Crystal structures of bacterial FabH suggest a molecular basis for the substrate specificity of the enzyme

Gajiwala, K.S.; Margosiak, Stephen A.; Lu, Jiajia; Cortez, Joseph; Su, Ying; Nie, Zhe; Appelt, K.

doi:10.1016/j.febslet.2009.08.001

Cited by 52 publications

(78 citation statements)

References 24 publications

(32 reference statements)

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“…If so, then the inability of the S36T mutant to inhibit the FAS would also suggest that the acyl-phosphopantetheine moiety contributes substantially to this mode of inhibition. Direct structural evidence for KS-ACP interactions has been reported (27,28,29), as have other in vitro assays revealing acyl-ACP inhibition of FabH (15,30). Similarly, changing concentrations of TesA could also alter the relative distribution of certain acyl-ACP species, which in turn could modulate the turnover frequency of the FAS.…”

Section: Discussionmentioning

confidence: 99%

In vitro reconstitution and steady-state analysis of the fatty acid synthase from Escherichia coli

Yu¹,

Liu

Zhu

et al. 2011

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

152

210

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Microbial fatty acid derivatives are emerging as promising alternatives to fossil fuel derived transportation fuels. Among bacterial fatty acid synthases (FAS), the Escherichia coli FAS is perhaps the most well studied, but little is known about its steady-state kinetic behavior. Here we describe the reconstitution of E. coli FAS using purified protein components and report detailed kinetic analysis of this reconstituted system. When all ketosynthases are present at 1 μM, the maximum rate of free fatty acid synthesis of the FAS exceeded 100 μM∕ min. The steady-state turnover frequency was not significantly inhibited at high concentrations of any substrate or cofactor. FAS activity was saturated with respect to most individual protein components when their concentrations exceeded 1 μM. The exceptions were FabI and FabZ, which increased FAS activity up to concentrations of 10 μM; FabH and FabF, which decreased FAS activity at concentrations higher than 1 μM; and holo-ACP and TesA, which gave maximum FAS activity at 30 μM concentrations. Analysis of the S36T mutant of the ACP revealed that the unusual dependence of FAS activity on holo-ACP concentration was due, at least in part, to the acyl-phosphopantetheine moiety. MALDI-TOF mass spectrometry analysis of the reaction mixture further revealed medium and long chain fatty acyl-ACP intermediates as predominant ACP species. We speculate that one or more of such intermediates are key allosteric regulators of FAS turnover. Our findings provide a new basis for assessing the scope and limitations of using E. coli as a biocatalyst for the production of diesel-like fuels.D ue to their high energy density and low water solubility, fatty acids are arguably the most appropriate biofuel precursors. Therefore, fatty acid synthases (FASs) have emerged as attractive engineering targets in society's recent quest for transportation fuels from renewable sources. Among different FASs, the Escherichia coli synthase is perhaps most well understood (1). However, notwithstanding extensive analysis of fatty acid biosynthesis and its regulation in E. coli (2-5), little is known about its steady-state kinetic properties. We therefore sought to undertake systematic kinetic analysis of the fully reconstituted E. coli FAS. It was anticipated that such analysis would provide a fundamentally new basis for assessing the scope and limitations of producing fatty acids using E. coli as a biocatalyst.Fatty acid biosynthesis in E. coli is catalyzed by an enzyme system consisting of nine distinct proteins-FabA, FabB, FabD, FabF, FabG, FabH, FabI, FabZ, and ACP (Fig. 1). Together, they convert one equivalent of acetyl-CoA and 6-8 equivalents of malonyl-CoA into C 14 -C 18 acyl-ACP species. One or two reducing equivalents of NAD(P)H are utilized in each round of chain elongation. Whereas most of the resulting fatty acyl chains are directly harnessed for phospholipid biosynthesis, the cytoplasmic mutant of the periplasmic thioesterase, TesA, is capable of releasing free fatty acids via hydrolysis of acyl-...

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Section: Discussionmentioning

confidence: 99%

In vitro reconstitution and steady-state analysis of the fatty acid synthase from Escherichia coli

Yu¹,

Liu

Zhu

et al. 2011

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

152

210

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“…With allyl bromide as the electrophile, also O-alkylation occurred to give enol ether (16) in 27% yield. Interestingly, upon GC analysis of the latter product, the thermal conditions efficiently promoted a Claisen rearrangement to give alkene (15) in the same dr of 10:3.…”

Section: Scheme 2 Proposed Mechanism For the Prins Cyclization Of Diementioning

confidence: 99%

“…Although the reaction proceeded in good yield, the presence of a small amount of the corresponding diastereoisomer (7:1) was somewhat unexpected. No diastereoisomer formation was reported for this step in the syntheses of platensimycin and platencin by Nicolaou et al In the second step, ketone (14) was allylated with allyl iodide to give alkene (15) in 76% as an inseparable mixture of diastereoisomers (dr 10:3). With allyl bromide as the electrophile, also O-alkylation occurred to give enol ether (16) in 27% yield.…”

Section: Scheme 2 Proposed Mechanism For the Prins Cyclization Of Diementioning

confidence: 99%

“…Conversely, the subsequent condensation of malonyl-ACP with acetyl-CoA to form -ketobutyryl-ACP, catalyzed by FabH, is a crucial initiating step in the fatty acid synthesis cycle. However, targeting this enzyme across a wide range of bacteria is challenging because of structural variations in the substrate binding site of FabH [15]. These architectural differences determine the fatty acid profile of an organism which causes e.g.…”

Section: Introductionmentioning

confidence: 99%

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Synthesis and Biological Activity of Novel

Waalboer

Rutjes

2011

itbj.sci.

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Abstract. The biological mode of action of platencin, a potential lead molecule for a new class of antibiotics, is detailed. Furthermore, enantiopure syntheses of several platencin derivatives are described, of which the core structure can be accessed in two exceedingly simple steps from commercially available starting materials. Furthermore, the antibiotic properties of the derivatives was evaluated.

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“…The energy minimization was carried out by MM2 and finally by RHF/3-21G. Automated docking was used to determine the orientation of inhibitors bound in the active site of bacterial beta-ketoacyl-acyl carrier protein synthase III (FabH; pdb id:3IL7) [29,38]. An incremental construction algorithm method, implemented in the program FlexX embedded LeadIT, was employed.…”

Section: Molecular Dockingmentioning

confidence: 99%

Synthesis, Molecular Modeling, and Quantitative Structure–activity Relationship Studies of Undec-10-Enehydrazide Derivatives as Antimicrobial Agents

Kumari

Narang

Nayak

et al. 2017

Asian J Pharm Clin Res

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Objective: In recent years, an increasing frequency and severity of antimicrobial resistance to different antimicrobial agents, demands new remedies for the treatment of infections. Therefore, in this study, a series of undec-10-enehydrazide derivatives were synthesized and screened for in vitro activity against selected pathogenic microbial strains. Methods:The synthesis of the intermediate and target compounds was performed by standard procedure. Synthesized compounds were screened for antimicrobial activity by tube dilution method. Molecular docking study of synthesized derivatives was also performed to find out their interaction with the target site of β-ketoacyl-acyl carrier protein synthase III, (FabH; pdb id:3IL7) by docking technique. Quantitative structure-activity relationship (QSAR) studies were also performed to correlate antimicrobial activity with structural properties of synthesized molecules.Results: Antimicrobial screening results showed that compound 8 having benzylidine moiety with methoxy groups at meta and para position and compound 16 having 3-chloro-2-(3-flourophenyl)-4-oxoazetidine moiety was found to be most potent. QSAR studies revealed the importance of Randic topology parameter (R) in describing the antimicrobial activity of synthesized derivatives. Molecular docking study indicated hydrophobic interaction of deeply inserted aliphatic side chain of the ligand with FabH. The N-atoms of hydrazide moiety interacts with Ala246 and Asn247 through H-bonding. The m-and p-methoxy groups form H-bond with water and side chain of Arg36, respectively. Conclusion:Compound 8 having benzylidine moiety with methoxy groups at meta and para position and compound 16 having 3-chloro-2-(3-flourophenyl)-4-oxoazetidine moiety was found to most potent antibacterial and antifungal compounds, respectively.

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Crystal structures of bacterial FabH suggest a molecular basis for the substrate specificity of the enzyme

Cited by 52 publications

References 24 publications

In vitro reconstitution and steady-state analysis of the fatty acid synthase from Escherichia coli

In vitro reconstitution and steady-state analysis of the fatty acid synthase from Escherichia coli

Synthesis and Biological Activity of Novel

Synthesis, Molecular Modeling, and Quantitative Structure–activity Relationship Studies of Undec-10-Enehydrazide Derivatives as Antimicrobial Agents

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