The high catalytic rate of the cold-active Vibrio alkaline phosphatase requires a hydrogen bonding network involving a large interface loop
| Autor: | Jens Guðmundur Hjörleifsson, Manuela Magnusdottir, Ronny Helland, Bjarni Ásgeirsson |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Reaction mechanism Dimer Acclimatization Crystallography X-Ray enzyme catalysis General Biochemistry Genetics and Molecular Biology Catalysis Enzyme catalysis 03 medical and health sciences chemistry.chemical_compound Residue (chemistry) 0302 clinical medicine psychrophilic enzyme Bacterial Proteins Catalytic Domain Enzyme Stability Amino Acid Sequence Research Articles Thermostability Vibrio biology VDP::Mathematics and natural science: 400::Chemistry: 440 Active site Hydrogen Bonding Alkaline Phosphatase intersubunit interactions thermostability Cold Temperature flexible loop 030104 developmental biology Monomer chemistry VDP::Matematikk og Naturvitenskap: 400::Kjemi: 440 030220 oncology & carcinogenesis biology.protein Biophysics Biocatalysis Research Article |
| Zdroj: | FEBS Open Bio |
| Popis: | This study highlights the importance of a hydrogen bonding network between the subunits in a homodimeric enzyme involving a large loop for maintaining a high catalytic rate. Although increased local flexibility in the opposite subunit increased when the network was broken by producing a loop variant, the catalytic rate was halved, contrary to expectations. The role of surface loops in mediating communication through residue networks is still a relatively poorly understood part in the study of cold adaptation of enzymes, especially in terms of their quaternary interactions. Alkaline phosphatase (AP) from the psychrophilic marine bacterium Vibrio splendidus (VAP) is characterized by an analogous large surface loop in each monomer, referred to as the large loop, that hovers over the active site of the other monomer. It presumably has a role in the high catalytic efficiency of VAP which accompanies its extremely low thermal stability. Here, we designed several different variants of VAP with the aim of removing intersubunit interactions at the dimer interface. Breaking the intersubunit contacts from one residue in particular (Arg336) reduced the temperature stability of the catalytically potent conformation and caused a 40% drop in catalytic rate. The high catalytic rates of enzymes from cold‐adapted organisms are often associated with increased dynamic flexibility. Comparison of the relative B‐factors of the R336L crystal structure to that of the wild‐type confirmed surface flexibility was increased in a loop on the opposite monomer, but not in the large loop. The increase in flexibility resulted in a reduced catalytic rate. The large loop increases the area of the interface between the subunits through its contacts and may facilitate an alternating structural cycle demanded by a half‐of‐sites reaction mechanism through stronger ties, as the dimer oscillates between high affinity (active) or low phosphoryl group affinity (inactive). |
| Databáze: | OpenAIRE |
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