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Hyperthermophilic Carbamate Kinase Stability and Anabolic In Vitro Activity at Alkaline pH

Hennessy, James E; Latter, Melissa; Philbrook, Amy; Bartkus, Daniel; Kim, Hye; Onagi, Hideki; Oakeshott, J.G.; Scott, Colin; Alissandratos, Apostolos; Easton, Christopher

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Carbamate kinases catalyze the conversion of carbamate to carbamoyl phosphate, which is readily transformed into other compounds. Carbamate forms spontaneously from ammonia and carbon dioxide in aqueous solutions, so the kinases have potential for sequestrative utilization of the latter compounds. Here, we compare seven carbamate kinases from mesophilic, thermophilic, and hyperthermophilic sources. In addition to the known enzymes from Enterococcus faecalis and Pyrococcus furiosus, the...[Show more]

dc.contributor.authorHennessy, James E
dc.contributor.authorLatter, Melissa
dc.contributor.authorPhilbrook, Amy
dc.contributor.authorBartkus, Daniel
dc.contributor.authorKim, Hye
dc.contributor.authorOnagi, Hideki
dc.contributor.authorOakeshott, J.G.
dc.contributor.authorScott, Colin
dc.contributor.authorAlissandratos, Apostolos
dc.contributor.authorEaston, Christopher
dc.date.accessioned2019-09-26T01:24:28Z
dc.identifier.issn0099-2240
dc.identifier.urihttp://hdl.handle.net/1885/171673
dc.description.abstractCarbamate kinases catalyze the conversion of carbamate to carbamoyl phosphate, which is readily transformed into other compounds. Carbamate forms spontaneously from ammonia and carbon dioxide in aqueous solutions, so the kinases have potential for sequestrative utilization of the latter compounds. Here, we compare seven carbamate kinases from mesophilic, thermophilic, and hyperthermophilic sources. In addition to the known enzymes from Enterococcus faecalis and Pyrococcus furiosus, the previously unreported enzymes from the hyperthermophiles Thermococcus sibiricus and Thermococcus barophilus, the thermophiles Fervidobacterium nodosum and Thermosipho melanesiensis, and the mesophile Clostridium tetani were all expressed recombinantly, each in high yield. Only the clostridial enzyme did not show catalysis. In direct assays of carbamate kinase activity, the three hyperthermophilic enzymes display higher specific activities at elevated temperatures, greater stability, and remarkable substrate turnover at alkaline pH (9.9 to 11.4). Thermococcus barophilus and Thermococcus sibiricus carbamate kinases were found to be the most active when the enzymes were tested at 80°C, and maintained activity over broad temperature and pH ranges. These robust thermococcal enzymes therefore represent ideal candidates for biotechnological applications involving aqueous ammonia solutions, since nonbuffered 0.0001 to 1.0 M solutions have pH values of approximately 9.8 to 11.8. As proof of concept, here we also show that carbamoyl phosphate produced by the Thermococcus barophilus kinase is efficiently converted in situ to carbamoyl aspartate by aspartate transcarbamoylase from the same source organism. Using acetyl phosphate to simultaneously recycle the kinase cofactor ATP, at pH 9.9 carbamoyl aspartate is produced in high yield and directly from solutions of ammonia, carbon dioxide, and aspartate.
dc.description.sponsorshipWe acknowledge financial support of this work by the Grains Research and Development Corporation (GRDC), the Australian Research Council (ARC), the Australian National University, and CSIRO.
dc.format.mimetypeapplication/pdf
dc.language.isoen_AU
dc.publisherAmerican Society for Microbiology
dc.rights© 2018 American Society for Microbiology
dc.sourceApplied and Environmental Microbiology
dc.titleHyperthermophilic Carbamate Kinase Stability and Anabolic In Vitro Activity at Alkaline pH
dc.typeJournal article
local.description.notesImported from ARIES
local.identifier.citationvolume84
dc.date.issued2018
local.identifier.absfor060113 - Synthetic Biology
local.identifier.ariespublicationa383154xPUB9280
local.publisher.urlhttps://www.asm.org/
local.type.statusPublished Version
local.contributor.affiliationHennessy, James E, College of Science, ANU
local.contributor.affiliationLatter, Melissa, College of Science, ANU
local.contributor.affiliationPhilbrook, Amy, College of Science, ANU
local.contributor.affiliationBartkus, Daniel, College of Science, ANU
local.contributor.affiliationKim, Hye, College of Science, ANU
local.contributor.affiliationOnagi, Hideki, College of Science, ANU
local.contributor.affiliationOakeshott, J.G., CSIRO
local.contributor.affiliationScott, Colin, CSIRO Land & Water
local.contributor.affiliationAlissandratos, Apostolos, College of Science, ANU
local.contributor.affiliationEaston, Christopher, College of Science, ANU
local.description.embargo2037-12-31
local.bibliographicCitation.issue3
local.bibliographicCitation.startpage1
local.bibliographicCitation.lastpage13
local.identifier.doi10.1128/AEM.02250-17
dc.date.updated2019-04-21T08:21:32Z
local.identifier.scopusID2-s2.0-85040661814
CollectionsANU Research Publications

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