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Characterization of feedback-resistant mevalonate kinases from the methanogenic archaeons Methanosaeta concilii and Methanocella paludicola.

ABSTRACT: The inhibition of mevalonate kinase (MVK) by downstream metabolites is an important mechanism in the regulation of isoprenoid production in a broad range of organisms. The first feedback-resistant MVK was previously discovered in the methanogenic archaeon Methanosarcinamazei. Here, we report the cloning, expression, purification, kinetic characterization and inhibition analysis of MVKs from two other methanogens, Methanosaetaconcilii and Methanocellapaludicola. Similar to the M. mazei MVK, these enzymes were not inhibited by diphosphomevalonate (DPM), dimethylallyl diphosphate (DMAPP), isopentenyldiphosphate (IPP), geranylpyrophosphate (GPP) or farnesylpyrophosphate (FPP). However, they exhibited significantly higher affinity to mevalonate and higher catalytic efficiency than the previously characterized enzyme.

SUBMITTER: Kazieva E

PROVIDER: S-EPMC5817203 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Characterization of feedback-resistant mevalonate kinases from the methanogenic archaeons Methanosaeta concilii and Methanocella paludicola.

Kazieva Ekaterina E Yamamoto Yoko Y Tajima Yoshinori Y Yokoyama Keiichi K Katashkina Joanna J Nishio Yousuke Y

Microbiology (Reading, England) 20170905 9

The inhibition of mevalonate kinase (MVK) by downstream metabolites is an important mechanism in the regulation of isoprenoid production in a broad range of organisms. The first feedback-resistant MVK was previously discovered in the methanogenic archaeon Methanosarcinamazei. Here, we report the cloning, expression, purification, kinetic characterization and inhibition analysis of MVKs from two other methanogens, Methanosaetaconcilii and Methanocellapaludicola. Similar to the M. mazei MVK, these ...[more]

PMID: 28869407

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Project description:The mevalonate pathway is a well-known metabolic route that provides biosynthetic precursors for myriad isoprenoids. An unexpected variety of the pathway has been discovered from recent studies on microorganisms, mainly on archaea. The most recently discovered example, called the "archaeal" mevalonate pathway, is a modified version of the canonical eukaryotic mevalonate pathway and was elucidated in our previous study using the hyperthermophilic archaeon Aeropyrum pernix This pathway comprises four known enzymes that can produce mevalonate 5-phosphate from acetyl coenzyme A, two recently discovered enzymes designated phosphomevalonate dehydratase and anhydromevalonate phosphate decarboxylase, and two more known enzymes, i.e., isopentenyl phosphate kinase and isopentenyl pyrophosphate:dimethylallyl pyrophosphate isomerase. To show its wide distribution in archaea and to confirm if its enzyme configuration is identical among species, the putative genes of a lower portion of the pathway-from mevalonate to isopentenyl pyrophosphate-were isolated from the methanogenic archaeon Methanosarcina mazei, which is taxonomically distant from A. pernix, and were introduced into an engineered Escherichia coli strain that produces lycopene, a red carotenoid pigment. Lycopene production, as a measure of isoprenoid productivity, was enhanced when the cells were grown semianaerobically with the supplementation of mevalonolactone, which demonstrates that the archaeal pathway can function in bacterial cells to convert mevalonate into isopentenyl pyrophosphate. Gene deletion and complementation analysis using the carotenogenic E. coli strain suggests that both phosphomevalonate dehydratase and anhydromevalonate phosphate decarboxylase from M. mazei are required for the enhancement of lycopene production.IMPORTANCE Two enzymes that have recently been identified from the hyperthermophilic archaeon A. pernix as components of the archaeal mevalonate pathway do not require ATP for their reactions. This pathway, therefore, might consume less energy than other mevalonate pathways to produce precursors for isoprenoids. Thus, the pathway might be applicable to metabolic engineering and production of valuable isoprenoids that have application as pharmaceuticals. The archaeal mevalonate pathway was successfully reconstructed in E. coli cells by introducing several genes from the methanogenic or hyperthermophilic archaeon, which demonstrated that the pathway requires the same components even in distantly related archaeal species and can function in bacterial cells.

Project description:Two-component signal transduction systems (TCSs) are a major mechanism used by bacteria in response to environmental changes. Although many sequenced archaeal genomes encode TCSs, they remain poorly understood. Previously, we reported that a methanogenic archaeon, Methanosaeta harundinacea, encodes FilI, which synthesizes carboxyl-acyl homoserine lactones, to regulate transitions of cellular morphology and carbon metabolic fluxes. Here, we report that filI, the cotranscribed filR2, and the adjacent filR1 constitute an archaeal TCS. FilI possesses a cytoplasmic kinase domain (histidine kinase A and histidine kinase-like ATPase) and its cognate response regulator. FilR1 carries a receiver (REC) domain coupled with an ArsR-related domain with potential DNA-binding ability, while FilR2 carries only a REC domain. In a phosphorelay assay, FilI was autophosphorylated and specifically transferred the phosphoryl group to FilR1 and FilR2, confirming that the three formed a cognate TCS. Through chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) using an anti-FilR1 antibody, FilR1 was shown to form in vivo associations with its own promoter and the promoter of the filI-filR2 operon, demonstrating a regulatory pattern common among TCSs. ChIP-qPCR also detected FilR1 associations with key genes involved in acetoclastic methanogenesis, acs4 and acs1. Electrophoretic mobility shift assays confirmed the in vitro tight binding of FilR1 to its own promoter and those of filI-filR2, acs4, and mtrABC. This also proves the DNA-binding ability of the ArsR-related domain, which is found primarily in Archaea. The archaeal promoters of acs4, filI, acs1, and mtrABC also initiated FilR1-modulated expression in an Escherichia coli lux reporter system, suggesting that FilR1 can up-regulate both archaeal and bacterial transcription. In conclusion, this work identifies an archaeal FilI/FilRs TCS that regulates the methanogenesis of M. harundinacea.

Dataset Information

Characterization of feedback-resistant mevalonate kinases from the methanogenic archaeons Methanosaeta concilii and Methanocella paludicola.

Publications

Characterization of feedback-resistant mevalonate kinases from the methanogenic archaeons Methanosaeta concilii and Methanocella paludicola.

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