Structure and Mechanism of a Cold-Adapted Bacterial Lipase

van der Ent, FlorianUppsala universitet,Beräkningsbiologi och bioinformatik (författare)

The structural origin of enzyme cold-adaptation has been the subject of considerable research efforts in recent years. Comparative studies of orthologous mesophilic-psychrophilic enzyme pairs found in nature are an obvious strategy for solving this problem, but they often suffer from relatively low sequence identity of the enzyme pairs. Small bacterial lipases adapted to distinctly different temperatures appear to provide an excellent model system for these types of studies, as they may show a very high degree of sequence conservation. Here, we report the first crystal structures of lipase A from the psychrophilic bacterium Bacillus pumilus, which confirm the high structural similarity to the mesophilic Bacillus subtilis enzyme, as indicated by their 81% sequence identity. We further employ extensive QM/MM calculations to delineate the catalytic reaction path and its energetics. The computational prediction of a rate-limiting deacylation step of the enzymatic ester hydrolysis reaction is verified by stopped-flow experiments, and steady-state kinetics confirms the psychrophilic nature of the B. pumilus enzyme. These results provide a useful benchmark for examining the structural basis of cold-adaptation and should now make it possible to disentangle the effects of the 34 mutations between the two enzymes on catalytic properties and thermal stability.

Lund, Bjarte A.Uppsala universitet,Institutionen för cell- och molekylärbiologi,Arctic Univ Norway, Univ Tromso, Dept Chem, Hylleraas Ctr Quantum Mol Sci, N-9037 Tromso, Norway. (författare)
Svalberg, LinnUppsala universitet,Institutionen för cell- och molekylärbiologi(Swepub:uu)linsv807 (författare)
Purg, MihaUppsala universitet,Institutionen för cell- och molekylärbiologi(Swepub:uu)mihpu392 (författare)
Chukwu, GhisleanUppsala universitet,Biokemi(Swepub:uu)ghich737 (författare)
Widersten, MikaelUppsala universitet,Biokemi(Swepub:uu)mikawide (författare)
V. Isaksen, GeirArctic Univ Norway, Univ Tromso, Dept Chem, Hylleraas Ctr Quantum Mol Sci, N-9037 Tromso, Norway. (författare)
Brandsdal, Bjørn O.Arctic Univ Norway, Univ Tromso, Dept Chem, Hylleraas Ctr Quantum Mol Sci, N-9037 Tromso, Norway. (författare)
Åqvist, JohanUppsala universitet,Beräkningsbiologi och bioinformatik,Arctic Univ Norway, Univ Tromso, Dept Chem, Hylleraas Ctr Quantum Mol Sci, N-9037 Tromso, Norway.(Swepub:uu)johanaq (författare)
Uppsala universitetBeräkningsbiologi och bioinformatik (creator_code:org_t)

Ingår i:Biochemistry: American Chemical Society (ACS)61:10, s. 933-9420006-29601520-4995

Av författaren/redakt...: van der Ent, Flo ...; Lund, Bjarte A.; Svalberg, Linn; Purg, Miha; Chukwu, Ghislean; Widersten, Mikae ...; visa fler...; V. Isaksen, Geir; Brandsdal, Bjørn ...; Åqvist, Johan; visa färre...

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