Identification and Analysis of the Acyl Carrier Protein (ACP) Docking Site on β-Ketoacyl-ACP Synthase III

Zhang, Yongmei; Rao, M. Srinivasa; Heath, Richard J.; Price, Allen C.; Olson, Arthur J.; Rock, Charles O.; White, Stephen W.

doi:10.1074/jbc.m008042200

Cited by 169 publications

(231 citation statements)

References 57 publications

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“…2-4). The idea that helix ␣2 of ACP binds to these surface features was derived from a computational docking model (11,12). Our chemical shift perturbation experiments validate the computational model by providing the first direct evidence for the involvement of helix ␣2 in Fab protein-ACP interactions.…”

Section: Discussionmentioning

confidence: 56%

“…This hypothesis was first explored with FabH where an electropositive/hydrophobic patch that was essential to bind ACP adjacent to the entrance to the FabH active site tunnel was identified (12). This surface charge distribution pattern is a ubiquitous feature of the crystal structures of type II enzymes (11,12). FabG has a predicted ACP-binding signature surface at the entrance to the active site opening (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…The analysis of the binding of ACP to FabH points to ionic interactions playing a determinant role in the interaction between these two proteins (12). This study used a computational algorithm to dock the NMR structure of E. coli ACP to FabH.…”

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confidence: 99%

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Key Residues Responsible for Acyl Carrier Protein and β-Ketoacyl-Acyl Carrier Protein Reductase (FabG) Interaction

Zhang¹,

Zheng

et al. 2003

Journal of Biological Chemistry

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133

148

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We tested this hypothesis by mutating two surface residues, Arg-129 and Arg-172, located in a hydrophobic patch adjacent to the active site entrance on ␤-ketoacyl-ACP reductase (FabG). Enzymatic analysis showed that the mutant enzymes were compromised in their ability to utilize ACP thioester substrates but were fully active in assays with a substrate analog. Direct binding assays and competitive inhibition experiments showed that the FabG mutant proteins had reduced affinities for ACP. Chemical shift perturbation protein NMR experiments showed that FabG-ACP interactions occurred along the length of ACP helix ␣2 and extended into the adjacent loop-2 region to involve Ile-54. These data confirm a role for the highly conserved electronegative/hydrophobic residues along ACP helix ␣2 in recognizing a constellation of Arg residues embedded in a hydrophobic patch on the surface of its partner enzymes, and reveal a role for the loop-2 region in the conformational change associated with ACP binding. The specific FabG-ACP interactions involve the most conserved ACP residues, which accounts for the ability of ACPs and the type II proteins from different species to function interchangeably.Fatty acid biosynthesis in bacteria and plants is carried out by a series of enzymes that are collectively known as the type II fatty-acid synthase and is most extensively studied in the Escherichia coli system (1, 2). ACP 1 is a small, acidic protein that functions as the central acyl group carrier in type II fatty-acid synthase systems. The ACPs are rod-shaped proteins with a preponderance of acidic residues organized into four ␣-helices (3-7). The acyl intermediates are attached to the terminal sulfhydryl of the 4Ј-phosphopantetheine prosthetic group (8), which is attached via a phosphodiester linkage to Ser-36 located at the beginning of the second helical segment. The primary gene product is an apoprotein that is converted to ACP by the transfer of the 4Ј-phosphopantetheine moiety of CoA to Ser-36 by [ACP]synthase (AcpS) (9, 10). ACP performs two functions. First, it sequesters the growing acyl chain from the aqueous environment, and second, upon binding to one of the type II proteins, it releases its grip on the fatty acid, which is inserted into the active site cavity of the enzyme. ACPs are widely distributed in nature and are easily recognized by their significant primary sequence identity, particularly at the prosthetic group attachment site and extending along helix ␣2 (11). These similarities are thought to underlie the observation that ACPs from virtually any source are substrates for AcpS and function with the fatty acid biosynthetic enzymes of E. coli (11). However, the individual enzymes of type II fatty acid synthesis do not share a primary structure motif that would define the presence of a common ACP-binding motif.The molecular details that govern the specific interactions between ACP and the type II enzymes are poorly known. The analysis of the binding of ACP to FabH points to ionic interactions playing a determinant ...

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Section: Discussionmentioning

confidence: 56%

Section: Discussionmentioning

confidence: 99%

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Key Residues Responsible for Acyl Carrier Protein and β-Ketoacyl-Acyl Carrier Protein Reductase (FabG) Interaction

Zhang¹,

Zheng

et al. 2003

Journal of Biological Chemistry

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133

148

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“…ACP is highly acidic and requires an electropositive surface on its cognate partners for binding. Chemical modification of arginine or lysine residues on LpxA compromises the acyl transferase activity (16), and, in FA biosynthesis, interactions of ACP with FabG, FabH, and AcpS are dependent on specific arginine residues (20)(21)(22). Like other ACP-targeted enzymes, LpxD presents an electropositive surface near the active site (SI Fig.…”

Section: Resultsmentioning

confidence: 99%

Structure and reactivity of LpxD, the N -acyltransferase of lipid A biosynthesis

Buetow

Smith

Dawson

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

162

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“…6A. The equivalent residue is arginine in several KAS III enzymes, where the guanidino side chain facilitates the binding of acyl carrier protein (46). It has been speculated that some bacterial type III PKSs may also have specificity for ACP-thioester substrates (11).…”

Section: Figmentioning

confidence: 99%

A New Family of Type III Polyketide Synthases in Mycobacterium tuberculosis

Saxena

Yadav²,

Mohanty

et al. 2003

Journal of Biological Chemistry

105

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The Mycobacterium tuberculosis genome has revealed a remarkable array of polyketide synthases (PKSs); however, no polyketide product has been isolated thus far. Most of the PKS genes have been implicated in the biosynthesis of complex lipids. We report here the characterization of two novel type III PKSs from M. tuberculosis that are involved in the biosynthesis of long-chain ␣-pyrones. Measurement of steady-state kinetic parameters demonstrated that the catalytic efficiency of PKS18 protein was severalfold higher for long-chain acyl-coenzyme A substrates as compared with the smallchain precursors. The specificity of PKS18 and PKS11 proteins toward long-chain aliphatic acyl-coenzyme A (C 12 to C 20 ) substrates is unprecedented in the chalcone synthase (CHS) family of condensing enzymes. Based on comparative modeling studies, we propose that these proteins might have evolved by fusing the catalytic machinery of CHS and ␤-ketoacyl synthases, the two evolutionarily related members with conserved thiolase fold. The mechanistic and structural importance of several active site residues, as predicted by our structural model, was investigated by performing site-directed mutagenesis. The functional identification of diverse catalytic activity in mycobacterial type III PKSs provide a fascinating example of metabolite divergence in CHSlike proteins.Mycobacteria are classified in the phylogeny of the Actinomycetes, along with the Streptomyces bacteria. Interestingly, these two actinomycete genera have received immense attention due to their contrasting effects on human society. Whereas Streptomyces have provided a rich source of antibiotics and other therapeutic products for human diseases, Mycobacterium tuberculosis and Mycobacterium leprae have been two of humankind's greatest scourges. Although the original phylogenic classifications of Mycobacterium and Streptomyces were based primarily on morphology, the genome sequences of these organisms have further established their common lineage (1, 2). These genome sequences have revealed several families of genes that are common between them, which include an unusually large number of gene clusters that are homologous to polyketide synthases (PKSs). 1 Although a polyketide producthas not yet been isolated from M. tuberculosis, recent studies have implicated some of its PKS genes in the biosynthesis of complex lipids (3). In this report, we have characterized two novel polyketide synthases from M. tuberculosis, which are involved in the biosynthesis of unusual long-chain ␣-pyrones.Polyketides are a diverse class of secondary metabolites that possess a broad range of biological activities (4). Despite structural diversity of these natural products, PKSs synthesize polyketides by a common chemical strategy. Initial priming by a starter molecule is followed by repetitive decarboxylative condensation of coenzyme A (CoA) analogues of simple carboxylic acids. PKS elongates the polyketide chain either by repetitively using a single active site to perform multiple condensation reaction...

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Identification and Analysis of the Acyl Carrier Protein (ACP) Docking Site on β-Ketoacyl-ACP Synthase III

Cited by 169 publications

References 57 publications

Key Residues Responsible for Acyl Carrier Protein and β-Ketoacyl-Acyl Carrier Protein Reductase (FabG) Interaction

Key Residues Responsible for Acyl Carrier Protein and β-Ketoacyl-Acyl Carrier Protein Reductase (FabG) Interaction

Structure and reactivity of LpxD, the N -acyltransferase of lipid A biosynthesis

A New Family of Type III Polyketide Synthases in Mycobacterium tuberculosis

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