Abstract:

Phytoplankton living in oligotrophic oceans have to deal with low nutrient availability. To acquire phosphate, these microorganisms express the enzyme alkaline phosphatase (AP) to hydrolyze organic phosphate and take up the inorganic moiety. To acquire CO2 , some phytoplankton have evolved carbon concentrating mechanisms, in which carbonic anhydrase (CA) is an essential component. These two enzymes usually use zinc as their cofactors. However, Zn availability is also very low in open ocean surface waters due to the depletion via biological uptake and complexation by organic ligands. Some phytoplankton species thus use cadmium and/or cobalt as a replacement for Zn. In this thesis, I investigate metal replacement in the coccolithophore, Emiliania huxleyi , report the isolation and characterization of a novel AP, EHAP1, in E. huxleyi , explore the genetic diversity and regulation of EHAP1 in various E. huxleyi strains, and examine the structure, catalytic mechanism and metal exchange of a novel CA, CDCA1 in the diatom, Thalassiosira weissflogii .

Zn can be fully replaced by Co but only partially by Cd in E. huxleyi . The relative use efficiencies of these metals are different with 75% efficiency for Zn and 66% for Cd compared with Co in E. huxleyi . The steady state uptake of Zn and Co is very fast, approaching the diffusion limit, but that of Cd is much slower.

EHAP1 is probably the major AP expressed in E. huxleyi . This enzyme has no sequence similarity to other known proteins. The expression of ehap1 at the transcript level is regulated by phosphate concentrations. Two forms of EHAP1 with virtually identical sequence are expressed on the cell surface of E. huxleyi , with the bigger form probably being the precursor of the smaller form since the AP activity correlates only with the abundance of the smaller form. The ehap1 gene is highly conserved in various E. huxleyi strains with less than 0.5% nucleic acid substitution. The hydrolytic kinetics of AP is similar among E. huxleyi strains but different from other five phytoplankton species tested. No other phytoplankton species seem to have this gene.

CDCA1 from T. weissflogii was overexpressed and crystallized. The crystal structure reveals that CDCA1 is a structural mimicry of a functional ?-CA dimer, with highly similar spatial organization of the active site residues, despite a lack of sequence homology between them. Although CDCA1 was originally isolated as a Cd enzyme, Cd at its active site is readily exchanged by Zn and vice versa. The conformational change of the active site pocket between metal-free and metal-bound forms provides the structural basis for such facile metal exchange. The Zn form of CDCA1 has higher catalytic efficiency than the Cd form, yet the latter can still satisfy the catalytic need for fast growing diatoms.

Overall, this thesis contributes to our understanding of the effect of metal replacement on phytoplankton physiology and ecology, and of the link between the biogeochemical cycles of major nutrients and trace elements in the oceans.