Bjarni Ásgeirsson

Science Institute, Department of Biochemistry,

Dunhaga 3, IS-107 Reykjavik.

   

 

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ORCID iD iconorcid.org/0000-0002-4275-2732
ResearcherID: E-3747-2015
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Cold-adaptation

Organisms living in the cold environments of the earth produce special enzymes. The enzymes from these so-called psychrophilic organisms must be able to perform their catalysis efficiently at very low temperatures where enzymes from mesophilic or thermophilic species are generally unable to sustain viable metabolism.  However, they often cannot survive at moderate and high temperatures.

Increased thermolability together with the increased catalytic efficiency at low temperatures is a common feature of these cold-active enzymes. Is there a link?

The sensitivity of the active conformation of cold-active enzymes to heating is believed to be a consequence of reduction in the weak bonds in the local sphere around the active site. This enhances structural flexibility so the catalytic mechanism does not freeze. 

Exactly how this flexibility is brought about is not fully understood. It is probably achieved with different strategies in different enzyme families. 

Cold-active alkaline phosphatase (AP) from a Vibrio sp. isolated at Grótta in Seltjarnarnes near Reykjavik (photo) is very thermolabile.  Our results have shown that a single amino acid substitution in the active site is sufficient to alter the structural stability as well as kinetic properties. Also, introducing a single disulfide bond results in higher stability and much reduced activity.   Similar chartacteristics are found with AP from Atlantic cod (Gadus morhua).   A new avenue for our research is to use electron paramagnetic resonance to monitor local flexibility.

Membrane Rafts

Alkaline phosphatase is an enzyme with a wide distribution in the biosphere. Yet, its role is unknown in many instances.  In animals, the enzyme is bound on the outside of cell membranes with a phosphatidylinositolglycan (GPI) anchor.  The enzyme is a glycoprotein but the benefit of the sugar chains is unknown. 

GPI-anchored enzymes are associated with lipid-rafts, a specific collection of certain types of lipid molecules and proteins in biological membranes that can be isolated specifically.  We intend to isolate the phosphatase with its associated membrane molecules and analyze those in the hope of gaining a better idea regarding its role.  We also intend to produce the non-glycosylated enzyme and measure if the sugars have effect on basic properties of the enzyme.  Mass spectrometry will be employed in the proteomics work and sequencing of glycans.  Nearest-neighbor relationships within the membrane proteins (and lipids) in the intestinal-lining of Atlantic cod are presently unknown and may have much wider releveance for membrane architecture and dynamic interactions related to transport and signalling.


Promiscuous hydrolysis of p-nitrophenyl sulfate by Vibrio alkaline phosphatase

Most modern enzymes are highly specific and catalyse reactions that they are optimized through evolution to facilitate. However, some of them have promiscuous activity which is usually several magnitudes lower than the native activity (1-4). 

Earlier results showed low sulfatase activity in the alkaline phosphatase from E. coli (ECAP) (2). Here, we compared a cold-active Vibrio alkaline phosphatase (VAP) with ECAP. We also performed four residue replacements in VAP in order to simulate the electrostatic potential in the active site of E. coli AP. Computational calculations were used in order to predict which mutations were necessary and that might enhance promiscuous activity in VAP.

The results showed that although initially there was no measurable arylsulfafatase activity in VAP, it appeared after 24 hours on storage at room temperature. Increased arylsulfatase activity was concomitant with reduced phosphatase activity over one day at room temperature. This was dependent on NaCl, being slowed-down by 500 mM NaCl.

The quadruple VAP variant, T112A/R113E/H116D/W274K, that brought VAP residues in line with those found in ECAP, had a similar sulfatase activity as the wild-type VAP, although heat stability of the active site (T50%) was significantly increased.

Metal ion analysis (Zn/Mg) suggested that Mg ions played a part in determining selective action of VAP. This ion is known to have lesser affinity for APs than the two zinc ions, and may leach out of the VAP with time under certain conditions.

(1) Jensen, R. A. (1976) Ann Rev Microbiol 30, 409-425; (2) O'Brien, P. J., and Herschlag, D. (1999) Chem & Biology 6, R91-R105; (3) Khersonsky, O., and Tawfik, D. S. (2010 Ann Rev Biochem 79, 471-505; (4) Pabis, A. and S. C. L. Kamerlin (2016). Curr Opin Structural Biol 37: 14-21.

 

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