Purification and Characterization of Fumarase from<i>Corynebacterium glutamicum</i>

Genda, Tomoko; Watabe, Shoji; Ozaki, Hachiro

doi:10.1271/bbb.70.1102

Cited by 23 publications

(20 citation statements)

References 1 publication

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“…The enzyme remained stable under aerobic conditions, and the incubation with Fe 2ϩ and DTT did not result in the increase of its activity. Both K m and V max values of the enzyme were close to the published values for other class II fumarases (28,29). In contrast to the class II enzyme, the class I fumarase of B. xenovorans, Bxe_A3136, was oxygen sensitive, and the aerobically measured activity of the (aerobically) purified protein was relatively low.…”

Section: Resultssupporting

confidence: 76%

Mesaconase Activity of Class I Fumarase Contributes to Mesaconate Utilization by Burkholderia xenovorans

Kronen

Sasikaran

Berg

2015

Appl Environ Microbiol

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bPseudomonas aeruginosa, Yersinia pestis, and many other bacteria are able to utilize the C 5 -dicarboxylic acid itaconate (methylenesuccinate). Itaconate degradation starts with its activation to itaconyl coenzyme A (itaconyl-CoA), which is further hydrated to (S)-citramalyl-CoA, and citramalyl-CoA is finally cleaved into acetyl-CoA and pyruvate. The xenobiotic-degrading betaproteobacterium Burkholderia xenovorans possesses a P. aeruginosa-like itaconate degradation gene cluster and is able to grow on itaconate and its isomer mesaconate (methylfumarate). Although itaconate degradation proceeds in B. xenovorans in the same way as in P. aeruginosa, the pathway of mesaconate utilization is not known. Here, we show that mesaconate is metabolized through its hydration to (S)-citramalate. The latter compound is then metabolized to acetyl-CoA and pyruvate with the participation of two enzymes of the itaconate degradation pathway, a promiscuous itaconate-CoA transferase able to activate (S)-citramalate in addition to itaconate and (S)-citramalyl-CoA lyase. The first reaction of the pathway, the mesaconate hydratase (mesaconase) reaction, is catalyzed by a class I fumarase. As this enzyme (Bxe_A3136) has similar efficiencies (k cat /K m ) for both fumarate and mesaconate hydration, we conclude that B. xenovorans class I fumarase is in fact a promiscuous fumarase/ mesaconase. This promiscuity is physiologically relevant, as it allows the growth of this bacterium on mesaconate as a sole carbon and energy source. I taconate (methylenesuccinate) is an industrially important fungal product and a common carbon source for various soil bacteria (1). Still, the importance of other C 5 -dicarboxylic acids such as ethylmalonate, methylsuccinate, citramalate (␣-methylmalate), and mesaconate (methylfumarate) was neglected for decades. Yet, the participation of these compounds in several central metabolic pathways has been shown in the last 15 years. Examples are the autotrophic 3-hydroxypropionate bi-cycle as well as the anaplerotic ethylmalonyl coenzyme A (ethylmalonyl-CoA) pathway and methylaspartate cycle functioning for the assimilation of C 2 units (2-6). Furthermore, mesaconate and citramalate are intermediates of the methylaspartate pathway of glutamate fermentation functioning in many (facultative) anaerobic bacteria (7).Itaconate was recently identified as a mammalian metabolite whose production is substantially induced during macrophage activation (8, 9). As itaconate is known as a potent inhibitor of the glyoxylate cycle, a metabolic pathway important for many pathogens during infection, itaconate production by macrophages is regarded as part of the antibacterial response of macrophages (9-11). Interestingly, many pathogens contain genes involved in itaconate assimilation (11). The capability to degrade itaconate promotes the survival and infectivity of pathogens inside the hosts (10-12). Bacterial itaconate degradation involves three steps, i.e., itaconate activation to itaconyl-CoA, its hydration (via mesaconyl-CoA) to ...

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Section: Resultssupporting

confidence: 76%

Mesaconase Activity of Class I Fumarase Contributes to Mesaconate Utilization by Burkholderia xenovorans

Kronen

Sasikaran

Berg

2015

Appl Environ Microbiol

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“…Determination of the specific activity of the fumarase Fum (EC 4.2.1.2) in crude extracts was performed as described previously (22). The assay mixture (500-l total volume) contained 100 mM sodium phosphate buffer, pH 7.3, and 0.5 to 2 l of crude extract.…”

Section: Methodsmentioning

confidence: 99%

The Global Repressor SugR Controls Expression of Genes of Glycolysis and of the l -Lactate Dehydrogenase LdhA in Corynebacterium glutamicum

Engels

Lindner²,

Wendisch³

2008

J Bacteriol

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The transcriptional regulator SugR from Corynebacterium glutamicum represses genes of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). Growth experiments revealed that the overexpression of sugR not only perturbed the growth of C. glutamicum on the PTS sugars glucose, fructose, and sucrose but also led to a significant growth inhibition on ribose, which is not taken up via the PTS. Chromatin immunoprecipitation combined with DNA microarray analysis and gel retardation experiments were performed to identify further target genes of SugR. Gel retardation analysis confirmed that SugR bound to the promoter regions of genes of the glycolytic enzymes 6-phosphofructokinase (pfkA), fructose-1,6-bisphosphate aldolase (fba), enolase (eno), pyruvate kinase (pyk), and NAD-dependent L-lactate dehydrogenase (ldhA). The deletion of sugR resulted in increased mRNA levels of eno, pyk, and ldhA in acetate medium. Enzyme activity measurements revealed that SugR-mediated repression affects the activities of PfkA, Fba, and LdhA in vivo. As the deletion of sugR led to increased LdhA activity under aerobic and under oxygen deprivation conditions, L-lactate production by C. glutamicum was determined. The overexpression of sugR reduced L-lactate production by about 25%, and sugR deletion increased L-lactate formation under oxygen deprivation conditions by threefold. Thus, SugR functions as a global repressor of genes of the PTS, glycolysis, and fermentative L-lactate dehydrogenase in C. glutamicum.Corynebacterium glutamicum, which was isolated as an Lglutamate-excreting soil bacterium (1, 39), is a predominantly aerobic, biotin-auxotrophic, gram-positive bacterium widely used for the industrial production of more than 2 million tons of amino acids per year, mainly L-glutamate and L-lysine (31,43). A general view of this nonpathogenic bacterium, which has become a model organism for the Corynebacterineae, a suborder of Actinomycetales that also comprises the genus Mycobacterium (54), can be found in two recent monographs (9, 17).C. glutamicum is able to grow on a variety of sugars, sugar alcohols, and organic acids as sole carbon and energy sources (64). As in many other gram-positive and gram-negative bacteria, the phosphoenolpyruvate-dependent phosphotransferase system (PTS) is the major sugar uptake system (15,37,45,47). The PTS-mediated glucose, fructose, and sucrose uptake in C. glutamicum operates by phosphoryl group transfer from phosphoenolpyruvate via EI (encoded by ptsI) and HPr (ptsH) to the sugar-specific permeases EII Glc , EII Fru , and EII Suc , respectively (ptsG, ptsF, and ptsS, respectively).

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“…The majority of enzymes of the TCA cycle of C. glutamicum have been characterised genetically and biochemically to some extent (Eikmanns, 2005), for instance citrate synthase (Eikmanns et al, 1994;Radmacher and Eggeling, 2007), isocitrate dehydrogenase (Bai et al, 1999;Chen and Yang, 2000;Eikmanns et al, 1995), the 2-oxoglutarate dehydrogenase complex (Niebisch et al, 2006;Usuda et al, 1996), succinate dehydrogenase (Kurokawa and Sakamoto, 2005), fumarase (Genda et al, 2006), malate dehydrogenase and malate:menaquinone oxidoreductase (Molenaar et al, 1998(Molenaar et al, , 2000. The investigation of the regulation of the TCA cycle started only a few years ago but already led to the identification of a complex regulatory network at the level of gene expression (Bussmann et al, 2009;Emer et al, 2009;Krug et al, 2005;Wennerhold and Bott, 2006;Wennerhold et al, 2005) and at the posttranscriptional level by protein phosphorylation (Niebisch et al, 2006;Schultz et al, 2009).…”

Section: Introductionmentioning

confidence: 99%