Identification and Preliminary Characterization of Phosphoenolpyruvate Carboxykinase from the Marine Alga, Emiliania huxleyi

Phosphoenolpyruvate carboxykinase catalyzes the nucleotide dependent reversible \ud decarboxylation of oxaloacetate to phosphoenolpyruvate. PEPCK plays a variety of \ud important roles in central and intermediary metabolism in organisms from all three \ud domains of life, and is the key enzyme in gluconeogenic and anapleurotic pathways. \ud In addition, this enzyme also participates in carbon concentrating mechanisms that \ud function to increase the availability of CO2 to RubisCO enzymes in many C4 and \ud CAM plants. However, there have been relatively few reports of PEPCK enzymes \ud from unicellular algae, or the metabolic role of this enzyme characterized in these \ud organisms. This is particularly relevant since marine algae, such as Emiliania \ud huxleyi, are considered to be carbon-limited in the ocean environment due to the low \ud concentration of dissolved C02 (~10 ??M), and thus require external and internal \ud carbonic anhydrases as a CCM. CAs are zinc-dependent metalloenzymes and the low \ud levels of trace metals in the oceans suggest that alternative CCM pathways, such as \ud those described in some plants, may also exist in unicellular algae. This hypothesis \ud was recently supported by the description of a C4 photosynthetic pathway in the \ud marine diatom, Thalassiosira weissflogii, involving the spatial separation of pep \ud carboxylase and pep carboxykinase enzyme activities. The research in this thesis is \ud the first identification of a phosphoenolpyruvate carboxykinase from a marine \ud coccolithophorid alga. Transcriptional profiles of this gene using qRT -PCR indicated \ud that there was no significant difference in the expression levels of this transcript in \ud response to variations in extracellular bicarbonate, providing important baseline data \ud for future studies on the expression of the gene under C02 and trace metal limitation \ud conditions. Recombinant E. huxleyi PEPCK was expressed in E. coli as an active \ud enzyme, and transformation of this clone into an E. coli PEPCK mutant \ud complimented this strain to wild type phenotype. This was the first report of genetic \ud complementation in marine coccolithophorids, and demonstrates that this technique \ud may provide a useful tool for identifying other genes involved in carbon metabolism \ud in this globally important marine alga.Biological Science