainly produces mycinamicins I and II, mycinamicin IV (M-IV),
      The abbreviations used are: M-IV
      mycinamicin IV
      M-III
      mycinamicin III
      M-V
      mycinamicin V
      PDB
      Protein Data Bank.
      and mycinamicin V (M-V) (
      • Satoi S.
      • Muto N.
      • Hayashi M.
      • Fujii T.
      • Otani M.
      Mycinamicins, new macrolide antibiotics. I. Taxonomy, production, isolation, characterization, and properties.
      ), whereas a high producing industrial strain produces mycinamicin I and mycinamicin II as its two major products (
      • Takenaka S.
      • Yoshida K.
      • Yamaguchi O.
      • Shimizu K.
      • Morohoshi T.
      • Kinoshita K.
      Enhancement of mycinamicin production by dotriacolide in Micromonospora griseorubida.
      ). Mycinamicin II, having strong antimicrobial activities against Gram-positive bacteria and mycoplasma (
      • Satoi S.
      • Muto N.
      • Hayashi M.
      • Fujii T.
      • Otani M.
      Mycinamicins, new macrolide antibiotics. I. Taxonomy, production, isolation, characterization, and properties.
      ), has been developed as a veterinary drug (
      • Takenaka S.
      • Yoshida K.
      • Yamaguchi O.
      • Shimizu K.
      • Morohoshi T.
      • Kinoshita K.
      Enhancement of mycinamicin production by dotriacolide in Micromonospora griseorubida.
      ). Details of the mycinamicin biosynthetic pathway have been established by the isolation and structural characterization of intermediates (
      • Kinoshita K.
      • Satoi S.
      • Hayashi M.
      • Harada K.
      • Suzuki M.
      • Nakatsu K.
      Mycinamicins, new macrolide antibiotics. VIII. Chemical degradation and absolute configuration of mycinamicins.
      ,
      • Kinoshita K.
      • Imura Y.
      • Takenaka S.
      • Hayashi M.
      Mycinamicins, new macrolide antibiotics. XI. Isolation and structure elucidation of a key intermediate in the biosynthesis of the mycinamicins, mycinamicin VIII.
      ,
      • Kinoshita K.
      • Satoi S.
      • Hayashi M.
      • Nakatsu K.
      Mycinamicins, new macrolide antibiotics. X. X-ray crystallography and the absolute configuration of mycinamicin IV.
      ) and by bioconversion studies of genetically modified strains (
      • Suzuki H.
      • Takenaka S.
      • Kinoshita K.
      • Morohoshi T.
      Biosynthesis of mycinamicins by a blocked mutant of Micromonospora griseorubida.
      ,
      • Kinoshita K.
      • Takenaka S.
      • Suzuki H.
      • Morohoshi T.
      • Hayashi M.
      Mycinamicins, new macrolide antibiotics. XIII. Isolation and structures of novel fermentation products from Micromonospora griseorubida (FERM BP-705).
      ,
      • Inouye M.
      • Takada Y.
      • Muto N.
      • Beppu T.
      • Horinouchi S.
      Characterization and expression of a P-450-like mycinamicin biosynthesis gene using a novel Micromonospora-Escherichia coli shuttle cosmid vector.
      ). More recently, the complete 62-kb nucleotide sequence of the mycinamicin biosynthetic gene cluster comprising 22 open reading frames has been determined (
      • Anzai Y.
      • Saito N.
      • Tanaka M.
      • Kinoshita K.
      • Koyama Y.
      • Kato F.
      Organization of the biosynthetic gene cluster for the polyketide macrolide mycinamicin in Micromonospora griseorubida.
      ). Two P450 enzymes, MycCI and MycG, were identified in this cluster. MycCI is the C-21 methyl hydroxylase of mycinamicin VIII, the earliest macrolide in the post-polyketide synthase tailoring pathway, whose optimal activity depends on its native redox partner ferredoxin MycCII (
      • Anzai Y.
      • Li S.
      • Chaulagain M.R.
      • Kinoshita K.
      • Kato F.
      • Montgomery J.
      • Sherman D.H.
      Functional analysis of MycCI and MycG, cytochrome P450 enzymes involved in biosynthesis of mycinamicin macrolide antibiotics.
      ). In the mid-1990s, dual hydroxylation and epoxidation functions were proposed for a second P450, MycG, based on genetic complementation analysis (
      • Inouye M.
      • Takada Y.
      • Muto N.
      • Beppu T.
      • Horinouchi S.
      Characterization and expression of a P-450-like mycinamicin biosynthesis gene using a novel Micromonospora-Escherichia coli shuttle cosmid vector.
      ). Its activity was characterized in detail using an in vitro system reconstituted with recombinant MycG, native substrates isolated from fermentation broths, and a surrogate commercial spinach ferredoxin/ferredoxin reductase redox system, which was employed because MycCII does not support the catalytic activity of MycG (
      • Anzai Y.
      • Li S.
      • Chaulagain M.R.
      • Kinoshita K.
      • Kato F.
      • Montgomery J.
      • Sherman D.H.
      Functional analysis of MycCI and MycG, cytochrome P450 enzymes involved in biosynthesis of mycinamicin macrolide antibiotics.
      ). Collectively, these studies demonstrated that MycG catalyzes sequential hydroxylation and epoxidation reactions at two distinct sites, a tertiary allylic C–H bond (C-14) and an olefin (C12–C13). Premature epoxidation at C12–C13 completely abolishes hydroxylation at C-14, thus terminating the pathway (Fig. 1). Interestingly, P450 Gfs4 in the biosynthesis of the macrolide antibiotic FD-891 in Streptomyces graminofaciens represents yet another example of a single P450 enzyme sequentially introducing both a hydroxyl and an epoxy group on the 16-membered ring macrolide scaffold, but it has reverse order reactivities compared with MycG (Fig. 2) (
      • Kudo F.
      • Motegi A.
      • Mizoue K.
      • Eguchi T.
      Cloning and characterization of the biosynthetic gene cluster of 16-membered macrolide antibiotic FD-891. Involvement of a dual functional cytochrome P450 monooxygenase catalyzing epoxidation and hydroxylation.
      ).
      Figure thumbnail gr1
      FIGURE 1Final steps of mycinamicin biosynthesis. The solid arrows indicate the major steps in mycinamicin biosynthesis. The dashed arrow demonstrates low conversion from M-III to mycinamicin IX catalyzed by MycG. M-I cannot be hydroxylated by MycG.
      Figure thumbnail gr2
      FIGURE 2Natural products decorated by bacterial multifunctional P450 enzymes. Oxygen atoms introduced by P450s are highlighted in red.
      Other characterized bacterial biosynthetic multifunctional P450s in Streptomyces secondary metabolome include TamI in the tirandamycin biosynthetic pathway of Streptomyces sp. 307-9 (
      • Carlson J.C.
      • Li S.
      • Burr D.A.
      • Sherman D.H.
      Isolation and characterization of tirandamycins from a marine-derived Streptomyces sp.
      ,
      • Carlson J.C.
      • Fortman J.L.
      • Anzai Y.
      • Li S.
      • Burr D.A.
      • Sherman D.H.
      Identification of the tirandamycin biosynthetic gene cluster from Streptomyces sp. 307-9.
      ,
      • Carlson J.C.
      • Li S.
      • Gunatilleke S.S.
      • Anzai Y.
      • Burr D.A.
      • Podust L.M.
      • Sherman D.H.
      Tirandamycin biosynthesis is mediated by co-dependent oxidative enzymes.
      ), and AurH in the biosynthetic pathway of aureothin in Streptomyces thioluteus (Fig. 2) (
      • Zocher G.
      • Richter M.E.
      • Mueller U.
      • Hertweck C.
      Structural fine-tuning of a multifunctional cytochrome P450 monooxygenase.
      ). TamI operates on a bicyclic ketal moiety of tirandamycin C to catalyze successive epoxidation and hydroxylation reactions in an iterative cascade with the flavin oxidase TamL (
      • Carlson J.C.
      • Li S.
      • Gunatilleke S.S.
      • Anzai Y.
      • Burr D.A.
      • Podust L.M.
      • Sherman D.H.
      Tirandamycin biosynthesis is mediated by co-dependent oxidative enzymes.
      ). AurH sequentially installs two C–O bonds into the polyketide backbone of aureothin to yield a tetrahydrofuran ring, a key pharmacophore of this antibiotic (
      • He J.
      • Hertweck C.
      Iteration as programmed event during polyketide assembly; molecular analysis of the aureothin biosynthesis gene cluster.
      ,
      • Müller M.
      • He J.
      • Hertweck C.
      Dissection of the late steps in aureothin biosynthesis.
      ). A critical difference that sets multifunctional P450s apart from substrate promiscuous enzymes is an apparent hierarchy in the sequence of catalytic steps, suggesting that each step may be a prerequisite for the one that follows (
      • Podust L.M.
      • Sherman D.H.
      Diversity of P450 enzymes in the biosynthesis of natural products.
      ).
      The current data reveal that many fungal P450s are multifunctional enzymes that catalyze up to four consecutive steps on the same substrate molecule (
      • Crešnar B.
      • Petrič S.
      Cytochrome P450 enzymes in the fungal kingdom.
      ). For instance, Tri4 (CYP58) in the plant pathogen Fusarium graminearum performs one epoxidation and three hydroxylation steps in the biosynthesis of trichothecenes (
      • Tokai T.
      • Koshino H.
      • Takahashi-Ando N.
      • Sato M.
      • Fujimura M.
      • Kimura M.
      Fusarium Tri4 encodes a key multifunctional cytochrome P450 monooxygenase for four consecutive oxygenation steps in trichothecene biosynthesis.
      ). The trichothecenes are sesquiterpenoid secondary metabolites that are potent mycotoxins of mold-contaminated cereal grains. Another example of a multifunctional fungal P450 is Fum6 (CYP505 family) in the biosynthesis of mycotoxins fumonisins from the maize pathogen Fusarium verticillioides (
      • Seo J.A.
      • Proctor R.H.
      • Plattner R.D.
      Characterization of four clustered and coregulated genes associated with fumonisin biosynthesis in Fusarium verticillioides.
      ,
      • Bojja R.S.
      • Cerny R.L.
      • Proctor R.H.
      • Du L.
      Determining the biosynthetic sequence in the early steps of the fumonisin pathway by use of three gene-disruption mutants of Fusarium verticillioides.
      ,
      • Proctor R.H.
      • Plattner R.D.
      • Desjardins A.E.
      • Busman M.
      • Butchko R.A.
      Fumonisin production in the maize pathogen Fusarium verticillioides. Genetic basis of naturally occurring chemical variation.
      ), which catalyzes two consecutive hydroxylations at adjacent carbon atoms. The biosynthesis of plant hormone gibberellins in the rice pathogen Fusarium fujikuroi involves four multifunctional P450 enzymes that catalyze 10 of the 15 biosynthetic steps (
      • Tudzynski B.
      • Rojas M.C.
      • Gaskin P.
      • Hedden P.
      The gibberellin 20-oxidase of Gibberella fujikuroi is a multifunctional monooxygenase.
      ,
      • Tudzynski B.
      • Hedden P.
      • Carrera E.
      • Gaskin P.
      The P450-4 gene of Gibberella fujikuroi encodes ent-kaurene oxidase in the gibberellin biosynthesis pathway.
      ,
      • Rojas M.C.
      • Hedden P.
      • Gaskin P.
      • Tudzynski B.
      The P450-1 gene of Gibberella fujikuroi encodes a multifunctional enzyme in gibberellin biosynthesis.
      ). Fungal P450s are integral membrane proteins, making structural and biophysical characterization challenging. In this regard, understanding the switch of function mechanism in the more accessible bacterial multifunctional P450s should bring considerable new insights to this versatile class of underexplored monooxygenases.
      Ongoing structural and functional studies are beginning to provide detailed insights into the molecular basis for sequential reactivity and the pattern of oxidation in multifunctional systems. Although no substrate-bound structure has been obtained for AurH, a hypothetical switch of function mechanism has been proposed based on computational docking of two consecutive substrates into x-ray structures of different AurH ligand-free conformers (
      • Zocher G.
      • Richter M.E.
      • Mueller U.
      • Hertweck C.
      Structural fine-tuning of a multifunctional cytochrome P450 monooxygenase.
      ). The key role is assigned to glutamine 91, which upon completion of the hydroxylation step changes the conformation to provide an H-bond to the newly installed C7-OH group. In the course of mutual conformational adjustments, the hydroxylated intermediate relocates deeper into the substrate binding pocket to enable the next attack by the activated oxygen species at C-9a, whereas the substrate backbone bends to facilitate tetrahydrofuran ring formation.
      Here, we have structurally characterized MycG in complex with each of its two consecutive native substrates, M-IV and M-V, and their much less reactive biosynthetic precursor, M-III, which bears the monomethoxy sugar javose instead of dimethoxylated mycinose (Fig. 1). Despite this single methyl group difference, M-III is only hydroxylated by MycG in trace amounts in vitro (
      • Anzai Y.
      • Li S.
      • Chaulagain M.R.
      • Kinoshita K.
      • Kato F.
      • Montgomery J.
      • Sherman D.H.
      Functional analysis of MycCI and MycG, cytochrome P450 enzymes involved in biosynthesis of mycinamicin macrolide antibiotics.
      ). Traces of the C14-hydroxylated M-III (mycinamicin IX) and M-III bearing a C12–C13 epoxide from culture broths of the mycF disruption mutant M. griseorubida TPMA0004 are also consistent with marginal activity of MycG against M-III in vivo (
      • Tsukada S.
      • Anzai Y.
      • Li S.
      • Kinoshita K.
      • Sherman D.H.
      • Kato F.
      Gene targeting for O-methyltransferase genes, mycE mycF, on the chromosome of Micromonospora griseorubida producing mycinamicin with a disruption cassette containing the bacteriophage phi C31 attB attachment site.
      ,
      • Anzai Y.
      • Tsukada S.
      • Sakai A.
      • Masuda R.
      • Harada C.
      • Domeki A.
      • Li S.
      • Kinoshita K.
      • Sherman D.H.
      • Kato F.
      Function of cytochrome P450 enzymes MycCI and MycG in Micromonospora griseorubida, a producer of the macrolide antibiotic mycinamicin.
      ). We provide structural information that suggests a complex recognition strategy by MycG in which the enzyme successfully distinguishes between substrates and the closely related M-III intermediate. However, the precise mechanism of discrimination between the two native substrates M-IV and M-V remained obscure in light of the crystal structures obtained in this work due to the large distances separating the reactive sites from the iron center. Paramagnetically induced 1H spin relaxation NMR measurements indicate that deeper penetration of the active site of MycG by M-IV may take place in solution, and the pattern of observed relaxation effects is consistent with the x-ray binding orientation as predominating in the presence of excess substrate.

      DISCUSSION

      Specificity in P450 catalysis is dictated by the local chemical environment of the enzyme active site, which results in precise positioning of the substrate with respect to the iron catalytic center. Within bonding distances of the activated oxygen species, steric and stereoelectronic factors, rather than chemical reactivity of the reaction sites, determine regio- and stereo-selectivity of the oxidative reactions (
      • Kells P.M.
      • Ouellet H.
      • Santos-Aberturas J.
      • Aparicio J.F.
      • Podust L.M.
      Structure of cytochrome P450 PimD suggests epoxidation of the polyene macrolide pimaricin occurs via a hydroperoxoferric intermediate.
      ,
      • Johnston J.B.
      • Ouellet H.
      • Podust L.M.
      • Ortiz de Montellano P.R.
      Structural control of cytochrome P450-catalyzed ω-hydroxylation.
      ,
      • Chen M.S.
      • White M.C.
      Combined effects on selectivity in Fe-catalyzed methylene oxidation.
      ). To determine whether the MycG substrates consecutively bind in two discrete positions while progressing through the hydroxylation/epoxidation cascade, the binding modes of M-IV and M-V and their biosynthetic precursor M-III were extensively explored in this study using the x-ray and NMR techniques. A complex recognition pattern has emerged from this analysis.
      The orthogonal mycinose-in desosamine-out substrate-binding mode consistently observed in multiple co-structures reported in this work could represent a preliminary step en route to a catalytically productive orientation. In favor of this hypothesis is the large void volume in the active site, the conformational ambiguity of the Thr-284–Ala-285 fragment and the patches of scattered electron density that suggest the possibility of alternative substrate-binding modes. Toward this end, the binding behavior of the biosynthetic precursor M-III demonstrates a very different desosamine-in javose-out orientation that is parallel to the heme co-factor. Finally, the NMR relaxation data indicate that in solution M-IV may penetrate the active site sufficiently to place the abstracted hydrogen atom at C-14 within 6 Å of the heme iron. However, the paramagnetic relaxation model did not reveal the basis for epoxidation across the C12–C13 double bond.
      As evidenced by the MycG-M-III structures, javose is less preferable than mycinose as an initial recognition marker. The unproductive desosamine-in javose-out orientation of M-III likely results from the failure of javose to establish effective interactions with the heme. For substrate to reach a catalytically productive mode, mycinose instead of desosamine should lead the way, which is consistent both with the x-ray structures and the relaxation behavior of M-IV substrate in solution. This is in turn consistent with desosamine being predominantly distal to the heme iron in the M-IV complex.
      Rapid binding of substrate in the surface-exposed recognition site apparently is followed by its translocation to the catalytically competent orientation. Attainment of the latter state is likely coupled with conformational fluctuations of the Thr-284–Phe-286 region, which demonstrates conformational ambiguity across the structures and may depend on Phe-286 due to its central location and slower rate of fluctuation compared with adjacent Thr-284–Ala-285. Behavior of the Phe-286 mutants indicates that neither additional space in the active site introduced by the Phe-286 substitutions to leucine, valine, or alanine nor protein flexibility gained in F286G mutant facilitated M-IV relocation deeper in the catalytic pocket.
      By contrast, mycinose in M-V has swung toward Val-286 in the F286V mutant, in the direction opposite to the one predicted by the proton relaxation NMR model (Fig. 6D). This suggests the possibility of translocation to the void space of the buried cavity. Thus, opening access to the buried space might be a necessary but not sufficient step en route to the epoxidation-compatible binding mode that would place the double bond parallel with respect to the heme iron. The possibility of substrate relocation in two different directions assumed by the NMR model and by the co-structure of the F286V mutant might provide a plausible explanation for both catalytic functions of MycG.
      Interestingly, work in our laboratories has recently shown that removal of substrate from CYP101A1 results in a significant inward displacement of the β3 sheet, which is structurally homologous to the Phe-286 region in MycG (
      • Asciutto E.K.
      • Young M.J.
      • Madura J.
      • Pochapsky S.S.
      • Pochapsky T.C.
      Solution structural ensembles of substrate-free cytochrome P450(cam).
      ). The current modeling results suggest that the same region is displaced out of the active site to accommodate the substrate in an orientation conducive to the observed chemistry. However, it is likely that further reorganization, perhaps driven by the binding of a redox partner as is the case with CYP101A1 (
      • Wei J.Y.
      • Pochapsky T.C.
      • Pochapsky S.S.
      Detection of a high barrier conformational change in the active site of cytochrome P450cam upon binding of putidaredoxin.
      ), is required to reach the catalytically competent conformation of the enzyme. Change in the heme redox state may be another plausible factor gating access to a catalytically productive orientation.
      Based on the combined efforts of crystallography and the paramagnetic NMR relaxation data described in this report, the hypothesis of sequential translocation of the substrate molecule from the recognition site to the catalytic site has emerged. We interpret this translocation as a synergism between conformational changes of MycG and limited flexibility of the substrate molecule. Mycinose rotation around the bond connecting it to the macrolactone appears significant for sampling conformations that eventually would enable the substrate molecule to translocate to the catalytic site. Transition of the sugar moiety between the transient and catalytic binding pockets resembles in principle the mechanism previously observed for PikC substrates (
      • Li S.
      • Ouellet H.
      • Sherman D.H.
      • Podust L.M.
      Analysis of transient and catalytic desosamine-binding pockets in cytochrome P-450 PikC from Streptomyces venezuelae.
      ). In that system a single D50N mutation in PikC releases electrostatic interactions in the transient site and facilitates relocation of the desosamine sugar moiety to the buried catalytic pocket. This observation has found practical application in a PikCD50N mutant fused to a surrogate electron transporter (
      • Li S.
      • Podust L.M.
      • Sherman D.H.
      In vitro characterization of a self-sufficient biosynthetic cytochrome P450 PikC fused to a heterologous reductase domain RhFRED.
      ), which has been developed as a highly effective C–H bond activation catalyst for hydroxylation of diverse macrolide and carbolide substrates with antibiotic properties (
      • Li S.
      • Chaulagain M.R.
      • Knauff A.R.
      • Podust L.M.
      • Montgomery J.
      • Sherman D.H.
      Selective oxidation of carbolide C–H bonds by an engineered macrolide P450 mono-oxygenase.
      ). Based on our knowledge of PikC, AurH, and now MycG, it would not be surprising if a multistep progression to the catalytically productive mode turns out to be a common feature of the bulky and conformationally restrained substrates such as glycosylated macrolactones. Evidence for multistep substrate binding has also been suggested for other P450 enzymes, including drug-metabolizing P450s from human liver, that feature large substrate-binding sites (reviewed in Ref.
      • Isin E.M.
      • Guengerich F.P.
      Substrate binding to cytochromes P450.
      ). In cases where substrate translocation is the limiting step, protein engineering strategies could be applied to facilitate substrate progression to the active site and thus enhance P450 catalysis.

      Acknowledgments

      We thank Potter Wickware for critical reading of the manuscript and the staff members James Holton, George Meigs, and Jane Tanamachi of beamline 8.3.1 at the Advanced Light Source, Lawrence Berkeley National Laboratory, for assistance with data collection. The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the United States Department of Energy under Contract DE-AC02-05CH11231. Molecular graphics images were produced in part using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by National Institutes of Health Grant P41 RR001081).

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