Abstract
Mesaconase catalyzes the hydration of mesaconate (methylfumarate) to (S)-citramalate. The enzyme participates in the methylaspartate pathway of glutamate fermentation as well as in the metabolism of various C5-dicarboxylic acids such as mesaconate or L-threo-β-methylmalate. We have recently shown that Burkholderia xenovorans uses a promiscuous class I fumarase to catalyze this reaction in the course of mesaconate utilization. Here we show that classical Escherichia coli class I fumarases A and B (FumA and FumB) are capable of hydrating mesaconate with 4% (FumA) and 19% (FumB) of the catalytic efficiency k cat/K m, compared to the physiological substrate fumarate. Furthermore, the genomes of 14.8% of sequenced Enterobacteriaceae (26.5% of E. coli, 90.6% of E. coli O157:H7 strains) possess an additional class I fumarase homologue which we designated as fumarase D (FumD). All these organisms are (opportunistic) pathogens. fumD is clustered with the key genes for two enzymes of the methylaspartate pathway of glutamate fermentation, glutamate mutase and methylaspartate ammonia lyase, converting glutamate to mesaconate. Heterologously produced FumD was a promiscuous mesaconase/fumarase with a 2- to 3-fold preference for mesaconate over fumarate. Therefore, these bacteria have the genetic potential to convert glutamate to (S)-citramalate, but the further fate of citramalate is still unclear. Our bioinformatic analysis identified several other putative mesaconase genes and revealed that mesaconases probably evolved several times from various class I fumarases independently. Most, if not all iron-dependent fumarases, are capable to catalyze mesaconate hydration.
Highlights
Fumarase (EC 4.2.1.2) catalyzes the reversible hydration of fumarate to (S)-malate (Fig 1A) and participates in the tricarboxylic acid (TCA) cycle and in a number of other metabolic processes
We have recently shown that B. xenovorans class I fumarase is efficient in the hydration of fumarate to (S)-malate and mesaconate to (S)-citramalate (L-citramalate in D/L nomenclature) (Fig 1C) and participates in mesaconate utilization in this bacterium (Fig 2) [10]
Analysis revealed the presence of a major band with an apparent molecular mass of 60 kDa and 50 kDa in the 50 mM or 300 mM imidazole fractions (Fig 3), which were used to study the catalytic properties of the enzymes
Summary
Fumarase (EC 4.2.1.2) catalyzes the reversible hydration of fumarate to (S)-malate (or L-malate in D/L nomenclature) (Fig 1A) and participates in the tricarboxylic acid (TCA) cycle and in a number of other metabolic processes. Promiscuity of Escherichia coli Class I Fumarases doi:10.1371/journal.pone.0145098.g001 activate a hydroxyl from the substrate (for elimination) or water (for addition) [2]. Escherichia coli possesses two class I enzymes, fumarases A and B (FumA and FumB), sharing a high degree of sequence similarity and having similar catalytic properties [6]. Class II fumarases like fumarase C (FumC) of E. coli are thermostable tetramers with four identical 50 kDa subunits that do not require Fe2+ for the activity [1]. They are oxygen tolerant enzymes catalyzing (S)-malate dehydration through the intermediate formation of an aci-carboxylate and can be found in many pro- and eukaryotic organisms [9]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
