Chemical Mechanism and Substrate Specificity of RhlI, an Acylhomoserine Lactone Synthase from <i>Pseudomonas aeruginosa</i>

Raychaudhuri, Aniruddha; Jerga, and Agoston; Tipton, Peter A.

doi:10.1021/bi048005m

Cited by 58 publications

(89 citation statements)

References 16 publications

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“…It appears, however, that molecular oxygen is not directly involved in the synthesis of HSL-based autoinducers. Synthesis of 3-oxo-C 12 -HSL and C 4 -HSL by their cognate autoinducer synthase (i.e., LasI and RhlI) requires S-adenosylmethionine (SAM) and 3-oxo-C 12 -acyl carrier protein or the N-butyryl acyl carrier protein, respectively (18,38). Our qRT-PCR analysis results shown in Fig.…”

Section: Discussionmentioning

confidence: 81%

Anaerobiosis-Induced Loss of Cytotoxicity Is Due to Inactivation of Quorum Sensing in Pseudomonas aeruginosa

et al. 2011

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Pseudomonas aeruginosa, an opportunistic pathogen of clinical importance, causes chronic airway infections in patients with cystic fibrosis (CF). Current literature suggests that pockets with reduced oxygen tension exist in the CF airway mucus. However, virulence features of this opportunistic pathogen under such conditions are largely unknown. Cell-free supernatant of the standard laboratory P. aeruginosa strain PAO1 obtained from anaerobic culture, but not aerobic culture, failed to kill A549 human airway epithelial cells. Further investigation revealed that this reduced cytotoxicity upon anaerobiosis was due to the suppressed secretion of elastase, a virulence factor controlled by P. aeruginosa quorum sensing (QS). Both a lacZ-reporter fusion assay and quantitative real-time PCR (RT-PCR) analysis demonstrated that transcription of the elastase-encoding lasB gene was substantially decreased during anaerobic growth compared with aerobic growth. Moreover, transcription of other genes controlled by the LasI/R QS system, such as rhlR, vqsR, mvfR, and rsaL, was also repressed under the same anaerobic growth conditions. Importantly, synthesis of 3-oxo-C 12 -HSL (PAI-1), an autoinducer molecule that mediates induction of the LasI/R QS system, was >22-fold decreased during anaerobic growth while C 4 -HSL (PAI-2), which mediates RhlI/R QS, was nondetectable under the same growth conditions. Transcription of the lasB gene was restored by exogenous supplementation with autoinducers, with PAI-2 more effective than PAI-1 or Pseudomonas quinolone signal (PQS) at restoring transcription of the lasB gene. Together, these results suggest that anaerobiosis deprives P. aeruginosa of the ability to regulate its virulence via QS and this misregulation attenuates the pathogenic potential of this important pathogen.

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Section: Discussionmentioning

confidence: 81%

Anaerobiosis-Induced Loss of Cytotoxicity Is Due to Inactivation of Quorum Sensing in Pseudomonas aeruginosa

et al. 2011

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“…A previous kinetic analysis of an acyl-HSL synthase, RhlI, suggested that the ionizing residue plays a part in the acylation reaction that leads to deprotonation of the α-amino group of SAM and subsequent nucleophilic attacks on the thioester bond of acyl-ACP (21). After acylation, lactonization appears to proceed via a direct attack of the carboxylate oxygen on the methylene carbon adjacent to the sulfonium ion within an N-acyl-SAM intermediate (21).…”

Section: Discussionmentioning

confidence: 99%

“…After acylation, lactonization appears to proceed via a direct attack of the carboxylate oxygen on the methylene carbon adjacent to the sulfonium ion within an N-acyl-SAM intermediate (21). In particular, various experiments consistently indicated that this lactonization step does not require a general acid-base catalysis (21).…”

Section: Discussionmentioning

confidence: 99%

Small-molecule inhibitor binding to an N -acyl-homoserine lactone synthase

Chung

Goo

et al. 2011

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

103

106

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Quorum sensing (QS) controls certain behaviors of bacteria in response to population density. In Gram-negative bacteria, QS is often mediated by N-acyl-L-homoserine lactones (acyl-HSLs). Because QS influences the virulence of many pathogenic bacteria, synthetic inhibitors of acyl-HSL synthases might be useful therapeutically for controlling pathogens. However, rational design of a potent QS antagonist has been thwarted by the lack of information concerning the binding interactions between acyl-HSL synthases and their ligands. In the Gram-negative bacterium Burkholderia glumae, QS controls virulence, motility, and protein secretion and is mediated by the binding of N-octanoyl-L-HSL (C8-HSL) to its cognate receptor, TofR. C8-HSL is synthesized by the acyl-HSL synthase TofI. In this study, we characterized two previously unknown QS inhibitors identified in a focused library of acyl-HSL analogs. Our functional and X-ray crystal structure analyses show that the first inhibitor, J8-C8, binds to TofI, occupying the binding site for the acyl chain of the TofI cognate substrate, acylated acyl-carrier protein.Moreover, the reaction byproduct, 5′-methylthioadenosine, independently binds to the binding site for a second substrate, Sadenosyl-L-methionine. Closer inspection of the mode of J8-C8 binding to TofI provides a likely molecular basis for the various substrate specificities of acyl-HSL synthases. The second inhibitor, E9C-3oxoC6, competitively inhibits C8-HSL binding to TofR. Our analysis of the binding of an inhibitor and a reaction byproduct to an acyl-HSL synthase may facilitate the design of a new class of QS-inhibiting therapeutic agents. Q uorum sensing (QS) is an intercellular signaling process that mediates certain behaviors of bacteria (including bioluminescence, biofilm formation, motility, and virulence factor production) in response to the bacterial cell population density (1-3). In Gram-negative bacteria, QS is often mediated by Nacyl-L-homoserine lactones (acyl-HSLs), which are synthesized by the LuxI family of acyl-HSL synthases from S-adenosyl-Lmethionine (SAM) and acylated acyl-carrier protein (acyl-ACP), with the release of holo-ACP and 5′-methylthioadenosine (MTA) as byproducts (SI Appendix, Fig. S1A) (4, 5). Compounds of the acyl-HSL class share a homoserine lactone ring moiety, but the acyl chains conjugated to the ring via an amide bond vary in length, oxidation state at C3, and amount of saturation (SI Appendix, Fig. S1A). The recent finding that p-coumarate is an alternative substrate for acyl-ACP has extended the known range of possible acyl-HSL substrates (6). On the other hand, the acyl-HSL receptor is a transcriptional regulator that controls the expression of target genes in response to acyl-HSL binding (1-3).Among the hundreds of genes regulated by QS, the most widely studied genes are those related to virulence; these genes are of particular interest because QS disruption is being investigated as a strategy for controlling virulent pathogens (7-9). QS inhibitors can act by suppressing a...

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“…5), arginines 19 and 41 interact with the phosphate, and the guanidinium group of Arg 41 stacks onto the adenine rings to fully enclose this end of the cofactor. Also, the backbone amides of Arg 41 , Asp 42 , and Leu 66 as well as the hydroxyl of Ser 18 mediate hydrogen bonding interactions. Finally, it may be significant that only one active site in the asymmetric unit has a bound cofactor because both active site clefts are accessible in the crystal packing arrangement.…”

Section: Resultsmentioning

confidence: 99%

Structure of RhlG, an Essential β-Ketoacyl Reductase in the Rhamnolipid Biosynthetic Pathway of Pseudomonas aeruginosa

Miller

Zhang

Rock

et al. 2006

Journal of Biological Chemistry

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Rhamnolipids are extracellular biosurfactants and virulence factors secreted by the opportunistic human pathogen Pseudomonas aeruginosa that are required for swarming motility. The rhlG gene is essential for rhamnolipid formation, and the RhlG enzyme is thought to divert fatty acid synthesis intermediates into the rhamnolipid biosynthetic pathway based on its similarity to FabG, the ␤- Pseudomonas aeruginosa is a Gram-negative bacterium that is capable of existing in multiple environmental niches and is an opportunistic pathogen that infects immunocompromised and cystic fibrosis patients (1, 2). It is a particularly serious pathogen in hospitals and is relatively resistant to many commonly used antibiotics. Key pathogenic features of the organism are that it produces an extracellular rhamnolipid surfactant that facilitates the acquisition of hydrophobic carbon sources (3), the development of biofilms (4, 5), and the dissemination of hydrophobic signaling molecules (6); it acts as a heat-stable hemolysin (7); and it is required for swarming motility (8, 9). P. aeruginosa swarming is a multicellular form of organized movement allowing the rapid colonization of surfaces (8, 10). Rhamnolipids are virulence factors that are found in the sputa of cystic fibrosis patients (11) and inflict damage on eukaryotic cells (12-15). Rhamnolipids also have potential industrial applications because of their ability to reduce the surface tension of water (16).Rhamnolipids are composed of molecules having either one or two rhamnose sugars linked to a dimer of ␤-hydroxyacids (primarily ␤-hydroxydecanoate) (17). The terminal stages of the rhamnolipid biosynthetic pathway are carried out by the dTDP-rhamnose transferases RhlB and RhlC that produce lipids with one or two rhamnose moieties, respectively (18,19). RhlA is essential for rhamnolipid synthesis, and although its biochemical activity has not been demonstrated, it is proposed to be involved in forming the ␤-hydroxydecanoyl-␤-hydroxydecanoate precursor (9,20). RhlG is an essential component in the rhamnolipid biosynthetic pathway because inactivation of the rhlG gene leads to the loss of rhamnolipid production (21). Based on the similarity of RhlG to FabG, 2 the NADPH-dependent ␤-ketoacyl-ACP reductase of type II fatty acid synthesis, RhlG is proposed to perform the same reaction as FabG and divert intermediates in fatty acid synthesis into the rhamnolipid pathway (21). A transacylase may be involved downstream of RhlG, and the mechanism for the transport of rhamnolipids out of the cell is unknown. This work reports the x-ray structure of RhlG and compares its structure and biochemical activities to the FabG of type II fatty acid biosynthesis. EXPERIMENTAL PROCEDURESExpression and Purification of RhlG-The P. aeruginosa rhlG gene was amplified by PCR and cloned into pET15b vector within NdeI/ BamHI restriction sites. The recombinant RhlG protein with a N-terminal His-tag was expressed in Escherichia coli strain BL21(DE3) and purified by nickel-affinity chromatography as described...

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Chemical Mechanism and Substrate Specificity of RhlI, an Acylhomoserine Lactone Synthase from Pseudomonas aeruginosa

Cited by 58 publications

References 16 publications

Anaerobiosis-Induced Loss of Cytotoxicity Is Due to Inactivation of Quorum Sensing in Pseudomonas aeruginosa

Anaerobiosis-Induced Loss of Cytotoxicity Is Due to Inactivation of Quorum Sensing in Pseudomonas aeruginosa

Small-molecule inhibitor binding to an N -acyl-homoserine lactone synthase

Structure of RhlG, an Essential β-Ketoacyl Reductase in the Rhamnolipid Biosynthetic Pathway of Pseudomonas aeruginosa

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