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J. Biol. Chem., Vol. 281, Issue 36, 26289-26297, September 8, 2006
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1
2
From the
Life Sciences Institute and Department of Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan, 48109, the Department of Biochemistry and Center in Structural Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, 37232, and the ¶Argonne National Laboratory, Structural Biology Center, Argonne, Illinois, 60439
Received for publication, June 7, 2006 , and in revised form, June 30, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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Most of the natural product macrolide antibiotics are produced by Streptomyces sp. and related bacteria, in which assembly of polyketides from simple carboxylic acid precursors is catalyzed by modular polyketide synthases. Over the past 15 years, advances in understanding the modular architecture of polyketide biosynthetic machinery has enabled development of metabolic engineering approaches for production of new antibiotics (4-7). However, significant additional structural variability of polyketide-derived natural products is due to postpolyketide synthase biosynthetic modifications that typically include hydroxylation/epoxidation and/or glycosylation. In most antibiotic biosynthetic pathways, hydroxylation(s) occur(s) at the late stages of assembly after formation of the natural product scaffold and often after glycosylation events. Such modifications are often necessary to impart or enhance biological activity (8). The 3-(dimethylamino)-3,4,6-trideoxy sugar desosamine (or mycaminose) confers biological activity to a number of macrolide antibiotics such as erythromycin, troleandomycin, mycinamicin, megalomicin (desosamine), tylosin, carbomycin, and spiramycin (mycaminose) and is the only glycoside present on pikromycin, methymycin, and the highly potent semisynthetic ketolide telithromycin (9).
Both macrolides and ketolides were shown crystallographically to bind in a specific pocket in the ribosomal tunnel via interactions with 23 S rRNA and act to block sterically egress of the nascent protein chains (10). The desosamine sugar (or mycaminose) attached to C5 of the 14-, 15-, and 16-membered ring macrolactones extends up the tunnel toward the peptidyl transferase center. Interactions between desosamine and the ribosome play a key role in both macrolide selectivity and macrolide resistance (11).
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A series of particularly intriguing observations were made several years ago regarding the substrate selectivity of PikC that appeared to correlate directly with hydrogen bond functionality at the C3' position of the glycoside, with no tolerance for modifications at C4' (14-17). Such a stringent requirement toward sugar structure contrasts with the apparent flexibility of PikC toward the macrolactone core of a substrate. Thus, PikC is able to catalyze mono-hydroxylation at either C10 or C12 of the 12-membered ring macrolactone of YC-17, giving rise to methymycin and neomethymycin, respectively, as well as at C12 of the 14-membered ring of narbomycin, giving rise to pikromycin (20). Hydroxylation at the C14 position of pikromycin, giving rise to neopikromycin, occurs with a very low yield, which precluded isolation and characterization of this metabolite until recently (21). Dihydroxylation of YC-17 results in novamethymycin (Fig. 1), a product that is found in small quantities in vivo and appears to be converted from methymycin in vitro (22). Dihydroxylation of narbomycin results in a very low but detectable production of novapikromycin (21).
Here we provide new information regarding the diverse hydroxylation pattern of the PikC monooxygenase by determining its crystal structure in a variety of forms, including ligand-free and bound to endogenous substrates YC-17 and narbomycin (see Table 1). This series of crystal structures has revealed that in the absence of a substrate, PikC adopts both "open" and "closed" conformations due to repositioning of the BC-loop and the F and G helices. This dynamic process appears to enable substrate access to the active site; however, it does not account for PikC substrate flexibility, which is largely due to the combination of a single macrolactone binding site with two alternative desosamine binding pockets. In both pockets, the desosamine C3' dimethylamino group is sandwiched between two carboxyl-containing (Glu, Asp) amino acids found in the BC-region, forming a salt bridge to the more proximal carboxyl group. Site-directed mutagenesis revealed that formation of this salt bridge is essential for PikC function.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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Crystallization and Diffraction Data Collection—Purified PikC with the His tag at 0.2 mM concentration was either subjected to crystallization alone or mixed with substrate at 2 mM concentration (1:10 ratio) and then subjected to crystallization. In all cases, prior to crystallization, the protein was incubated for 1 h on ice with 1 mM dithiothreitol to prevent dimerization via Cys-375, which is readily observed during prolonged storage of PikC. Co-crystallization with ligands, either inhibitor or substrates, resulted in crystals of different size and morphology grown by vapor diffusion hanging drop method from a variety of crystallization conditions as indicated in Table 1. Ligand-free PikC crystals grew in the presence of 5 mM azole inhibitor 4-phenylimidazole (not found in the final crystal structure). All diffraction data were collected at 100-110 K at the Southeast Regional Collaborative Access Team (SER-CAT) 22ID and Structural Biology Center 19ID beamlines, Advanced Photon Source, Argonne National Laboratory, Argonne, IL. Glycerol mixed with the reservoir solution was used as a cryoprotectant at concentrations ranging from 18 to 25%, which have been determined empirically for each crystal type. The images were integrated and the intensities were merged by using HKL2000 (24).
Crystal Structure Determination—Molecular replacement was used to determine all crystal structures (Table 1). The ligand-free form of PikC was initially determined using the program CNS (25) and EryF (46% sequence identity) coordinates (26) as a search model. Crystal structures of YC-17- and narbomycin-bound PikC were similarly determined by molecular replacement using CNS (25), with ligand-free PikC coordinates as a search model. All reflections were used for refinement. The final atomic models (Table 1) were obtained after a number of iterations of refinement (CNS (25)) and manual model building with the program O (27). Ramachandran statistics (28) (Table 1) indicate no outliers with an exception of one residue, Pro-181, in the narbomycin-bound PikC structure (Protein Data Bank ID 2C7X). From 10 to 13 N-terminal residues, depending on the crystal form, are missing in all protein chains due to insufficient electron density in this region. Also, six BC-loop residues are missing in an open ligand-free form due to the same reason.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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X-ray Structure of Narbomycin-bound PikC—The 14-membered ring macrolide narbomycin is one of the two endogenous substrates for PikC (20). Hydroxylation of narbomycin at C12 (Fig. 1) of the macrolactone ring gives rise to the antibiotic pikromycin. In the co-crystal structure (1.75 Å resolution), narbomycin is unambiguously positioned in the active site, and its electron density is well defined (Fig. 3A). Of particular significance are interactions between the C3' dimethylamino group of the desosamine sugar and two carboxyl-containing residues localized in the BC-region (Glu-85 and Asp-50) (Fig. 4A). Although proximal Glu-85 (3.2 Å distance) provides a salt bridge contact, distal Asp-50 (5.3 Å distance) may compensate for a partial positive charge of the protonated tertiary amine. A calculated pKa of 8.85 for the desosamine nitrogen atom (calculated by using the on-line program SPARC (33)) is in good agreement with the values experimentally obtained for erythromycin (34), suggesting that at neutral pH, desosamine exists primarily in the protonated form. Interestingly, electrostatic interactions involving the C3' dimethylamino group are also involved in macrolide recognition in the ribosome tunnel where a positive charge is neutralized by a negative charge of an rRNA phosphate group invariantly positioned within 5 Å in structurally defined macrolide/ribosome complexes (35-38); however, no salt bridge is formed (Fig. 4C). In this regard, macrolide resistance in bacterial pathogens is often attributed to mono- or dimethylation at the A2058 N6 position (E. coli numbering) or to mutations that change nucleotide identity, both eliminating interactions with desosamine (11). In addition, PikC amino acid side chains Phe-178, Ala-187, Gln-188, Met-191, and Tyr-295 are within4Åofthe desosamine moiety (Figs. 3A and 4A). Although substrate anchoring by a biosynthetic P450 involving desosamine is unique, another type of anchoring mechanism was observed previously for the epothilone P450 EpoK from Sorangium cellulosum. In EpoK, a pendant thiazole moiety linked to the macrolactone ring system is involved in 
- interactions with two aromatic amino acids that determine substrate specificity (39).
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40:1 ratio in vivo) (21). The structure suggests that unfavorable stereochemistry of the C-H bonds at C14 (pointing away from the oxygen scission site) is the basis for this, which could be due to rotation of the C14-C15 bond toward the heme iron, possibly to avoid steric interference with the side chains of amino acids Leu-93 and Thr-294 (Fig. 3A). In contrast, a C-H bond at C12 is positioned favorably toward the oxygen scission site. Whether hydroxylation is controlled by regio- and stereochemistry of the C-H bond, by distance to the heme iron, or by the iron-oxo reactive intermediate (or a combination of these variables) remains to be elucidated. It is also possible that Fe+3 reduction followed by oxygen binding might result in repositioning of narbomycin, as is indicated by NMR paramagnetic relaxation studies on the P450BM3-laurate complex, in which 6 Å movement of the substrate into the correct position for hydroxylation has been detected (41).
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X-ray Structure of YC-17-bound PikC—The smaller 12-membered ring macrolide YC-17 is the second endogenous substrate of PikC (20). In contrast to narbomycin, it is hydroxylated in vivo and in vitro at two different positions (1:1 ratio). Modification at allylic C10 results in methymycin, whereas modification at methylene C12 results in formation of neomethymycin (Fig. 1). In the co-crystal structure determined to 2.35 Å resolution, YC-17 is unambiguously positioned in one orientation within the active site, and its electron density is well defined (Fig. 3B). As observed with narbomycin, the macrolactone ring of YC-17 is bound almost entirely via hydrophobic interactions projecting the more hydrophilic surface of the lactone toward the I helix and is devoid of hydrogen bonding. The binding site for the 12-membered ring lactone overlaps the entire binding site for the 14-membered ring macrocycle plus an extra amino acid residue, Val-290. As observed for narbomycin, the C3' dimethylamino group of the desosamine moiety in YC-17 is sandwiched between two carboxyl groups (Glu-85 and Glu-94) in a different binding pocket (Fig. 5) that is more buried in the protein interior when compared with the narbomycin desosamine binding site (Figs. 3B and 4B). Although proximal Glu-94 (2.7 Å distance) provides a salt bridge, distal Glu-85 (5.8 Å distance) may potentially compensate for the partial positive charge at the desosamine tertiary amine. Trp-74, Asn-91, and Glu-94 participate in a network of hydrogen bond contacts with the desosamine moiety (Fig. 4B). The BC-loop is more extensively involved in interactions with desosamine in this binding pocket, providing additional hydrophobic contacts via Trp-74, Leu-81, and Leu-88. The remaining desosamine-protein interactions are via Met-191 and His-238. Interestingly, the desosamine interaction pattern with amino acid residues in the PikC active site mimics interactions of desosamine with the bacterial ribosome drug target, although these involve specific RNA nucleotides and the phosphate backbone (Fig. 4C).
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2 Å) in positioning of the FG-region, which is most dynamic in the absence of substrate (supplemental movies 1 and 2). Overall r.m.s. deviation between the two complexes is 0.51 Å and is almost the same (0.6 Å) for carboxyl residues anchoring desosamine. This indicates that specific substrate-anchoring modes largely account for the diversity of PikC products rather than an induced fit mechanism. The two YC-17 hydroxylation sites, allylic C10 and methylene C12 atoms, are within 7.5 and 5.3 Å from the heme iron, respectively (Figs. 3B and 4B). However, the C12-C13 bond of YC-17 is rotated slightly away from the heme iron, thus presenting a C-H bond at C12 for catalysis. This favorable stereochemical arrangement in combination with the 5.3 Å distance to the heme iron apparently leads to hydroxylation of YC-17 at C12, giving rise to neomethymycin (Fig. 1). However, the question remains whether the C10 position in YC-17 is hydroxylated from this same binding orientation and/or via the same mechanism since it is separated by >7 Å from the heme iron atom.
Site-directed Mutagenesis in Desosamine Binding Pockets—To investigate directly the role of carboxyl functionalities in the desosamine binding pockets, site-directed mutagenesis was performed to replace Asp-50, Glu-85, and Glu-94 with alanine or glutamine/asparagines (Fig. 6). Functional activity of the mutant enzymes was assessed in vitro. Reaction products were analyzed by high pressure liquid chromatography. Substitution of the proximal carboxyl group forming a salt bridge to the C3' dimethylamino group, Glu-94 for YC-17 and Glu-85 for narbomycin, almost entirely abolishes conversion of the corresponding substrate, indicating that both charge and hydrogen-bonding capacities in the proximal position are essential for desosamine anchoring. This result is in agreement with a stringent requirement of hydrogen-bonding functionality in the C3' position in genetically modified sugars (14-17). Substitution of the carboxyl at distal amino acid groups (Glu-85 for YC-17 and Asp-50 for narbomycin) generated PikC mutants that retain partial to complete catalytic activity, indicating that the negative charge of the distal carboxyl is dispensable. Interestingly, the E85Q mutant, which lacks a negative charge but retains a hydrogen-bonding capability, shifts the product ratio of YC-17 hydroxylation toward methymycin (Fig. 6). This change in product profile does not rule out the possibility of interchangeable roles of proximal and distal carboxyl groups in desosamine binding, which may result in realignment of YC-17 in the active site, bringing C10 closer to the heme iron. Substitution of a third residue not participating in binding, Asp-50 for YC-17 and Glu-94 for narbomycin, allows full (YC-17) or significant residual (narbomycin) conversion to occur. It is particularly interesting that the D50N mutant provides more efficient hydroxylation of both macrolide substrates when compared with native PikC. Double mutant E85A/E94A is catalytically incompetent toward both endogenous macrolactone substrates (not shown).
In summary, we have determined four x-ray structures for the PikC P450 monooxygenase that catalyzes hydroxylation of two macrolide substrates, leading to four dominant reaction products. Based on these data, a unique anchoring mechanism has been identified that involves a specific salt bridge of glutamate amino acid residues to the C3' dimethylamino group of the deoxysugar substituent. Site-directed mutagenesis of Glu-85, Glu-94, and Asp-50 amino acid residues in the substrate binding pocket were conducted to confirm the role of each in desosamine anchoring and their impact on the product ratio. Formation of a salt bridge between the protein carboxyl group and desosamine dimethylamino group is essential for catalytic conversion of each substrate. The data suggest that substrate tolerance and diverse product distribution occur from two specific anchoring orientations rather than induced fit mechanisms. The 12- and 14-membered ring macrolactone portions of both macrolide substrates bind in the active site, utilizing virtually the same set of protein-substrate interactions. In contrast, the desosamine group binds in two alternative binding pockets, each providing two carboxyl residues, one of which involves a salt bridge to a C3' dimethylamino group of desosamine. Moreover, the presence of two desosamine binding pockets provides a unique opportunity to develop rational design of unnatural glycosylated substrates for PikC. It also enables selection of one of the two desosamine binding sites by site-directed mutagenesis to shift reaction product distribution toward a single desirable bioactive metabolite. It is particularly intriguing that the "acceptor" carbon atoms are located in non-equivalent positions with respect to the heme iron. Based on the x-ray structure, there is a 5 Å distance between the heme iron atom and the corresponding reactive methylene C atom that results in hydroxylation of analogous positions, C12 in YC-17, leading to neomethymycin, and C14 in narbomycin, leading to neopikromycin (Fig. 1). The latter hydroxylation product is formed in very low amounts, which might be due to the unfavorable stereochemistry of the C14-C15 bond that points toward the heme iron with both C14-H bonds directed away from the oxygen scission site. Two other analogous allylic hydroxylation sites, C10 in YC-17 and C12 in narbomycin, are both within 7.5 Å from the heme iron. This is far enough to raise questions about the ability of these positions to be hydroxylated (i) from this same substrate orientation as opposed to an alternative, as yet uncovered substrate orientation or (ii) via an oxo-ferryl P450 intermediate as opposed to a peroxy-iron species.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants GM37942, GM69970, ES00627 (to M. R. W.), and GM078553 (to D. H. S.), by grants from the Vanderbilt-Meharry Center for AIDS Research, Vanderbilt University Medical Center (VUMC) Discovery Grant Program, and U. S. Civilian Research and Development Foundation (to L. M. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
The on-line version of this article (available at http://www.jbc.org) contains two supplemental movies.
1 To whom correspondence may be addressed. Tel.: 734-615-9907; Fax: 734-615-3641; E-mail: davidhs{at}umich.edu. 2 To whom correspondence may be addressed. Tel.: 615-343-0943; Fax: 615-322-4349; E-mail: larissa.m.podust{at}vanderbilt.edu.
3 The abbreviations used are: Pik, pikromycin; r.m.s., root mean square; MES, 4-morpholineethanesulfonic acid.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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atoms to catalytically relevant YC-17- and narbomycin-bound forms. The heme co-factor is shown in red.
are in blue. To avoid excessive cluttering of stereo views with the electron density bulks, a number of residues, including heme and those that project on top of the substrate or each other, are excluded from the map calculation. Excluded in addition to heme in panel A are: Val-179, Ile-239, Val-242, Asn-392, and Ile-395; Excluded in addition to heme in panel B are: His-238, Val-242, Ala-243, and Met-394.
