Structural and Functional Characterization of Malate Synthase G from Opportunistic Pathogen <i>Pseudomonas aeruginosa</i>

McVey, Alyssa C.; Medarametla, Prasanthi; Chee, Xavier; Bartlett, Sean; Poso, Antti; Spring, David R.; Rahman, Taufiq; Welch, Martin

doi:10.1021/acs.biochem.7b00852

Cited by 13 publications

(12 citation statements)

References 41 publications

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“…Growth on acetate as a sole carbon source elicited a similarly informative set of changes. There was a strong induction of the glyoxylate shunt enzymes (AceA and GlcB), which are known to be essential for growth on acetate [32,33], and of enzymes directly involved in acetate activation (such as AcsA, AckA and Pta) and acetate uptake (PA3234) [34]. The TCA cycle-associated enzymes were almost all up-regulated during growth on acetate, as was the membrane-bound malate-quinone oxidoreductase, MqoB (Figure 2).…”

Section: Resultsmentioning

confidence: 99%

Contextual flexibility inPseudomonas aeruginosacentral carbon metabolism during growth in single carbon sources

Dolan

Kohlstedt

Trigg

et al. 2019

Preprint

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Pseudomonas aeruginosa is an opportunistic human pathogen, particularly noted for causing infections in the lungs of people with cystic fibrosis (CF). Previous studies have shown that the gene expression profile of P. aeruginosa appears to converge towards a common metabolic program as the organism adapts to the CF airway environment. However, at a systems level, we still have only a limited understanding of how these transcriptional changes impact on metabolic flux. To address this, we analysed the transcriptome, proteome and fluxome of P. aeruginosa grown on glycerol or acetate. These carbon sources were chosen because they are the primary breakdown products of airway surfactant, phosphatidylcholine, which is known to be a major carbon source for P. aeruginosa in the CF airways. We show that the flux of carbon through central metabolism is radically different on each carbon source. For example, the newly-recognised EDEMP cycle plays an important role in supplying NADPH during growth on glycerol. By contrast, the EDEMP cycle is attenuated during growth on acetate, and instead, NADPH is primarily supplied by the isocitrate dehydrogenase(s)-catalyzed reaction. Perhaps more importantly, our proteomic and transcriptomic analyses reveal a global remodelling of gene expression during growth on the different carbon sources, with unanticipated impacts on aerobic denitrification, electron transport chain architecture, and the redox economy of the cell. Collectively, these data highlight the remarkable metabolic plasticity of P. aeruginosa; a plasticity which allows the organism to seamlessly segue between different carbon sources, maximising the energetic yield from each. Importance Pseudomonas aeruginosa is an opportunistic human pathogen, well-known for causing infections in the airways of people with cystic fibrosis. Although it is clear that P. aeruginosa is metabolically well-adapted to life in the CF lung, little is currently known about how the organism metabolises the nutrients available in the airways. In this work, we use a combination of gene expression and isotope tracer (“fluxomic”) analyses to find out exactly where the input carbon goes during growth on two CF-relevant carbon sources, acetate and glycerol (derived from the breakdown of lung surfactant). We find that carbon is routed (“fluxed”) through very different pathways during growth on these substrates, and that this is accompanied by an unexpected remodelling of the cell’s electron transfer pathways. Having access to this “blueprint” is important because the metabolism of P. aeruginosa is increasingly being recognised as a target for the development of much-needed antimicrobial agents.

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Section: Resultsmentioning

confidence: 99%

Contextual flexibility inPseudomonas aeruginosacentral carbon metabolism during growth in single carbon sources

Dolan

Kohlstedt

Trigg

et al. 2019

Preprint

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Section: Discussionmentioning

confidence: 99%

“…In 2012, Fahnoe et al reported that a pair of 2-aminopyridine (2-AP) derivatives, discovered following a high-throughput screen, could apparently inhibit both enzymes (ICL Pa and MS Pa ) of the glyoxylate shunt [13]. This was a remarkable observation since ICL Pa and MS Pa exhibit very different structures and activities [9,10]. However, no detailed SAR was carried out, and, even for the hits, the mode of action, enzyme binding properties, cytotoxicity profiles, drug-drug interactions, and drug clearance properties of the compounds were not investigated further by Fahnoe et al [13] In the current work, we rectified this and also carried out a limited SAR analysis of the 2-AP derivatives.…”

Section: Discussionmentioning

confidence: 99%

“…The resulting calculated inhibition constants were Ki = 4.5 µM (SB002 binding to the free enzyme) and Ki' = 3.9 µM (SB002 binding to the ES complex). MSPa is known to exhibit ordered binding kinetics, with glyoxylate binding a necessary prelude to acetyl-CoA binding [10]. At a saturating concentration of glyoxylate, SB002 elicited a change in the kinetic profile from Michaelis-Menten (hyperbolic kinetics) to sigmoidal, with a large apparent increase in KM with increasing inhibitor concentrations ( Figure 3C).…”

Section: Mode Of Actionmentioning

confidence: 99%

“…MS from P. aeruginosa (MS Pa ) has also been characterized. The enzyme is a monomer comprised of an 8α/8β TIM barrel fold and an α-helical C-terminal domain, which borders the active site along with the TIM barrel [10].…”

Section: Introductionmentioning

confidence: 99%

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2-Aminopyridine Analogs Inhibit Both Enzymes of the Glyoxylate Shunt in Pseudomonas aeruginosa

McVey

Bartlett

Kajbaf

et al. 2020

IJMS

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Pseudomonas aeruginosa is an opportunistic pathogen responsible for many hospital-acquired infections. P. aeruginosa can thrive in diverse infection scenarios by rewiring its central metabolism. An example of this is the production of biomass from C2 nutrient sources such as acetate via the glyoxylate shunt when glucose is not available. The glyoxylate shunt is comprised of two enzymes, isocitrate lyase (ICL) and malate synthase G (MS), and flux through the shunt is essential for the survival of the organism in mammalian systems. In this study, we characterized the mode of action and cytotoxicity of structural analogs of 2-aminopyridines, which have been identified by earlier work as being inhibitory to both shunt enzymes. Two of these analogs were able to inhibit ICL and MS in vitro and prevented growth of P. aeruginosa on acetate (indicating cell permeability). Moreover, the compounds exerted negligible cytotoxicity against three human cell lines and showed promising in vitro drug metabolism and safety profiles. Isothermal titration calorimetry was used to confirm binding of one of the analogs to ICL and MS, and the mode of enzyme inhibition was determined. Our data suggest that these 2-aminopyridine analogs have potential as anti-pseudomonal agents.

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Detection and Inhibition of Bacteria on a Dual‐Functional Silver Platform

Vedarethinam

Huang

et al. 2018

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Structural and Functional Characterization of Malate Synthase G from Opportunistic Pathogen Pseudomonas aeruginosa

Cited by 13 publications

References 41 publications

Contextual flexibility inPseudomonas aeruginosacentral carbon metabolism during growth in single carbon sources

Contextual flexibility inPseudomonas aeruginosacentral carbon metabolism during growth in single carbon sources

2-Aminopyridine Analogs Inhibit Both Enzymes of the Glyoxylate Shunt in Pseudomonas aeruginosa

Detection and Inhibition of Bacteria on a Dual‐Functional Silver Platform

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