Crystallization of the NADP-dependent β-keto acyl carrier protein reductase from <i>Escherichia coli</i>

Rafferty, John B.; Fisher, M.; Langridge, S.J.; Martindale, Wayne; Thomas, N.C.; Simon, Jörg; Bithell, S.; Slabas, Antoni R.; Rice, David W.

doi:10.1107/s0907444997013668

Cited by 5 publications

(4 citation statements)

References 16 publications

(16 reference statements)

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“…Evidence for this comes from lipid biosynthesis studies with permeabilised chloroplasts [5]and from immunogold localisation studies [6]. No structural details of the molecular association of FAS components are known but structural details on several of the individual enzymes of FAS are emerging at near atomic resolution, as the genes are being cloned and overexpressed [7, 8]. It is our intention to clone and overexpress all the components of plant FAS in order to facilitate the study of the direct interaction of individual components.…”

Section: Introductionmentioning

confidence: 99%

cDNA cloning of Brassica napus malonyl‐CoA:ACP transacylase (MCAT) (fab D) and complementation of an E. coli MCAT mutant

Simon

Slabas

1998

FEBS Letters

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The GenBank database was searched using the E. coli malonyl CoA:ACP transacylase (MCAT) sequence, for plant protein/cDNA sequences corresponding to MCAT, a component of plant fatty acid synthetase (FAS), for which the plant cDNA has not been isolated. A 272-bp Zea mays EST sequence (GenBank accession number: AA030706) was identified which has strong homology to the E. coli MCAT. A PCR derived cDNA probe from Zea mays was used to screen a Brassica napus (rape) cDNA library. This resulted in the isolation of a 1200-bp cDNA clone which encodes an open reading frame corresponding to a protein of 351 amino acids. The protein shows 47% homology to the E. coli MCAT amino acid sequence in the coding region for the mature protein. Expression of a plasmid (pMCATrap2) containing the plant cDNA sequence in Fab D89, an E. coli mutant, in MCAT activity restores growth demonstrating functional complementation and direct function of the cloned cDNA. This is the first functional evidence supporting the identification of a plant cDNA for MCAT.z 1998 Federation of European Biochemical Societies.

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Section: Introductionmentioning

confidence: 99%

cDNA cloning of Brassica napus malonyl‐CoA:ACP transacylase (MCAT) (fab D) and complementation of an E. coli MCAT mutant

Simon

Slabas

1998

FEBS Letters

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“…1c ). Sequence alignments indicated that RsFabG1 and RsFabG2 are 65 % and 43 % identical to E. coli FabG, respectively, and showed that the catalytically active short chain dehydrogenase/reductase (SDR) family triad (Ser, Tyr and Lys) and the N-terminal cofactor binding sequence (Gly motif [GlyXXXGlyXGly]) defined by the X-ray crystal structures of E. coli FabG [ 16 , 19 , 20 ] are present in both R. solanacearum proteins (Fig. 1b ).…”

Section: Resultsmentioning

confidence: 99%

“…Although the reason for this failure is unknown, it suggests that RsFabG2 and RsFabG1 are designed for distinct physiological roles. Although the catalytically active SDR triad, the N-terminal cofactor binding sequence [ 16 , 19 , 20 ], and two Arg residues important in binding the ACP moiety of the physiological substrate [ 21 ] are conserved in the two proteins (Fig. 1c ), RsFabG2 is only 41 % identical to RsFabG1.…”

Section: Discussionmentioning

confidence: 99%

Ralstonia solanacearum fatty acid composition is determined by interaction of two 3-ketoacyl-acyl carrier protein reductases encoded on separate replicons

Feng

Yang

et al. 2015

BMC Microbiol

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BackgroundFabG is the only known enzyme that catalyzes reduction of the 3-ketoacyl-ACP intermediates of bacterial fatty acid synthetic pathways. However, there are two Ralstonia solanacearum genes, RSc1052 (fabG1) and RSp0359 (fabG2), annotated as encoding putative 3-ketoacyl-ACP reductases. Both FabG homologues possess the conserved catalytic triad and the N-terminal cofactor binding sequence of the short chain dehydrogenase/reductase (SDR) family. Thus, it seems reasonable to hypothesize that RsfabG1 and RsfabG2 both encode functional 3-ketoacyl-ACP reductases and play important roles in R. solanacearum fatty acid synthesis and growth.MethodsComplementation of Escherichia colifabG temperature-sensitive mutant with R. solanacearum fabGs encoded plasmids was carried out to test the function of RsfabGs in fatty acid biosynthesis. RsFabGs proteins were purified by nickel chelate chromatography and fatty acid biosynthetic reaction was reconstituted to investigate the 3-ketoacyl-ACP reductase activity of RsFabGs in vitro. Disruption of both RsfabG genes was done via DNA homologous recombination to test the function of both RsfabG in vivo. And more we also carried out pathogenicity tests on tomato plants using RsfabG mutant strains. ResultsWe report that expression of either of the two proteins (RsFabG1 and RsFabG2) restores growth of the E. coli fabG temperature-sensitive mutant CL104 under non-permissive conditions. In vitro assays demonstrate that both proteins restore fatty acid synthetic ability to extracts of the E. coli strain. The RsfabG1 gene carried on the R. solanacearum chromosome is essential for growth of the bacterium, as is the case for fabG in E. coli. In contrast, the null mutant strain with the megaplasmid-encoded RsfabG2 gene is viable but has a fatty acid composition that differs significantly from that of the wild type strain. Our study also shows that RsFabG2 plays a role in adaptation to high salt concentration and low pH, and in pathogenesis of disease in tomato plants.ConclusionR. solanacearum encodes two 3-ketoacyl-ACP reductases that both have functions in fatty acid synthesis. We supply the first evidence that, like other enzymes in the bacterial fatty acid biosynthetic pathway, one bacterium may simultaneously possess two or more 3-oxoacyl-ACP reductase isozymes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0554-x) contains supplementary material, which is available to authorized users.

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“…Xcc RpfF, the key enzyme for the biosynthesis of the DSF family signals, utilizes 3‐hydroxyacyl‐ACPs as substrates (Zhou et al , ), while 3‐hydroxyacyl‐ACPs are the products of OAR in the bacterial fatty acid synthetic pathway (Rafferty et al , ). Thus, the production of DSF family signals is closely associated with OAR activity.…”

Section: Discussionmentioning

confidence: 99%

A novel 3‐oxoacyl‐ACP reductase (FabG3) is involved in the xanthomonadin biosynthesis of Xanthomonas campestris pv. campestris

Jianrong

Guo

et al. 2019

Molecular Plant Pathology

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Summary Xanthomonas campestris pv. campestris (Xcc), the causal agent of black rot in crucifers, produces a membrane‐bound yellow pigment called xanthomonadin to protect against photobiological and peroxidative damage, and uses a quorum‐sensing mechanism mediated by the diffusible signal factor (DSF) family signals to regulate virulence factors production. The Xcc gene XCC4003, annotated as Xcc fabG3, is located in the pig cluster, which may be responsible for xanthomonadin synthesis. We report that fabG3 expression restored the growth of the Escherichia coli fabG temperature‐sensitive mutant CL104 under non‐permissive conditions. In vitro assays demonstrated that FabG3 catalyses the reduction of 3‐oxoacyl‐acyl carrier protein (ACP) intermediates in fatty acid synthetic reactions, although FabG3 had a lower activity than FabG1. Moreover, the fabG3 deletion did not affect growth or fatty acid composition. These results indicate that Xcc fabG3 encodes a 3‐oxoacyl‐ACP reductase, but is not essential for growth or fatty acid synthesis. However, the Xcc fabG3 knock‐out mutant abolished xanthomonadin production, which could be only restored by wild‐type fabG3, but not by other 3‐oxoacyl‐ACP reductase‐encoding genes, indicating that Xcc FabG3 is specifically involved in xanthomonadin biosynthesis. Additionally, our study also shows that the Xcc fabG3‐disrupted mutant affects Xcc virulence in host plants.

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Crystallization of the NADP-dependent β-keto acyl carrier protein reductase from Escherichia coli

Cited by 5 publications

References 16 publications

cDNA cloning of Brassica napus malonyl‐CoA:ACP transacylase (MCAT) (fab D) and complementation of an E. coli MCAT mutant

cDNA cloning of Brassica napus malonyl‐CoA:ACP transacylase (MCAT) (fab D) and complementation of an E. coli MCAT mutant

Ralstonia solanacearum fatty acid composition is determined by interaction of two 3-ketoacyl-acyl carrier protein reductases encoded on separate replicons

A novel 3‐oxoacyl‐ACP reductase (FabG3) is involved in the xanthomonadin biosynthesis of Xanthomonas campestris pv. campestris

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