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Asgeirsson

Manuela Magnúsdóttir

Alkaline phosphatase

Life at extreme conditions, such as at high or low temperatures, calls for proteins that can work properly in those particular environmental conditions. Cold-adapted enzymes are interesting because of their high catalytic activity compared to mesophilic and thermophilic homologes.

Alkaline phosphatase (AP) from a particular Vibrio psychrophilic marine bacteria catalyzes the hydrolysis of various phosphate esters. The family of alkaline phosphatases is especially interesting for its diversity in the biosphere. The enzyme is a dimer but each monomer is quite different in length, from 355 residues in an Antarctic variant to 521 in the variant we are studying. Since the core structure is very similar, the differences appear in the form of longer surface loops. In the Vibrio variant, each monomer has a very long surface loop that holds onto the next monomer.

In this project, we are studying the role of some of these surface loops. In many cold-adapted enzymes, the surface loops are found to be longer compared to proteins of mesophilic organisms. This cannot be the whole story in the case of alkaline phosphatase, since the longest and shortest versions known are both cold-adapted.

Two point mutations (Y346F and R336L) and two different deletions of residues have been performed on the largest surface loop of Vibrio AP. Y346F caused insignificant changes in kinetic rate constants and heat-stability, as only one hydrogen bond had been broken. In constrast, four hydrogens bonds were broken with the R336L mutation, and there was a drastic decrease in heat-stability accompanied by higher kcat. No big changes were observed in Km values. Heat tolerance of the total conformation as monitored by circular dichroism was similar for both point mutations and the wild-type enzyme.

Overall conclusions so far ...

• Point-mutations in the large loop did not make a major difference for global stability of the protein but clearly affected active site integrity and catalytic efficiency.

• Removing four hydrogen bonds connecting the major overlapping loop to the other subunit´s protein surface produced higher catalytic activity and, therefore, showed functional connections between the loop and the active site that are being studied further.

• We also believe that here is some cooperation between subunits in catalysis that Arg336 can affect. Further mutants are being studied to pinpoint this more accurately.

OVERVIEW

Y346F removes one hydrogen bond.
- Pink shows residues that are part of the large surface loop on Vibrio alkaline phosphatase.

R336L removes four bonds.
- Pink shows residues that are part of the large surface loop on Vibrio alkaline phosphatase.
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Overview of the Vibrio alkaline phosphatase structure.
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Helland, R., R. L. Larsen, Asgeirsson, B. (2009). "The 1.4 Å crystal structure of the large and cold-active Vibrio sp. alkaline phosphatase." Biochim Biophys Acta 1794(2): 297-308.
Alkaline phosphatase (AP) from the cold-adapted Vibrio strain G15-21 is among the AP variants with the highest known k(cat) value. Here the structure of the enzyme at 1.4 A resolution is reported and compared to APs from E. coli, human placenta, shrimp and the Antarctic bacterium strain TAB5. The Vibrio AP is a dimer although its monomers are without the long N-terminal helix that embraces the other subunit in many other APs. The long insertion loop, previously noted as a special feature of the Vibrio AP, serves a similar function. The surface does not have the high negative charge density as observed in shrimp AP, but a positively charged patch is observed around the active site that may be favourable for substrate binding. The dimer interface has a similar number of non-covalent interactions as other APs and the "crown"-domain is the largest observed in known APs. Part of it slopes over the catalytic site suggesting that the substrates may be small molecules. The catalytic serines are refined with multiple conformations in both monomers. One of the ligands to the catalytic zinc ion in binding site M1 is directly connected to the crown-domain and is closest to the dimer interface. Subtle movements in metal ligands may help in the release of the product and/or facilitate prior dephosphorylation of the covalent intermediate. Intersubunit interactions may be a major factor for promoting active site geometries that lead to the high catalytic activity of Vibrio AP at low temperatures."

100305/Básg