The application of computational methods to explore the diversity and structure of bacterial fatty acid synthase
2003; Elsevier BV; Volume: 44; Issue: 1 Linguagem: Inglês
10.1194/jlr.r200016-jlr200
ISSN1539-7262
AutoresYongmei Zhang, Hédia Marrakchi, Stephen W. White, Charles O. Rock,
Tópico(s)Bacteriophages and microbial interactions
ResumoAcyl carrier protein (A111111111222222206) is a central element in the bacterial, type II dissociated fatty acid synthase (FAS II) system. ACP delivers the fatty acyl intermediates to a variety of enzymes with different biochemical functions and 3-dimensional (3-D) structures. Computational techniques have proved invaluable in guiding the experimental designs that have uncovered the recognition helix on ACP and the common features on its target enzymes responsible for specific protein•protein interactions. Escherichia coli has been the model organism for the study of FAS II, but the availability of complete genomic sequences of a growing number of bacteria coupled with computational bioinformatics has led to new discoveries on the mechanisms that regulate E. coli FAS II and allowed the differences between the E. coli paradigm and major groups of pathogens to be identified and experimentally addressed. Computational methods facilitated the discovery of the E. coli fatty acid synthesis transcriptional regulator, FabR, and led to the identification of novel bacterial FAS II proteins in Gram-positive pathogens, including enoyl-ACP reductases (FabK and FabL) and trans-2-cis-3-decenoyl-ACP isomerase FabM.As more genomic sequences and 3-D coordinates are added to the databases, the power and resolution of the computational approaches will increase to offer deeper insight into the structure, diversity and function of lipid metabolic pathways. Acyl carrier protein (A111111111222222206) is a central element in the bacterial, type II dissociated fatty acid synthase (FAS II) system. ACP delivers the fatty acyl intermediates to a variety of enzymes with different biochemical functions and 3-dimensional (3-D) structures. Computational techniques have proved invaluable in guiding the experimental designs that have uncovered the recognition helix on ACP and the common features on its target enzymes responsible for specific protein•protein interactions. Escherichia coli has been the model organism for the study of FAS II, but the availability of complete genomic sequences of a growing number of bacteria coupled with computational bioinformatics has led to new discoveries on the mechanisms that regulate E. coli FAS II and allowed the differences between the E. coli paradigm and major groups of pathogens to be identified and experimentally addressed. Computational methods facilitated the discovery of the E. coli fatty acid synthesis transcriptional regulator, FabR, and led to the identification of novel bacterial FAS II proteins in Gram-positive pathogens, including enoyl-ACP reductases (FabK and FabL) and trans-2-cis-3-decenoyl-ACP isomerase FabM. As more genomic sequences and 3-D coordinates are added to the databases, the power and resolution of the computational approaches will increase to offer deeper insight into the structure, diversity and function of lipid metabolic pathways. Fatty acid metabolism is a fundamental component of the cellular metabolic network. Fatty acids are the essential building blocks for membrane phospholipid formation. Most bacteria synthesize fatty acids using a series of discrete monofunctional proteins, each catalyzing one reaction in the pathway [reviewed in ref. (1Cronan Jr., J.E. Rock. C.O. Biosynthesis of membrane lipids.in: Neidhardt F.C. Curtis R. Gross C.A. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W. Riley M. Schaechter M. Umbarger H.E. In Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. American Society for Microbiology, Washington, D.C.1996: 612-636Google Scholar, 2Rock C.O. Cronan Jr., J.E. Escherichia coli as a model for the regulation of dissociable (type II) fatty acid biosynthesis.Biochim. Biophys. Acta. 1996; 1302: 1-16Google Scholar, 3Jackowski S. Rock C.O. Forty years of fatty acid biosynthesis.Biochem. Biophys. Res. Commun. 2002; 292: 1155-1166Google Scholar)]. The bacterial system, also known as the dissociated, type II fatty acid synthase (FAS II), contrasts with the yeast and animal type I fatty acid synthases (FAS I). The type II system is a collection of individual enzymes encoded by separate genes, whereas the type I system is a polypeptide about 260 kDa in size with multiple active sites that perform all the catalytic reactions in the pathway. Although the structural organizations of FAS I and FAS II are different, the chemical reactions and the catalytic mechanisms for fatty acid synthesis are essentially the same. Escherichia coli FAS II has been extensively studied and the biochemical properties of the individual enzymes are the paradigm for type II fatty acid synthase (1Cronan Jr., J.E. Rock. C.O. Biosynthesis of membrane lipids.in: Neidhardt F.C. Curtis R. Gross C.A. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W. Riley M. Schaechter M. Umbarger H.E. In Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. American Society for Microbiology, Washington, D.C.1996: 612-636Google Scholar). Figure 1outlines the major steps in the bacterial FAS II. Acetyl-CoA carboxylase catalyzes the first committed reaction of fatty acid biosynthesis. The product of the reaction is malonyl-CoA and the malonate group is then transferred to ACP by malonyl-CoA:ACP transacylase (FabD) to form malonyl-ACP. Fatty acid synthesis is initiated by the Claisen condensation of malonyl-ACP with acetyl-CoA catalyzed by β-ketoacyl-ACP synthase III (FabH) to form β-ketobutyryl-ACP. Four enzymes catalyze each cycle of elongation. The β-keto group is reduced by the NADPH-dependent β-ketoacyl-ACP reductase (FabG), and the resulting β-hydroxy intermediate is dehydrated by the β-hydroxyacyl-ACP dehydratase (FabA or FabZ) to an enoyl-ACP. Next, the reduction of the enoyl chain by the NAD(P)H-dependent enoyl-ACP reductase (FabI, FabK, or FabL) produces an acyl-ACP. Additional cycles of elongation are initiated by the β-ketoacyl-ACP synthase (FabB or FabF), which elongates the acyl-ACP by two carbons to form a β-ketoacyl-ACP. Elongation ends when the fatty acyl chain reaches the appropriate length that can be used for membrane phospholipid or lipopolysaccharide synthesis. A central feature of the bacterial FAS II is that all the fatty acyl intermediates are covalently attached to a small, acidic, and highly conserved ACP. The intermediates are covalently attached to ACP by a thioester bond to the sulfhydryl of the 4′-phosphopantetheine prosthetic group. The prosthetic group is in turn covalently attached to an invariant serine residue via a phosphodiester linkage. Apo-ACP is inactive in fatty acid synthesis, and is converted to the active protein by holo-ACP synthase (AcpS), which transfers the 4′-phosphopantetheine from CoA to apo-ACP (4Flugel R.S. Hwangbo Y. Lambalot R.H. Cronan Jr., J.E. Walsh C.T. Holo-(Acyl carrier protein) synthase and phosphopantetheinyl transfer in Escherichia coli.J. Biol. Chem. 2000; 275: 959-968Google Scholar). On the other hand, the prosthetic group is cleaved from ACP by [ACP]phosphodiesterase, although not much is known about this enzyme (5Fischl A.S. Kennedy E.P. Isolation and properties of acyl carrier protein phosphodiesterase of Escherichia coli.J. Bacteriol. 1990; 172: 5445-5449Google Scholar, 6Nakanishi M. Yatome C. Ishida N. Kitade Y. Putative ACP phosphodiesterase gene (acpD) encodes an azoreductase.J. Biol. Chem. 2001; 276: 46394-46399Google Scholar). There are over a hundred ACP sequences deposited in the molecular sequence databases. Multiple sequence alignment analysis reveals a high degree of homology for ACPs across species centered around the serine where 4′-phosphopantetheine is attached (Fig. 2A).Fig. 2Highly conserved negatively charged and hydrophobic residues in the helix II of ACP are important for protein recognition. A: Multiple alignment of ACP amino acid sequences within the helix II region. Helix II is predicted to be the recognition site on ACP in protein•ACP complexes. Primary sequences in the helix II region of various bacterial ACPs are compared using the program Clustal W (67Thompson J.D. Higgins D.G. Gibson T.J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.Nucleic Acids Res. 1994; 22: 4673-4680Google Scholar). The completely conserved residues are highlighted in black and residues that are conservatively substituted are highlighted in gray. The serine residue to which the prosthetic group is attached is indicated with an asterisk. The numbers refer to the portions of the last amino acids in the sequences. B: A ribbon diagram of the helix II of the E. coli ACP. Twenty-one residues (Ala34 to Ile54) were used to generate the structure using the program MOLSCRIPT (68Kraulis P.J. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures.J. Appl. Crystallogr. 1991; 24: 946-950Google Scholar), and the graph was rendered with Raster 3D (69Merritt E.A. Murphy M.E.P. Raster3D version 2.0. A program for photorealistic molecular graphics.Acta Crystallogr. 1994; D50: 869-873Google Scholar). The side chains of the conserved negatively charged residues, E41, E47, E48, and E49 (shown in CPK style), protrude from the surface along helix II to interact with positively charged residues on the complementary proteins. Highly conserved hydrophobic residues that are important for FabH/ACP interactions, M44 and A45, are also indicated. C: Electrostatic potential surface of the E. coli ACP helix II region. Red indicates negative charge, blue indicates positive charge, and white is hydrophobic. Negatively charged and hydrophobic residues corresponding to those shown in B are labeled. The extreme ranges of red and blue represent electrostatic potential of <−12 to >+3 kbT, where kb is the Boltzmann constant and the T is the temperature. The figure was calculated using the GRASP program (70Nicholls A. Sharp K.A. Honig B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons.Proteins. 1991; 11: 281-296Google Scholar).View Large Image Figure ViewerDownload (PPT)Fig. 2Highly conserved negatively charged and hydrophobic residues in the helix II of ACP are important for protein recognition. A: Multiple alignment of ACP amino acid sequences within the helix II region. Helix II is predicted to be the recognition site on ACP in protein•ACP complexes. Primary sequences in the helix II region of various bacterial ACPs are compared using the program Clustal W (67Thompson J.D. Higgins D.G. Gibson T.J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.Nucleic Acids Res. 1994; 22: 4673-4680Google Scholar). The completely conserved residues are highlighted in black and residues that are conservatively substituted are highlighted in gray. The serine residue to which the prosthetic group is attached is indicated with an asterisk. The numbers refer to the portions of the last amino acids in the sequences. B: A ribbon diagram of the helix II of the E. coli ACP. Twenty-one residues (Ala34 to Ile54) were used to generate the structure using the program MOLSCRIPT (68Kraulis P.J. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures.J. Appl. Crystallogr. 1991; 24: 946-950Google Scholar), and the graph was rendered with Raster 3D (69Merritt E.A. Murphy M.E.P. Raster3D version 2.0. A program for photorealistic molecular graphics.Acta Crystallogr. 1994; D50: 869-873Google Scholar). The side chains of the conserved negatively charged residues, E41, E47, E48, and E49 (shown in CPK style), protrude from the surface along helix II to interact with positively charged residues on the complementary proteins. Highly conserved hydrophobic residues that are important for FabH/ACP interactions, M44 and A45, are also indicated. C: Electrostatic potential surface of the E. coli ACP helix II region. Red indicates negative charge, blue indicates positive charge, and white is hydrophobic. Negatively charged and hydrophobic residues corresponding to those shown in B are labeled. The extreme ranges of red and blue represent electrostatic potential of <−12 to >+3 kbT, where kb is the Boltzmann constant and the T is the temperature. The figure was calculated using the GRASP program (70Nicholls A. Sharp K.A. Honig B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons.Proteins. 1991; 11: 281-296Google Scholar).View Large Image Figure ViewerDownload (PPT) ACP structure and function has received considerable attention over the past several decades in light of its central role in fatty acid and polyketide synthesis. ACP also supplies acyl groups for the synthesis of lipid A (7Raetz C.R.H. Bacterial lipopolysaccharides: A remarkable family of bioactive macroamphiphiles. In Escherichia coli and Salmonella typhimurium.in: Neidhardt F.C. Curtiss R. Gross C.A. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W. Riley M. Schaechter M. Umbarger H.E. American Society for Microbiology, Washington, D.C.1996: 1035-1063Google Scholar), glycerolipids (2Rock C.O. Cronan Jr., J.E. Escherichia coli as a model for the regulation of dissociable (type II) fatty acid biosynthesis.Biochim. Biophys. Acta. 1996; 1302: 1-16Google Scholar), and quorum sensing compounds (8Moré, M. I. Finger D. Stryker J.L. Fuqua C. Eberhard A. Winans S.C. Enzymatic synthesis of a quorum-sensing autoinducer through use of defined substrates.Science. 1996; 272: 1655-1658Google Scholar). Expression of ACPs from a variety of bacteria, plants, and animals in an E. coli host strain produces the respective ACP proteins in three forms, apo-ACP, holo-ACP, and acyl-ACP (9Revill W.P. Bibb M.J. Hopwood D.A. Relationships between fatty acid and polyketide synthases from Streptomyces coelicolor A3(2): Characterization of the fatty acid synthase acyl carrier protein.J. Bacteriol. 1996; 178: 5660-5667Google Scholar, 10Schaeffer M.L. Agnihotri G. Kallender H. Brennan P.J. Lonsdale J.T. Expression, purification, and characterization of the Mycobacterium tuberculosis acyl carrier protein, AcpM.Biochim. Biophys. Acta. 2001; 1532: 67-78Google Scholar, 11Kremer L. Nampoothiri K.M. Lesjean S. Dover L.G. Graham S. Betts J. Brennan P.J. Minnikin D.E. Locht C. Besra G.S. Biochemical characterization of acyl carrier protein (AcpM) and malonyl-CoA:AcpM transacylase (mtFabD), two major components of Mycobacterium tuberculosis fatty acid synthase II.J. Biol. Chem. 2001; 276: 27967-27974Google Scholar, 12Ohlrogge J. Savage L. Jaworski J. Voelker T. Post-Beittenmiller D. Alteration of acyl-acyl carrier protein pools and acetyl-CoA carboxylase expression in Escherichia coli by a plant medium chain acyl-acyl carrier protein thioesterase.Arch. Biochem. Biophys. 1995; 317: 185-190Google Scholar, 13Tropf S. Revill W.P. Bibb M.J. Hopwood D.A. Schweizer M. Heterologously expressed acyl carrier protein domain of rat fatty acid synthase functions in Escherichia coli fatty acid synthase and Streptomyces coelicolor polyketide synthase systems.Chem. Biol. 1998; 5: 135-146Google Scholar). These observations, along with numerous in vitro enzymatic experiments, illustrate that ACPs from highly diverse organisms function interchangeably in the E. coli FAS II system. This conclusion predicts that the protein• ACP interactions are achieved through a conserved set of electrostatic and/or hydrophobic contacts. This hypothesis is also supported by the fact that domains of ACPs from distantly related organisms (E. coli and Rhizobium) can be interchanged without affecting the protein's conformation and function (14Ritsema T. Gehring A.M. Stuitje A.R. van der Drift K.M. Dandal I. Lambalot R.H. Walsh C.T. Thomas-Oates J.E. Lugtenberg B.J. Spaink H.P. Functional analysis of an interspecies chimera of acyl carrier proteins indicates a specialized domain for protein recognition.Mol. Gen. Genet. 1998; 257: 641-648Google Scholar). Consistent with this, the primary sequence analysis of the ACPs identifies a motif that is conserved in ACP family members (Fig. 2A). Several amino acids in this region of the protein, Asp35, Ser36, Leu37, Glu41, and Glu47 (E. coli numbering), are completely conserved in the over 50 ACP sequences analyzed in preparation for this article. The 3-dimensional (3-D) structures of several bacterial ACPs clearly define the similar structural characteristics within the protein family. The ACPs are asymmetric monomers consisting of four α-helices packed into a bundle held together by interhelical hydrophobic interactions (15Wong H.C. Liu G. Zhang Y.M. Rock C.O. Zheng J. The solution structure of acyl carrier protein from Mycobacterium tuberculosis.J. Biol. Chem. 2002; 277: 15874-15880Google Scholar, 16Holak T.A. Kearsley S.K. Kim Y. Prestegard J.H. Three-dimensional structure of acyl carrier protein determined by NMR pseudoenergy and distance geometry calculations.Biochemistry. 1988; 27: 6135-6142Google Scholar, 17Xu G-Y. Tam A. Lin L. Hixon J. Fritz C.C. Powers R. Solution structure of B. subtilis acyl carrier protein.Structure. 2001; 9: 277-287Google Scholar, 18Crump M.P. Crosby J. Dempsey C.E. Parkinson J.A. Murray M. Hopwood D.A. Simpson T.J. Solution structure of the actinorhodin polyketide synthase acyl carrier protein from Streptomyces coelicolor A3(2).Biochemistry. 1997; 36: 6000-6008Google Scholar, 19Roujeinikova A. Baldock C. Simon W.J. Gilroy J. Baker P.J. Stuitje A.R. Rice D.W. Slabas A.R. Rafferty J.B. X-ray crystallographic studies on butyryl-ACP reveal flexibility of the structure around a putative acyl chain binding site.Structure. 2002; 10: 825-835Google Scholar). The conserved acidic residues (Fig. 2B, C) are arrayed along helix II with Ser36, the site of prosthetic group attachment, located on a loop at the N-terminus of helix II. This conserved region of the protein is postulated to represent a "recognition helix" that accounts for the ability of the ACPs from widely diverse organisms to interact with target enzymes and to function interchangeably in FAS II systems. As the carrier of intermediates in FAS II, ACP must interact with the proteins in the pathway to specifically deliver the appropriate substrate to each enzyme with a different biochemical function. At the same time, protein/ACP interactions must be transient (and relatively weak) to allow rapid on and off rates for the substrates and products. Unlike ACP itself (Fig. 2A), and many groups of enzymes that bind a common substrate, ACP-interacting proteins do not share a primary signature sequence that defines an ACP binding motif. Instead, recent experiments support the hypothesis that the enzymes of the type II system share 3-D surface features that account for their specific recognition of ACP and its thioesters. This hypothesis was first explored with FabH, the condensing enzyme that catalyzes the initial step in the elon-gation of fatty acids. Its two substrates, acetyl-CoA and malonyl-ACP, bind sequentially in a ping-pong enzyme mechanism to produce acetoacetyl-ACP (20Tsay J-T. Oh W. Larson T.J. Jackowski S. Rock C.O. Isolation and characterization of the β-ketoacyl-acyl carrier protein synthase III gene (fabH) from Escherichia coli K-12.J. Biol. Chem. 1992; 267: 6807-6814Google Scholar, 21Jackowski S. Rock C.O. Acetoacetyl-acyl carrier protein synthase, a potential regulator of fatty acid biosynthesis in bacteria.J. Biol. Chem. 1987; 262: 7927-7931Google Scholar, 22Jackowski S. Murphy C.M. Cronan Jr., J.E. Rock C.O. Acetoacetyl-acyl carrier protein synthase: a target for the antibiotic thiolactomycin.J. Biol. Chem. 1989; 264: 7624-7629Google Scholar). The study of E. coli FabH•ACP interactions was guided by an automated protein•protein docking program, SurfDock (23Ruf W. Shobe J. Rao S.M. Dickinson C.D. Olson A. Edgington T.S. Importance of factor VIIa Gla-domain residue Arg-36 for recognition of the macromolecular substrate factor X Gla-domain.Biochemistry. 1999; 38: 1957-1966Google Scholar), which was used to predict the FabH•ACP complex using the X-ray structure of FabH and the NMR structure of ACP (24Zhang Y-M. Rao M.S. Heath R.J. Price A.C. Olson A.J. Rock C.O. White S.W. Identification and analysis of the acyl carrier protein (ACP) docking site on β-ketoacyl-ACP synthase III.J. Biol. Chem. 2001; 276: 8231-8238Google Scholar). The predicted interaction surface on FabH is located adjacent to the active site tunnel. It consists of a pattern of positively charged residues imbedded in a hydrophobic patch (Fig. 3B), a pattern observed in several other protein•protein interfaces (25Larsen T.A. Olson A.J. Goodsell D.S. Morphology of protein-protein interfaces.Structure. 1998; 6: 421-427Google Scholar, 26Hu Z. Ma B. Wolfson H. Nussinov R. Conservation of polar residues as hot spots at protein interfaces.Proteins. 2000; 39: 331-342Google Scholar). Significantly, the computational predictions identified helix II of ACP (Fig. 3A) as the partner with FabH, which placed the prosthetic group attachment site at the entrance of the active site tunnel. The computational predictions were verified experimentally by site-directed mutagenesis of FabH. All of the positively charged residues adjacent to the active site tunnel contribute to ACP binding, but Arg249 on FabH provides the most important electrostatic interaction, possibly with Glu41 on ACP (24Zhang Y-M. Rao M.S. Heath R.J. Price A.C. Olson A.J. Rock C.O. White S.W. Identification and analysis of the acyl carrier protein (ACP) docking site on β-ketoacyl-ACP synthase III.J. Biol. Chem. 2001; 276: 8231-8238Google Scholar). The FabH from Mycobacterium tuberculosis (mtFabH) has a markedly different substrate specificity than E. coli FabH (ecFabH), but it is similar to its E. coli counterpart in terms of topology (27Scarsdale J.N. Kazanina G. He X. Reynolds K.A. Wright H.T. Crystal structure of the Mycobacterium tuberculosis β-ketoacyl-acyl carrier protein synthase III.J. Biol. Chem. 2001; 276: 20516-20522Google Scholar). There are only a few amino acid residue alterations in the active site pocket that are postulated to account for the differences in substrate specificity between the two proteins. Like ecFabH, the surface of mtFabH is highly electronegative with the exception of the electropositive/hydrophobic patch adjacent to the active site tunnel (Fig. 3D). This surface feature of mtFabH is complementary to the electronegative/hydrophobic surface of the mycobacterial acyl carrier protein, AcpM, along helix II (Fig. 3C). A survey of six other 3-D structures of E. coli FAS II enzymes indicates that they all contain a region adjacent to their active entrances that resembles the electropositive/hydrophobic FabH• ACP binding surface (24Zhang Y-M. Rao M.S. Heath R.J. Price A.C. Olson A.J. Rock C.O. White S.W. Identification and analysis of the acyl carrier protein (ACP) docking site on β-ketoacyl-ACP synthase III.J. Biol. Chem. 2001; 276: 8231-8238Google Scholar). These similarities between the helix IIs of ACPs and the surface features of the FabH enzymes explain the ability of both ACP and AcpM to interchangeably interact with ecFabH and mtFabH. In fact, all of the FAS II components of known structure have a electropositive/hydrophobic feature adjacent to their active sites, which likely represents the ACP binding sites on these proteins and accounts for their ability to interact with ACP from multiple species (24Zhang Y-M. Rao M.S. Heath R.J. Price A.C. Olson A.J. Rock C.O. White S.W. Identification and analysis of the acyl carrier protein (ACP) docking site on β-ketoacyl-ACP synthase III.J. Biol. Chem. 2001; 276: 8231-8238Google Scholar). Despite considerable effort, there are no cocrystal structures of complexes between FAS II enzymes and ACP, perhaps due to the low affinity of the complexes (μM). However, AcpS forms a high-affinity complex with ACP, and the recent structure determination of the Bacillus subtilis AcpS•ACP binary complex provides strong support for the idea that ACP-binding proteins interact with helix II on ACP (28Parris K.D. Lin L. Tam A. Mathew R. Hixon J. Stahl M. Fritz C.C. Seehra J. Somers W.S. Crystal structures of substrate binding to Bacillus subtilis holo-(acyl carrier protein) synthase reveal a novel trimeric arrangement of molecules resulting in three active sites.Structure. 2000; 8: 883-895Google Scholar). The interactions between AcpS and ACP are predominately electrostatic with two hydrophobic contacts. The interacting residues are located on ACP helix II, which contacts a complementary helix on AcpS. As noted in the primary sequence alignment (Fig. 2A), the amino acids that contact AcpS are highly conserved along helix II of ACP, including Asp35, Glu41, and Glu47 (E. coli numbering). The side chains of the negatively charged residues (Glu or Asp) situated along ACP helix II protrude from the surface to interact with positively charged Arg residues on a corresponding helix of AcpS (28Parris K.D. Lin L. Tam A. Mathew R. Hixon J. Stahl M. Fritz C.C. Seehra J. Somers W.S. Crystal structures of substrate binding to Bacillus subtilis holo-(acyl carrier protein) synthase reveal a novel trimeric arrangement of molecules resulting in three active sites.Structure. 2000; 8: 883-895Google Scholar). These data support a general role for helix II as the recognition helix that dictates the specific binding interactions between ACP and its complementary enzymes. However, considerably more research is needed to validate this hypothesis and it is anticipated that NMR spectroscopy will play an important role in forwarding this area of investigation, since this technique is particularly well suited to study weak protein•protein interactions. In contrast to E. coli, some bacteria have additional specialized ACPs. For example, there are four different ACPs in Rhizobia, AcpP, NodF, RkpF, and AcpXL (29Geiger O. Lopez-Lara I.M. Rhizobial acyl carrier proteins and their roles in the formation of bacterial cell-surface components that are required for the development of nitrogen-fixing root nodules on legume hosts.FEMS Microbiol. Lett. 2002; 208: 153-162Google Scholar, 30Lopez-Lara I.M. Geiger O. Expression and purification of four different rhizobial acyl carrier proteins.Microbiology. 2000; 146: 839-849Google Scholar). M. tuberculosis has a biosynthetic AcpM and two other putative ACPs (11Kremer L. Nampoothiri K.M. Lesjean S. Dover L.G. Graham S. Betts J. Brennan P.J. Minnikin D.E. Locht C. Besra G.S. Biochemical characterization of acyl carrier protein (AcpM) and malonyl-CoA:AcpM transacylase (mtFabD), two major components of Mycobacterium tuberculosis fatty acid synthase II.J. Biol. Chem. 2001; 276: 27967-27974Google Scholar). Streptomyces coelicolor A3(2) contains the regular ACP for FAS II and two other ACPs involved in polyketide synthesis (9Revill W.P. Bibb M.J. Hopwood D.A. Relationships between fatty acid and polyketide synthases from Streptomyces coelicolor A3(2): Characterization of the fatty acid synthase acyl carrier protein.J. Bacteriol. 1996; 178: 5660-5667Google Scholar). Although these ACPs show a lower level of homology in their overall amino acid sequences, the amino terminal 4-helix bundle and the primary sequence along helix II are highly conserved. The working hypothesis is that these ACPs have acquired additional novel structural features, outside the protein recognition site, to accommodate the different types of acyl chains they carry. For example, the C-terminal half of Rhizobial NodF may be specialized for sequestering polyunsaturated fatty acyl chains (14Ritsema T. Gehring A.M. Stuitje A.R. van der Drift K.M. Dandal I. Lambalot R.H. Walsh C.T. Thomas-Oates J.E. Lugtenberg B.J. Spaink H.P. Functional analysis of an interspecies chimera of acyl carrier proteins indicates a specialized domain for protein recognition.Mol. Gen. Genet. 1998; 257: 641-648Google Scholar). The extended carboxy-terminus in M. tuberculosis AcpM is proposed to protect the extremely long-chain fatty acyl chains in mycolic acid biosynthesis from the hydrophilic environment (10Schaeffer M.L. Agnihotri G. Kallender H. Brennan P.J. Lonsdale J.T. Expression, purification, and characterization of the Mycobacterium tuberculosis acyl carrier protein, AcpM.Biochim. Biophys. Acta. 2001; 1532: 67-78Google Scholar). These hypotheses await experimental validation through the structural determination of these acyl-ACP derivatives, and/or their complexes with FAS II enzymes. In addition to shedding light on the biochemical mechanism of FAS II, these computational predictions and structural studies may have practical value in the development of new drugs. ACP is an integral feature of the multifunctional subunit of vertebrate FAS I (31Wakil S.J. Fatty acid synthase, a proficient multifunctional enzyme.Biochemistry. 1989; 28: 4523-4530Google Scholar), in contrast to the dissociable ACPs in the bacterial FAS II systems. An exciting idea for future development of the protein•ACP interaction study is to design compounds that bind to the recognition helix of ACP and block its interaction with target enzymes. Compounds with this property represent candidate molecules toward the development of novel broad-spectrum antimicrobials. Recently, there has been success in the design and synthesis of molecules, which are capable of blocking protein•protein interactions between protease and proteinaceous inhibitors (32Park H.S. Lin Q. Hamilton A.D. Modulation of protein-protein interactions by synthetic receptors: design of molecules that disrupt serine protease-proteinaceous inhibitor interaction.Proc. Natl. Acad. Sci. USA. 2002; 99: 5105-51
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