Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M404011200 on May 3, 2004

J. Biol. Chem., Vol. 279, Issue 29, 30634-30642, July 16, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/29/30634    most recent
M404011200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Buglino, J.
Right arrow Articles by Lima, C. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Buglino, J.
Right arrow Articles by Lima, C. D.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Crystal Structure of PapA5, a Phthiocerol Dimycocerosyl Transferase from Mycobacterium tuberculosis*

John Buglino{ddagger}§, Kenolisa C. Onwueme¶||, Julian A. Ferreras¶, Luis E. N. Quadri¶**, and Christopher D. Lima, Supported in part by National Institutes of Health Grant GM62529. Also acknowledges additional support from the Rita Allen Foundation and the Arnold and Mabel Beckman Foundation{ddagger}{ddagger}{ddagger}

From the {ddagger}Structural Biology Program, Sloan-Kettering Institute, New York, New York 10021 and the Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York 10021

Received for publication, April 12, 2004 , and in revised form, April 26, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Polyketide-associated protein A5 (PapA5) is an acyltransferase that is involved in production of phthiocerol and phthiodiolone dimycocerosate esters, a class of virulence-enhancing lipids produced by Mycobacterium tuberculosis. Structural analysis of PapA5 at 2.75-Å resolution reveals a two-domain structure that shares unexpected similarity to structures of chloramphenicol acetyltransferase, dihydrolipoyl transacetylase, carnitine acetyltransferase, and VibH, a non-ribosomal peptide synthesis condensation enzyme. The PapA5 active site includes conserved histidine and aspartic acid residues that are critical to PapA5 acyltransferase activity. PapA5 catalyzes acyl transfer reactions on model substrates that contain long aliphatic carbon chains, and two hydrophobic channels were observed linking the PapA5 surface to the active site with properties consistent with these biochemical activities and substrate preferences. An additional {alpha} helix not observed in other acyltransferase structures blocks the putative entrance into the PapA5 active site, indicating that conformational changes may be associated with PapA5 activity. PapA5 represents the first structure solved for a protein involved in polyketide synthesis in Mycobacteria.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mycobacterium tuberculosis produces polyketides, a complex family of lipids (1) that includes compounds associated with mycobacterial virulence. Up to 24 genes are predicted to encode proteins with polyketide synthase activity in the M. tuberculosis genome (H37Rv) (2). Although several of these and associated genes have been directly implicated with Mycobacterium virulence (3–11), several others remain less well characterized. One such group includes a family of five polyketide-associated proteins (Paps)1 that were suspected to encode activities associated with polyketide biosynthesis or transport (12–15). Additional Pap orthologs have been uncovered in polyketide synthase loci in Mycobacterium leprae, Mycobacterium bovis, and other Mycobacterium species (16), suggesting that Paps may contribute activities to conserved pathways across Mycobacterium species.

Phthiocerol dimycocerosate esters and their congeners, otherwise known as PDIMs, comprise a polyketide family that has been shown to be directly involved in mycobacterial virulence (3, 4). M. tuberculosis mutants deficient in PDIM production are attenuated in mice and PDIMs produced by M. leprae promote Schwann cell tropism (17, 18). PDIM biosynthesis has been proposed to involve the activities of at least three polyketide synthase gene systems. The first and second systems include ppsA-E and mas, genes involved in phthiocerol/phthiodiolone synthesis and mycocerosic acid synthesis, respectively (10). The third includes pks15/1, a gene that has been associated with the incorporation of the phenolic group into mycoside PDIM variants (19), and possibly other pks genes to produce early biosynthetic precursors (6, 8, 21).

The genes associated with diesterification of phthiocerol and phthiodiolone to mycocerosic acid have remained unclear, but recent progress has been made through the genetic and functional characterization of M. tuberculosis PapA5, a Pap family member located within the PDIM synthesis gene cluster (16). Deletion and complementation of the gene encoding PapA5 indicated that PapA5 was essential for PDIM production in M. tuberculosis, and although the involvement of PapA5 in the diesterification of phthiocerol and phthiodiolone could not be tested directly due to the unavailability of these compounds, PapA5 was assayed for CoA-dependent acyltransferase activities in an effort to define PapA5 substrate specificities for a variety of model lipid compounds. Taken together, these results suggested that papA5 encoded a protein capable of catalyzing acyl transfer chemistry. Although protein sequences of Pap family members include several amino acid motifs associated with other proteins that catalyze acyltransferase activities, the Pap family shares little sequence identity with acyltransferases outside of these regions. To elucidate structure-activity relationships for the Pap family of proteins, we determined the 2.75-Å crystal structure of PapA5 from M. tuberculosis. The structure reveals that PapA5 is related to the family of CoA-dependent acyltransferases. Further structural analysis combined with previously reported acyltransferase activities on defined lipid substrates suggests a model for PapA5 function.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Purification of M. tuberculosis PapA5—PapA5-(1–422) was cloned, expressed, and purified from Escherichia coli as a N-terminal His6-Smt3 fusion protein (22). The pET-based plasmid was transformed into E. coli BL21(DE3) CodonPlus RIL (Stratagene). A 5-liter culture was grown by fermentation at 37 °C to an A600 of 2, adjusted to 30 °C and 1 mM isopropyl-1-thio-{beta}-D-galactopyranoside, and incubated for 4 h. Cells were harvested by centrifugation and resuspended in 20 mM Tris-HCl (pH 8.0), 350 mM NaCl, 10 mM imidazole, 20% sucrose, 1 mM {beta}-mercaptoethanol, and 20 µg/ml lysozyme and sonicated. After insoluble material was removed by centrifugation, His6-Smt3-PapA5 was purified by metal affinity and gel filtration chromatography (Superdex 200). The His-Smt3 tag was removed by the Smt3-specific protease Ulp1, and PapA5 was further purified by gel filtration (Superdex 75). PapA5 was obtained at 10 mg/L of E. coli culture and appeared homogeneous by SDS-PAGE and Coommassie Blue staining. PapA5 was concentrated to 10 mg/ml, flash-frozen in liquid nitrogen, and stored at -80 °C.

Crystallographic Analysis—PapA5 crystals were obtained by vapor diffusion against a well solution containing 5–10% polyethylene glycol 4000, 0.2 M ammonium acetate, 5% glycerol, 0.1 M sodium acetate (pH 5), and 20 mM dithiothreitol. Crystals were cryo-protected by addition of 15% glycerol. Crystals of native protein diffracted X-rays to 2.5 Å, although data were only processed to 2.75 Å due to problems associated with crystal mosaicity (P3121 a = b = 172.98 Å, c = 80.54 Å, {alpha} = {beta} = 90°, {gamma} = 120°). Native data were collected at National Synchrotron Light Source beamline X4A (Brookhaven, NY), and mercury data were collected from a crystal using a laboratory copper K{alpha} source (Rigaku RU200) equipped with a confocal Osmic multilayer system and a Raxis-IV imaging plate detector. Data were reduced with DENZO, SCALEPACK (23), and CCP4 (24). 2.75 Å phases were calculated with SOLVE and RESOLVE (25) using three mercury positions and non-crystallographic symmetry (NCS) averaging using the two molecules of PapA5 in the asymmetric unit. Subsequent electron density maps were traced by hand with the program O to produce atomic models for each PapA5 protomer (26). The model was released from NCS restraints, each monomer was rebuilt, and the model was refined to 2.75 Å with CNS (27). The deposited model consists of resides 1–81, 94–175, 181–191, and 205–418 for monomer A and residues 3–81, 94–175, and 181–418 for monomer B. The model has excellent geometry with no Ramachandran outliers in disallowed regions (See Table I for crystallographic statistics).


View this table:
[in this window]
[in a new window]
  		TABLE I
Crystallographic data and refinement statistics Data indicated within parentheses indicate statistics for data in the highest resolution bin. mc, main chain; sc, side chain.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Overview of the PapA5 Structure—Purified recombinant PapA5 containing amino acids 1–422 was purified and crystallized. Crystals of PapA5 belonged to space group P3121 and contained two independent monomers per asymmetric unit. Phases were calculated to 2.75 Å using 2-fold NCS averaging with data obtained from a native protein crystal and a native crystal into which thimerosal was soaked (see "Experimental Procedures"; Table I). Both PapA5 monomers contain segments of polypeptide that did not have sufficient electron density to permit model building. These regions include residues 82–93, 176–180, 192–204, and 419–422 for monomer A and 1–2, 82–93, 176–180, and 419–422 for monomer B. The overall average Bfactor for the final coordinate set was ~68 Å2. The termini that demarcate the disordered segments (marked by asterisks in Fig. 1) had Bfactor values of nearly twice that value, suggesting that segments not observed in the electron density maps were due to thermal motion in the crystal lattice. Despite the high overall Bfactor and disordered segments, the final model was successfully refined without NCS restraints to an Rfactor of 23.6 and Rfree of 29.5 with excellent geometry and no outliers in the Ramachandran plot. Monomer B will be referred to in subsequent discussions as few differences were observed between monomers, and monomer B contained a larger number of ordered amino acids.



View larger version (44K):
[in this window]
[in a new window]
  		FIG. 1.
Structure of PapA5. A and B, orthogonal views of the PapA5 structure as depicted by ribbon representation. N and C (in red) demarcate the termini of PapA5. Italicized numbers and asterisks (in red) demarcate the amino acid number and ends of segments that were not observed in the electron density maps, respectively. Helices and strands are lettered or numbered, and domains 1 and 2 are labeled. The conserved amino acid residues His124 and Asp128 are labeled and shown in bond representation. Images were generated with SETOR or PyMOL unless noted otherwise (41, 42).

 
The PapA5 structure can most easily be described by dividing the protein into two domains (Fig. 1). Domain 1 is composed of secondary structural elements that include {beta}-strands 1–8 and 13 and {alpha}-helices A through D. Domain 2 includes {beta}-strains 9–12 and 14–15 and {alpha}-helices E through I. Domains 1 and 2 are self-contained with a few noted exceptions. Domain 1 includes {beta}13, a strand that emanates from domain 2 to complete the four-stranded anti-parallel {beta}-sheet in domain 1 ({beta}6, {beta}7, {beta}2, and {beta}13). In addition, a loop from domain 2 between {beta}-strands 10 and 11 extends into domain 1 and contacts portions of helix C and D. A large crossover loop is also observed between helix D and {beta}-strand 8 that spans nearly 50 Å between the two domains. Monomer B amino acids 192–204 within the crossover loop have Bfactor values nearly twice that of the average model Bfactor, while the same region in monomer A is disordered and not present in the electron density maps. Despite these interdomain contacts, the connectivity between domains would not restrict movements of domain 1 and 2 with respect to one another. Domains 1 and 2 are structurally related and can be aligned to within 4.1 Å r.m.s.d. over 104 amino acids with 7% sequence identity. While similar, domain 1 contains the only known catalytic amino acid residues (His124 and Asp128) that have been directly implicated in PapA5 activity (16). His124 and Asp128 are located between strand 7 and helix C in the interface between domains 1 and 2 (Figs. 1 and 2).



View larger version (75K):
[in this window]
[in a new window]
  		FIG. 2.
Sequence alignment and structural similarity among Pap family members and chloramphenicol acetyltransferase. The amino acid sequence of M. tuberculosis PapA5 (Mt) is aligned with PapA5 orthologs from M. bovis (Mb), M. leprae (Ml), and Mycobacterium marinum (Mm) and to Rif20 from Amycolatopsis mediterranei S699, as well as two tandem structure-based alignments to chloramphenicol acetyltransferase (Cla1 and Cla2). Gaps are denoted by a period. PapA5 amino acid numbers and secondary structure elements are indicated above the aligned sequences; {beta}-strands are depicted as red arrows and {alpha}-helices as green bars; intervening polypeptide segments are depicted as a line, disordered N- and C-terminal segments are depicted as dashed lines for monomer B. Side chain identity shared between Pap orthologs and Rif20 sequences is shown above the alignment, while side chain identity between PapA5 and chloramphenicol acetyltransferase is shown below the alignment. Regions of structural similarity observed between chloramphenicol acetyltransferase and PapA5 are highlighted by a blue background in the alignment. Positions where mutation resulted in loss of function for M. tuberculosis PapA5 are denoted by red circles above the PapA5 sequence (16).

 
PapA5 Is Related to CoA-dependent Acyltransferases—A structural alignment between PapA5 and the Protein Data Bank using DALI shows that PapA5 contains structural and sequence motifs that are characteristic of the CoA-dependent acyltransferase family (Fig. 2) (28). In rank order, proteins that could be aligned to PapA5 include the condensation domain from VibH (Protein Data Bank code 1l5a [PDB] ; 336 amino acids aligned to 4.0 Å r.m.s.d. with 11% sequence identity; Z-score 23.1), carnitine acetyltransferase (Protein Data Bank code 1ndf [PDB] ; 285 amino acids aligned to 3.8 Å r.m.s.d. with 10% sequence identity; Z-score 12.1), chloramphenicol acetyltransferase (CAT) (Protein Data Bank code 3cla [PDB] ; 127 amino acids aligned to 3.9 Å r.m.s.d. with 12% sequence identity; Z-score 6.9), and dihydrolipoyl transacetylase (Protein Data Bank code 1eaf [PDB] ; 108 amino acids aligned to 2.9 Å r.m.s.d. with 17% sequence identity; Z-score 5.6). As stated above, domains 1 and 2 from Papa5 are related to each other, so each can be aligned to a single protomer from chloramphenicol acetyltransferase. In addition, PapA5 domains 1 and 2 are oriented in a similar manner to that observed for two of the three chloramphenicol acetyltransferase protomers as observed in the intact chloramphenicol acetyltransferase trimer (29). Although structural alignments show PapA5 domain 2 to be more similar based on the number of amino acids that could be aligned, the alignments to domain 1 reveal higher sequence identity between structures, including the known HHX3DG catalytic amino acid motif that is conserved and observed in many CoA-dependent acyltransferase family members (Fig. 2).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Chloramphenicol acetyltransferase catalyzes CoA-dependent acetyl transfer in a reaction that is dependent on the second conserved histidine (His195) in the active site HHX3DG sequence motif (29). His195 has been proposed to be a general base that promotes deprotonation of the chloramphenicol hydroxyl prior to the transfer of the acetyl group, and mutation of this residue results in a severely defective enzyme. While the aspartic acid has also been shown to be critical for activity, it appears to play a structural role in the organization of the active site. His124 and Asp128 are the corresponding histidine and aspartic acid in the PapA5 sequence and structure (Figs. 2 and 3), and as observed for chloramphenicol acetyltransferase, His124 is essential for PapA5 CoA-dependent acyltransferase activity (see below; Ref. 16). Although conserved, the second histidine in the HHX3DG motif does not perform similar catalytic roles in all CAT family members insomuch as mutation of this residue in the yeast dihydrolipoyl transacetylase results in no observable catalytic defect (30).



View larger version (38K):
[in this window]
[in a new window]
  		FIG. 3.
Structure of the PapA5 active site. A and B are shown in stereo. Side chains are depicted by solid bond rendering, while the backbone path is illustrated by a blue worm representation. Potential hydrogen-bonding interactions shown by dashed lines, and waters were omitted from the figure for clarity. The relative positions of channels 1 and 2 are indicated by arrows and labels (see "Discussion"). A, the PapA5 active site as observed in our structure with no ligands bound. B, chloramphenicol and CoA ligands superimposed onto the PapA5 active site based on structural alignments obtained with chloramphenicol acetyltransferase and dihydrolipoyl transacetylase, respectively.

 
PapA5 exhibits more structural similarity to VibH and carnitine acetyltransferase, since PapA5, VibH, and carnitine acetyltransferase each contain two tandem CAT-like domains with one active site located within the N-terminal CAT-like domain (31, 32). Carnitine acetyltransferase catalyzes the acyl transfer between carnitine and acetyl-CoA utilizing a similar mechanism to that proposed for CAT. In addition, the structural analysis of carnitine acetyltransferase and respective ligand complexes combined with previous biochemical data support a general base mechanism for His343 in deprotonation of the carnitine hydroxyl group prior to acyl transfer (32).

VibH belongs to a large family of condensation domains that are associated with non-ribosomal peptide synthetases, large multidomain enzymes that catalyze the synthesis of a number of compounds such as antibiotics and virulence factors (33–35). VibH catalyzes amide bond formation in the synthesis of vibriobactin (36, 37). VibH also contains a conserved HHX3DG catalytic amino acid motif, although structural and mutational analysis revealed that VibH utilizes a mechanism distinct from CAT and carnitine acetyltransferase in that mutation of the second histidine did not result in severe catalytic defects but mutation of the Asp residue did. These data suggest that the tandem CAT-like domain architecture can be utilized in alternative ways to achieve a variety of chemical reactions.

CoA-dependent acyltransferase family members have been categorized on the structural level in the SCOP data base by virtue of their oligomeric state (38). Some CAT family members such as chloramphenicol acetyltransferase and dihydrolipoyl transacetylase are oligomeric and form their respective active sites in the intersubunit interfaces between protomers. Carnitine acetyltransferase and VibH are monomeric, but contain two tandem CAT-like domains. In both instances, VibH and carnitine acetyltransferase share similarity with the CAT intersubunit active site organization insomuch as each has its active site positioned within the interface between the two tandem CAT-like domains. PapA5 also contains two CAT-like domains and is organized in a similar manner to that observed for VibH and carnitine acetyltransferase (Fig. 1).

Analysis of the aligned structures for PapA5 and carnitine acetyltransferase revealed similar locations for the active site histidine and aspartic acid residues located within domain 1 between domains 1 and 2 (Fig. 4). The carnitine acetyltransferase structure utilized for this alignment also included the substrate carnitine (32), and comparison of the PapA5 and carnitine acetyltransferase active site clefts revealed a deep substrate cleft for carnitine acetyltransferase (Fig. 4D), whereas the analogous PapA5 cleft is occluded by helix H (Fig. 4C). Helix H is unique to the Pap family (Fig. 2), and its potential role in substrate coordination is discussed further below.



View larger version (59K):
[in this window]
[in a new window]
  		FIG. 4.
Comparison of PapA5 to the structure of carnitine acetyltransferase bound to carnitine. A, ribbon representation of PapA5 as in Fig. 1 but oriented to look into the active site through helix H (labeled with an arrow). N and C (in red) demarcate the termini of PapA5 and His124 and Asp128 are labeled and shown in bond representation. B, ribbon representation of carnitine acetyltransferase with the substrate carnitine indicated in bond representation and by an arrow (Ref. 32; Protein Data Bank code 1NDF [PDB] ). N and C (in red) demarcate the termini of carnitine acetyltransferase, and His343 and Glu347 are labeled and shown in bond representation. C, surface representation of PapA5 in the same orientation to that presented in A. Residues and surfaces corresponding to helix H (Fig. 2) are indicated by an arrow and colored green. D, surface representation of carnitine acetyltransferase in the same orientation to that presented in B. Carnitine is depicted by an arrow and in bond representation at the base of a deep cleft within the protein. Surfaces were calculated with GRASP (20).

 
PapA5 Active Site and Substrate Selectivity—PapA5 has been proposed to catalyze the diesterification of phthiocerol and phthiodiolone with mycocerosate, possibly through a mechanism that is dependent on the activation of mycocerosic acids as thioesters (16). The possible mechanisms by which PapA5 might participate in this reaction were previously explored by measuring PapA5 acyltransferase activity using palmitoyl-CoA and several model substrates that included short-, medium-, and long-chain alcohols, diols, hydroxy esters, acids, amines, and thiols (16). A subset of those substrates tested is depicted in Fig. 5. PapA5 exhibited a preference for saturated medium chain alcohols in reactions that were dependent on the presence of amino acid residues His124 and Asp128, suggesting that PapA5 utilizes a similar acyl transfer mechanism to that observed for several CAT family members including carnitine acetyltransferase.



View larger version (30K):
[in this window]
[in a new window]
  		FIG. 5.
In vitro structure-activity relationships for PapA5. A, schematic structures of several ligands used to test PapA5 acyltransferase activity as reported in Ref. 16. R represents the location of various functional groups as outlined in B. B, list of attributes for each of the ligands presented in A including functional groups (R), name, approximate physical lengths, and respective activities as measured in a palmitoyl-CoA-dependent reaction. me, monoester; de, diester. C, schematic structure for phthiocerol and phthiodiolone. R1 and R2 groups are listed below the structure for either compound. Note the overall architectural similarity to the scaffold for compounds 1–14 in A.

 
While CAT and dihydrolipoyl transacetylase utilize intersubunit interfaces to interact with respective ligands, large solvent-exposed channels were observed between the two tandem CAT-like domains in both VibH and carnitine acetyltransferase (Fig. 4, C and D) (31, 32). To gain insight into how PapA5 might organize its active site with respect to these other CAT family members, the PapA5 active site was superimposed to crystal structures of CAT, dihydrolipoyl transacetylase, and carnitine acetyltransferase to enable modeling of chloramphenicol (29), carnitine (32), and CoA (32, 39) into the PapA5 active site (Fig. 3B). Only CoA and chloramphenicol are depicted in Fig. 3B, since the ligand positions observed in carnitine acetyltransferase superimpose well to a first approximation with those ligands observed in CAT and dihydrolipoyl transacetylase (32).

A similar modeling exercise was undertaken using VibH and respective ligands from CAT and dihydrolipoyl transacetylase (31). These alignments revealed both ligand binding sites to be accessible to solvent within the VibH structure, suggesting that VibH utilizes similar surfaces and substrate clefts to interact with its substrates. Experimental structures of carnitine acetyltransferase in complex with carnitine and CoA also showed a similar arrangement of ligands, suggesting that it too utilizes the same solvent-exposed channels to bind and coordinate respective ligands (32). Inspection of the modeled ligands in the PapA5 active site shows the CoA ligand coordinated between {beta}-strands 10 and 12 in an orientation similar to that observed in all CAT-CoA complexes, suggesting that PapA5 would interact with CoA ligands in a similar manner (Figs. 1 and 3B).

The modeled positions of carnitine and chloramphenicol within the PapA5 active site show the respective hydroxyl moieties directly over catalytic His124 in PapA5, suggesting that the basic mechanisms employed in CAT activity are likely conserved in the PapA5 structure (Fig. 3B, carnitine shown in Fig. 4). Despite proper placement of the hydroxyl group, both carnitine and chloramphenicol encounter significant steric clashes with amino acid residues emanating from helix H, namely Phe327 and Phe331 (Fig. 3B). Helix H is unique to PapA5 and is not observed in either VibH, carnitine acetyltransferase, or other CAT family members (Fig. 2). Helix H effectively blocks access to one of the solvent exposed channels into the active sites observed in either VibH or carnitine acetyltransferase (Figs. 1, 3, and 4).

Further inspection of the PapA5 molecular surface reveals two channels that lead into the PapA5 active site (Fig. 6). Channel 1 is ~20 Å deep and is fully exposed via a solvent channel to the exterior of PapA5 (Figs. 3 and 6), whereas channel 2 is constricted by helix H and is ~15 Å deep, indicating that if PapA5 were to utilize channel 2 in a manner reminiscent to that observed in carnitine acetyltransferase, it must undergo conformational changes to move helix H away from the channel to enable substrate interaction (Fig. 4). Interestingly, PapA5 exhibited substrate preference for octanol in acyltransferase activity assays in addition to other observed activities on substrates with aliphatic chains of ~10 Å in length (Fig. 5; Ref. 16). While the measured depth of either channel would accommodate the preferred substrate lengths, they differ with respect to hydrophobicity and sequence conservation (Fig. 6, C and D). Channel 1 includes few conserved residues between Pap family members but is very hydrophobic in nature, while channel 2 includes several conserved residues but is slightly more hydrophilic in character.



View larger version (81K):
[in this window]
[in a new window]
  		FIG. 6.
Surface representations of PapA5 depicting the channels that lead to the active site. A, surface representation highlighting conserved residues in the PapA5 family and two potential entry points into two channels that lead to the active site histidine residue, His124. B, surface representation as in A rotated 180° to highlight the diminished degree of conservation on this side of the protein. C, a view of the PapA5 surface sectioned to show the surface of channels 1 and 2 and location of the active site histidine. Surfaces are color-coded for sequence similarity between the sequences of Pap family members as described in the legend to Fig. 2. A 20-Å path between the active site and the PapA5 surface is provided by channel 1, while a 15-Å path is provided by channel 2 between the active site and the PapA5 surface. D, same view as in C but color-coded to represent the locations of hydrophobic and hydrophilic amino acid side chains.

 
The preferred substrate lengths exhibited by PapA5 in vitro suggest a possible mode of interaction with phthiocerol and phthiodiolone, the proposed in vivo substrates for PapA5 (Fig. 5). Phthiocerol and phthiodiolone include two hydroxyl moieties located at positions 9 and 11 along the aliphatic chain (Fig. 5C). The approximate length (9–11 carbon units) of the remaining aliphatic chain is architecturally similar in many respects to that observed for several of the preferred model substrates, namely octanol (Fig. 5, compare A and C). If analogous to carnitine acetyltransferase and chloramphenicol acetyltransferase, channel 2 would provide the most likely binding site for octanol and the analogous portions of either phthiocerol and phthiodiolone. In addition, the long aliphatic chains associated with the remaining portions of either phthiocerol and phthiodiolone could be accommodated in channel 1 (17–21 carbon units; Figs. 5C and 6).

Phthiocerol or phthiodiolone are not commercially available or easily purified from natural sources, so it is currently implausible to obtain the relevant complexes to enable a more detailed study of PapA5 in complex with its physiological ligands, a necessary step to provide the basis for development of structure-based inhibitors of this enzyme. Although natural ligand complexes are currently beyond the scope of this study, we do plan to obtain complexes between PapA5 and some of the model compounds previously reported (16) and represented in Fig. 5. However, the structural and biochemical characterization of PapA5 combined with the structures of carnitine acetyltransferase and VibH suggest putative roles for the catalytic residues observed in PapA5. In addition, structural and functional similarity between these protein families has likely identified the surfaces and channels utilized by PapA5 in its interactions with respective substrates, the exact details of which await further investigation.

It has been previously noted that the Pap family shares weak similarity to Rif20 (16), a gene encoded within the rifamycin gene cluster (40). The PapA5 structure and the structure-based sequence alignment between PapA5 and Rif20 and the conserved catalytic elements observed in these proteins support our earlier speculation (16) that Rif20 encodes a protein with similar catalytic properties to that observed for PapA5 and is responsible for catalyzing the as yet unidentified acyltransferase activity required for C25 O-acetylation during rifamycin biosynthesis.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 1Q9J [PDB] ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* 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. Back

§ Supported in part by National Institutes of Health Grant GM62529. Back

|| Supported by National Institutes of Health Grant 1 F31 AI054326 [GenBank] -01 and Medical Scientist Training Program Grant GM07739. Back

** Supported in part by The Niarchos, The William Randolph Hearst, and The Potts Memorial Foundations. Back

{ddagger}{ddagger} To whom correspondence should be addressed: Structural Biology Program, Sloan-Kettering Inst., Box 414, 1275 York Ave., New York, NY 10021. E-mail: lima{at}limalab.org.

1 The abbreviations used are: Pap, polyketide-associated protein; PDIM, phthiocerol dimycocerosate ester and its congener; NCS, non-crystallographic symmetry; r.m.s.d., root mean square deviation; CAT, chloramphenicol acetyltransferase. Back


   ACKNOWLEDGMENTS
 
We thank the staff of beamline X4A at the National Synchrotron Light Source.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bentley, R., and Bennett, J. W. (1999) Annu. Rev. Microbiol. 53, 411-446[CrossRef][Medline] [Order article via Infotrieve]
  2. Cole, S. T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Harris, D., Gordon, S. V., Eiglmeier, K., Gas, S., Barry, C. E., III, Tekaia, F., Badcock, K., Basham, D., Brown, D., Chillingworth, T., Connor, R., Davies, R., Devlin, K., Feltwell, T., Gentles, S., Hamlin, N., Holroyd, S., Hornsby, T., Jagels, K., Krogh, A., McLean, J., Moule, S., Murphy, L., Oliver, K., Osborne, J., Quail, M. A., Rajandream, M.-A., Rogers, J., Rutter, S., Seeger, K., Skelton, J., Squares, R., Squares, S., Sulston, J. E., Taylor, K., Whitehead, S., and Barrell, B. G. (1998) Nature 393, 537-544[CrossRef][Medline] [Order article via Infotrieve]
  3. Cox, J. S., Chen, B., McNeil, M., and Jacobs, W. R. (1999) Nature 402, 79-83[CrossRef][Medline] [Order article via Infotrieve]
  4. Camacho, L. R., Ensergueix, D., Perez, E., Gicquel, B., and Guilhot, C. (1999) Mol. Microbiol. 34, 257-267[CrossRef][Medline] [Order article via Infotrieve]
  5. Dubey, V. S., Sirakova, T. D., Cynamon, M. H., and Kolattukudy, P. E. (2003) J. Bacteriol. 185, 4620-4625[Abstract/Free Full Text]
  6. Sirakova, T. D., Dubey, V. S., Cynamon, M. H., and Kolattukudy, P. E. (2003) J. Bacteriol. 185, 2999-3008[Abstract/Free Full Text]
  7. Sirakova, T. D., Dubey, V. S., Kim, H.-J., Cynamon, M. H., and Kolattukudy, P., E. (2003) Infect. Immun. 71, 3794-3801[Abstract/Free Full Text]
  8. Rousseau, C., Sirakova, T. D., Dubey, V. S., Bordat, Y., Kolattukudy, P. E., Gicquel, B., and Jackson, M. (2003) Microbiology 149, 1837-1847[Abstract/Free Full Text]
  9. Mathur, M., and Kolattukudy, P. E. (1992) J. Biol. Chem. 267, 19388-19395[Abstract/Free Full Text]
  10. Kolattukudy, P. E., Fernandes, N. D., Azad, A. K., Fitzmaurice, A. M., and Sirakova, T. D. (1997) Mol. Microbiol. 24, 263-270[CrossRef][Medline] [Order article via Infotrieve]
  11. Azad, A. K., Sirakova, T. D., Fernandes, N. D., and Kolattukudy, P. E. (1997) J. Biol. Chem. 272, 16741-16745[Abstract/Free Full Text]
  12. Garnier, T., Eiglmeier, K., Camus, J. C., Medina, N., Mansoor, H., Pryor, M., Duthoy, S., Grondin, S., Lacroix, C., Monsempe, C., Simon, S., Harris, B., Atkin, R., Doggett, J., Mayes, R., Keating, L., Wheeler, P. R., Parkhill, J., Barrell, B. G., Cole, S. T., Gordon, S. V., and Hewinson, R. G. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 7877-7882[Abstract/Free Full Text]
  13. Cole, S. T., Eiglmeier, K., Parkhill, J., James, K. D., Thomson, N. R., Wheeler, P. R., Honore, N., Garnier, T., Churcher, C., Harris, D., Mungall, K., Basham, D., Brown, D., Chillingworth, T., Connor, R., Davies, R. M., Devlin, K., Duthoy, S., Feltwell, T., Fraser, A., Hamlin, N., Holroyd, S., Hornsby, T., Jagels, K., Lacroix, C., Maclean, J., Moule, S., Murphy, L., Oliver, K., Quail, M. A., Rajandream, M. A., Rutherford, K. M., Rutter, S., Seeger, K., Simon, S., Simmonds, M., Skelton, J., Squares, R., Squares, S., Stevens, K., Taylor, K., Whitehead, S., Woodward, J. R., and Barrell, B. G. (2001) Nature 409, 1007-1011[CrossRef][Medline] [Order article via Infotrieve]
  14. Brennan, P. J., and Vissa, V. D. (2001) Lepr. Rev. 72, 415-428[Medline] [Order article via Infotrieve]
  15. de Crécy-Lagard, V. (1999) in Amino Acids, Peptides, Porphyrins, and Alkaloids (Kelly, J. W., ed) Vol. 4, pp. 221-238, Elsevier Science Publishers B.V., Amsterdam
  16. Onwueme, K. C., Ferreras, J. A., Buglino, J., Lima, C. D., and Quadri, L. E. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 4608-4613[Abstract/Free Full Text]
  17. Ng, V., Zanazzi, G., Timpl, R., Talts, J. F., Salzer, J. L., and Brennan, P. J., Rambukkana, A. (2000) Cell 103, 511-524[CrossRef][Medline] [Order article via Infotrieve]
  18. Rambukkana, A., Zanazzi, G., Tapinos, N., and Salzer, J. L. (2002) Science 296, 927-931[Abstract/Free Full Text]
  19. Constant, P., Perez, E., Malaga, W., Laneelle, M. A., Saurel, O., Daffe, M., and Guilhot, C. (2002) J. Biol. Chem. 277, 38148-38158[Abstract/Free Full Text]
  20. Nicholls, A., Sharp, K. A., and Honig, B. (1991) Proteins Struct. Funct. Genet. 11, 281-296[CrossRef][Medline] [Order article via Infotrieve]
  21. Sirakova, T. D., Thirumala, A. J., Dubey, V. S., Sprecher, H., and Kolattukudy, P. E. (2001) J. Biol. Chem. 276, 16833-16839[Abstract/Free Full Text]
  22. Mossessova, E., and Lima, C. D. (2000) Mol. Cell 5, 865-876[CrossRef][Medline] [Order article via Infotrieve]
  23. Otwinowski, Z., and Minor, W. (1997) Methods Enzymol. 276, 307-326
  24. Collaborative Computational Project (1994) Acta Crystallogr. Sect. D Biol. Crystallogr. 50, 760-763[CrossRef][Medline] [Order article via Infotrieve]
  25. Terwilliger, T. C., and Berendzen, J. (1999) Acta Crystallogr. Sect. D Biol. Crystallogr. 55, 849-861[CrossRef][Medline] [Order article via Infotrieve]
  26. Jones, T. A., Zou, J. Y., Cowan, S. W., and Kjeldgaard, M. (1991) Acta Crystallogr. Sect. A 47, 110-118
  27. Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L. (1998) Acta Crystallogr. Sect. D Biol. Crystallogr. 54, 905-921[CrossRef][Medline] [Order article via Infotrieve]
  28. Holm, L., and Sander, C. (1993) J. Mol. Biol. 233, 123-138[CrossRef][Medline] [Order article via Infotrieve]
  29. Leslie, A. G. W., Moody, P. C., and Shaw, W. V. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 4133-4137[Abstract/Free Full Text]
  30. Niu, X. D., Stoops, J. K., and Reed, L. J. (1990) Biochemistry 29, 8614-8619[CrossRef][Medline] [Order article via Infotrieve]
  31. Keating, T. A., Marshall, C. G., Walsh, C. T., and Keating, A. E. (2002) Nat. Struct. Biol. 9, 522-526[Medline] [Order article via Infotrieve]
  32. Jogl, G., and Tong, L. (2003) Cell 112, 113-122[CrossRef][Medline] [Order article via Infotrieve]
  33. Marahiel, M. A., Stachelhaus, T., and Mootz, H. D. (1997) Chem. Rev. 97, 2651-2674[CrossRef][Medline] [Order article via Infotrieve]
  34. Quadri, L. E., Keating, T. A., Patel, H. M., and Walsh, C. T. (1999) Biochemistry 38, 14941-14954[CrossRef][Medline] [Order article via Infotrieve]
  35. Quadri, L. E. (2000) Mol. Microbiol. 37, 1-12[CrossRef][Medline] [Order article via Infotrieve]
  36. Keating, T. A., Marshall, C. G., and Walsh, C. T. (2000) Biochemistry 39, 15513-15521[CrossRef][Medline] [Order article via Infotrieve]
  37. Keating, T. A., Marshall, C. G., and Walsh, C. T. (2000) Biochemistry 39, 15522-15530[CrossRef][Medline] [Order article via Infotrieve]
  38. Hubbard, T. J., Murzin, A. G., Brenner, S. E., and Chothia, C. (1997) Nucleic Acids Res. 25, 236-239[Abstract/Free Full Text]
  39. Mattevi, A., Obmolova, G., Schulze, E., Kalk, K. H., Westphal, A. H., de Kok, A., and Hol, W. G. J. (1992) Science 255, 1544-1550[Abstract/Free Full Text]
  40. Floss, H. G., and Yu, T. W. (1999) Curr. Opin. Chem. Biol. 3, 592-597[CrossRef][Medline] [Order article via Infotrieve]
  41. Evans, S. V. (1993) J. Mol. Graph. 11, 134-138[CrossRef][Medline] [Order article via Infotrieve]
  42. DeLano, W. L. (2002) PyMOL: An Open-source Molecular Graphic Tool, DeLano Scientific, San Carlos, CA (www.pymol.org)

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
G. S. Garvey, S. P. McCormick, and I. Rayment
Structural and Functional Characterization of the TRI101 Trichothecene 3-O-Acetyltransferase from Fusarium sporotrichioides and Fusarium graminearum: KINETIC INSIGHTS TO COMBATING FUSARIUM HEAD BLIGHT
J. Biol. Chem., January 18, 2008; 283(3): 1660 - 1669.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
P. Kumar, M. W. Schelle, M. Jain, F. L. Lin, C. J. Petzold, M. D. Leavell, J. A. Leary, J. S. Cox, and C. R. Bertozzi
PapA1 and PapA2 are acyltransferases essential for the biosynthesis of the Mycobacterium tuberculosis virulence factor Sulfolipid-1
PNAS, July 3, 2007; 104(27): 11221 - 11226.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
H. Unno, F. Ichimaida, H. Suzuki, S. Takahashi, Y. Tanaka, A. Saito, T. Nishino, M. Kusunoki, and T. Nakayama
Structural and Mutational Studies of Anthocyanin Malonyltransferases Establish the Features of BAHD Enzyme Catalysis
J. Biol. Chem., May 25, 2007; 282(21): 15812 - 15822.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
J.-M. Yang and C.-H. Tung
Protein structure database search and evolutionary classification
Nucleic Acids Res., August 2, 2006; 34(13): 3646 - 3659.
[Abstract] [Full Text] [PDF]

Home page
 J. Bacteriol.Home page
K. C. Onwueme, C. J. Vos, J. Zurita, C. E. Soll, and L. E. N. Quadri
Identification of Phthiodiolone Ketoreductase, an Enzyme Required for Production of Mycobacterial Diacyl Phthiocerol Virulence Factors
J. Bacteriol., July 15, 2005; 187(14): 4760 - 4766.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
X. Ma, J. Koepke, S. Panjikar, G. Fritzsch, and J. Stockigt
Crystal Structure of Vinorine Synthase, the First Representative of the BAHD Superfamily
J. Biol. Chem., April 8, 2005; 280(14): 13576 - 13583.
[Abstract] [Full Text] [PDF]

Home page
 J. Bacteriol.Home page
G. S. Hotter, B. J. Wards, P. Mouat, G. S. Besra, J. Gomes, M. Singh, S. Bassett, P. Kawakami, P. R. Wheeler, G. W. de Lisle, et al.
Transposon Mutagenesis of Mb0100 at the ppe1-nrp Locus in Mycobacterium bovis Disrupts Phthiocerol Dimycocerosate (PDIM) and Glycosylphenol-PDIM Biosynthesis, Producing an Avirulent Strain with Vaccine Properties At Least Equal to Those of M. bovis BCG
J. Bacteriol., April 1, 2005; 187(7): 2267 - 2277.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/29/30634    most recent
M404011200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Buglino, J.
Right arrow Articles by Lima, C. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Buglino, J.
Right arrow Articles by Lima, C. D.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement