title: Cloning, Expression and Characterization of the δ-carbonic Anhydrase of Thalassiosira weissflogii (Bacillariophyceae).
abstract: Carbonic anhydrase (CA) is a ubiquitous metalloenzyme responsible for accelerating the interconversion of CO2 and bicarbonate. Although CAs are involved in a broad range of biochemical processes involving carboxylation or decarboxylation reactions, they are of special interest due to their role in photosynthetic CO2 assimilation in marine phytoplankton, especially under low-CO2 conditions. Several phylogenetically independent classes of CAs have been identified in a variety of marine phytoplankton. TWCA1, first discovered in Thalassiosira weissflogii (Grunow) G. Fryxell & Hasle, is the founding member of the δ-class of CAs; these appear to be extracellular enzymes, but are still relatively poorly characterized. To date, it has remained uncertain whether TWCA1 possesses true CA activity due to the difficulty in producing a functional protein in a heterologous expression system. Herein we describe the fusion of a full-length open reading frame of TWCA1 to the coding sequence of a self-splicing intein in a pTWIN2 expression vector that has allowed successful production of a functional enzyme in Escherichia coli. Assay of the recombinant protein shows that TWCA1 is a catalytically active δ-CA possessing both CO2 hydration and esterase activity.
authors.affiliation: Department of Earth Sciences, University of Oxford, South Parks Road, Oxford, OX1 3AN, UK.
authors.forename: Renee Bee Yong
authors.initials: RB
authors.lastname: Lee
authors.affiliation: Department of Plant Sciences, University of Oxford, South Parks Road, Oxford, OX1 3RB, UK.
authors.forename: J Andrew C
authors.initials: JA
authors.lastname: Smith
authors.affiliation: Department of Earth Sciences, University of Oxford, South Parks Road, Oxford, OX1 3AN, UK.
authors.forename: Rosalind E M
authors.initials: RE
authors.lastname: Rickaby
keywords: Thalassiosira weissflogii; carbon-concentrating mechanism; esterase activity; marine diatom; photosynthesis; δ-carbonic anhydrase