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Comparative Study
. 2013;8(2):e55549.
doi: 10.1371/journal.pone.0055549. Epub 2013 Feb 6.

Biochemical similarities and differences between the catalytic [4Fe-4S] cluster containing fumarases FumA and FumB from Escherichia coli

Barbara M A van Vugt-Lussenburg  1 , Laura van der Weel, Wilfred R Hagen, Peter-Leon Hagedoorn
Affiliations
Comparative Study

Biochemical similarities and differences between the catalytic [4Fe-4S] cluster containing fumarases FumA and FumB from Escherichia coli

Barbara M A van Vugt-Lussenburg et al. PLoS One. 2013.

Abstract

Background: The highly homologous [4Fe-4S] containing fumarases FumA and FumB, sharing 90% amino acid sequence identity, from Escherichia coli are differentially regulated, which suggests a difference in their physiological function. The ratio of FumB over FumA expression levels increases by one to two orders of magnitude upon change from aerobic to anaerobic growth conditions.

Methodology/principal findings: To understand this difference in terms of structure-function relations, catalytic and thermodynamic properties were determined for the two enzymes obtained from homologous overexpression systems. FumA and FumB are essentially identical in their Michaelis-Menten kinetics of the reversible fumarate to L-malate conversion; however, FumB has a significantly greater catalytic efficiency for the conversion of D-tartrate to oxaloacetate consistent with the requirement of the fumB gene for growth on D-tartrate. Reduction potentials of the [4Fe-4S](2+) Lewis acid active centre were determined in mediated bulk titrations in the presence of added substrate and were found to be approximately -290 mV for both FumA and FumB.

Conclusions/significance: This study contradicts previously published claims that FumA and FumB exhibit different catalytic preferences for the natural substrates L-malate and fumarate. FumA and FumB differ significantly only in the catalytic efficiency for the conversion of D-tartrate, a supposedly non-natural substrate. The reduction potential of the substrate-bound [4Fe-4S] active centre is, contrary to previously reported values, close to the cellular redox potential.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Reaction scheme of the L-malate dehydration (top) and the D-tartrate dehydration reactions (bottom) catalyzed by fumarase.
Figure 2
Figure 2. EPR spectra of FumA (red) and FumB (blue).
a FumA [3Fe-4S]1+ clusters; as isolated, not regenerated. b FumB [3Fe-4S]1+ clusters; as isolated, not regenerated. c FumA [4Fe-4S]1+ clusters; regenerated, reduced and in the presence of 5 mM fumarate. d FumB [4Fe-4S]1+ clusters; regenerated, reduced and in the presence of 5 mM fumarate. The g-values are 2.032, 1.914 and 1.822 for FumA and 2.032, 1.916 and 1.821 for FumB. EPR parameters: microwave frequency a 9.630 GHz, b 9.631 GHz, c 9.407 GHz, d 9.631 GHz; microwave power a 8.0 mW, b 8.0 mW, c 20 mW, d 20 mW; modulation frequency 100 kHz; modulation amplitude a 0.63 mT, b 0.63 mT, c 1.25 mT, d 1.25 mT; temperature a 14.5 K, b 14.5 K, c 16K, d 14.5 K.
Figure 3
Figure 3. EPR-monitored redox titration curves of FumA (•, red) and FumB (□, blue).
The two points at low, undefined potential represent samples reduced with excess dithionite (10 mM). The solid lines represents fits to the Nernst equation: [Image: see text]. Fit parameters for FumA: Em = −300±6 mV; and for FumB: Em = −283±9 mV.
Figure 4
Figure 4. Oxygen sensitivity of FumA (•, red) and FumB (□, blue).
Fumarase activity was measured after air oxidation for 0, 0.5, 1 or 2 minutes. The residual activity was plotted as a percentage of the initial activity. The solid lines represent fits to the following equation:[Image: see text]. The fit parameters were as follows, for FumA: A = 5±6%; kinact = (1.8±0.5)·102 M−1s−1, for FumB: A = 6±6%; kinact = (1.6±0.4)·102 M−1s−1.
Figure 5
Figure 5. Amino acid sequence alignment of E. coli FumA and FumB. FumA and FumB share 90% sequence identity and 95% sequence similarity.
*Cysteine residues strictly conserved in multiple sequence alignment, using the ‘Cobalt’ program, from the 500 homologs exhibiting >72% sequence identity with E. coli FumA.
See this image and copyright information in PMC

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