TY - JOUR
T1 - Cold-Active Starch-Degrading Enzymes from a Cold and Alkaline Greenland Environment
T2 - Role of Ca2+ Ions and Conformational Dynamics in Psychrophilicity
AU - Bendtsen, Malthe Kjær
AU - Nowak, Jan Stanislaw
AU - Paiva, Pedro
AU - López Hernández, Marcos
AU - Ferreira, Pedro
AU - Pedersen, Jan Skov
AU - Bekker, Nicolai Sundgaard
AU - Viezzi, Elia
AU - Bisiak, Francesco
AU - Brodersen, Ditlev E
AU - Pedersen, Lars Haastrup
AU - Zervas, Athanasios
AU - Fernandes, Pedro A
AU - Ramos, Maria Joao
AU - Stougaard, Peter
AU - Thøgersen, Mariane Schmidt
AU - Otzen, Daniel E
PY - 2025/3/14
Y1 - 2025/3/14
N2 - Cold-active enzymes hold promise for energy-efficient processes. Amylases are widely used in household and industrial applications, but only a few are cold-active. Here we describe three novel secreted amylases, Rho13, Ika2 and I3C6, all from bacteria growing in the cold and alkaline ikaite columns in Greenland. They all hydrolyzed starch to smaller malto-oligomers, but only Rho13 and Ika2 hydrolyzed cyclodextrins, and only Ika2 displayed transglycosylation activity. Ika2 forms a stable dimer, while both Rho13 and I3C6 are mainly monomeric. They all have optimal active temperatures around 30-35 °C and significant enzymatic activity below 20 °C, but Rho13 and I3C6 had an alkaline optimal pH, while Ika2 was markedly acidophilic. They showed complex dependence on Ca
2+ concentration, with the activity of Rho13 and I3C6 following a bell-shaped curve and Ika2 being unaffected; however, removal of Ca
2+ reduced the stability of all three enzymes. Loss of structure occurred well above the temperature of optimal activity, showing the characteristic psychrophilic divorce between activity and stability. MD simulations showed that Ika2 did not have a well-defined Ca
2+ binding site, while Rho13 and I3C6 both maintained one stably bound Ca
2+ ion. We identified psychrophilic features as higher levels of backbone fluctuations compared to mesophilic counterparts, based on a lower number of internal hydrogen bonds and salt bridges. This increased fluctuation was also found in regions outside the active site and may provide easier substrate access and accommodation, as well as faster barrier transitions. Our work sheds further light on the many ways in which psychrophilic enzymes adapt to increased catalysis at lower temperatures.
AB - Cold-active enzymes hold promise for energy-efficient processes. Amylases are widely used in household and industrial applications, but only a few are cold-active. Here we describe three novel secreted amylases, Rho13, Ika2 and I3C6, all from bacteria growing in the cold and alkaline ikaite columns in Greenland. They all hydrolyzed starch to smaller malto-oligomers, but only Rho13 and Ika2 hydrolyzed cyclodextrins, and only Ika2 displayed transglycosylation activity. Ika2 forms a stable dimer, while both Rho13 and I3C6 are mainly monomeric. They all have optimal active temperatures around 30-35 °C and significant enzymatic activity below 20 °C, but Rho13 and I3C6 had an alkaline optimal pH, while Ika2 was markedly acidophilic. They showed complex dependence on Ca
2+ concentration, with the activity of Rho13 and I3C6 following a bell-shaped curve and Ika2 being unaffected; however, removal of Ca
2+ reduced the stability of all three enzymes. Loss of structure occurred well above the temperature of optimal activity, showing the characteristic psychrophilic divorce between activity and stability. MD simulations showed that Ika2 did not have a well-defined Ca
2+ binding site, while Rho13 and I3C6 both maintained one stably bound Ca
2+ ion. We identified psychrophilic features as higher levels of backbone fluctuations compared to mesophilic counterparts, based on a lower number of internal hydrogen bonds and salt bridges. This increased fluctuation was also found in regions outside the active site and may provide easier substrate access and accommodation, as well as faster barrier transitions. Our work sheds further light on the many ways in which psychrophilic enzymes adapt to increased catalysis at lower temperatures.
KW - Bacterial Proteins/chemistry
KW - Calcium/metabolism
KW - Cold Temperature
KW - Enzyme Stability
KW - Greenland
KW - Hydrogen-Ion Concentration
KW - Hydrolysis
KW - Molecular Dynamics Simulation
KW - Protein Conformation
KW - Starch/metabolism
KW - molecular dynamics
KW - protein stability
KW - psychrophilic enzymes
UR - http://www.scopus.com/inward/record.url?scp=105001386587&partnerID=8YFLogxK
U2 - 10.3390/biom15030415
DO - 10.3390/biom15030415
M3 - Journal article
C2 - 40149951
SN - 2218-273X
VL - 15
JO - Biomolecules
JF - Biomolecules
IS - 3
M1 - 415
ER -