Role Of NC-loop In Catalytic Activity, Stability And Divergent Evolution In Lipase From Fervidobacterium Changbaicum

Loop was the important element of secondary structure. Flexible loops play importantroles in protein functions. The elegant catalytic mechanism of enzymes relies on mobility andflexibility of functional loops in many aspects, such as orientating important catalytic residues,participating in the substrate binding, and regulating the entrance of substrates and the releaseof products. It has been shown that the members of α/β hydrolase superfamily (lipase Vfamily, epoxide hydrolases, haloalkane dehalogenases, and perhydrolases) possessed theNC-loop connecting the N-terminal of the catalytic domain and the Cap domain. NC-loopswere diverse in the sequence, length, flexibility, and conformation. In this work, we took thethermostable lipase FClip1from Fervidobacterium changbaicum and belonging to lipase Vfamily as a research model, investigated systemically the role of NC-loop of the lipase FClip1on catalytic process, stability, and divergent evolution by protein engineering.The model of FClip1possesses a catalytic domain and a cap domain. NC-loop contains21residues (Asp131-Lys151). Based on the solvent accessibility of residues, NC-loop can beroughly divided into two parts. The N-terminal (Asp131-Ser138) is located in the proteininterior and participates in the formation of the substrate binding pocket, while the C-terminal(Glu139-Lys151) is a highly surface-exposed region. We designed and constructed a series ofmutants by systematically truncating the NC-loop from its N-terminal or C-terminal.Deletions from the N-terminal or C-terminal part of NC-loop had different effects on activity.Deletions from the N-terminal caused the enzyme to retain no more than1.5%activity of wildtype. Removing as small as three residues could greatly inactivate the enzyme. TheN-terminal was very important for catalytic activity of the enzyme. However, deletions fromthe C-terminal improved the catalytic activity, the thermal stability at78oC, and the resistanceagainst GdnHCl. It was suggested that the C-terminal was not involved in the enzyme catalysis.Ala-scanning mutagenesis on the N-terminal and saturation mutagenesis at the Tyr134were carried out to determine the important residues for enzyme catalysis. Enzyme assayexhibited that the introduction of alanine did not change the enzyme activity of FClip1. Thissuggested that the side chains of N-terminal residues retaining only a methyl group did notlose the catalytic activity of the enzyme. Besides, the enzymatic activity of the mutant Y134Ereduced about22-fold. It indicated that the importance of NC-loop for enzyme catalysis notonly reflected in the confomational integrity, but also was related with the amino acidcomposition. We also used circular permutation to probe the flexibility and plasticity ofNC-loop. The permutants CP146and CP152maintained high enzymatic activity. It showedthat the C-terminal of NC-loop was flexible and plastic, and might be a target region forenzyme redesign to optimize further the enzymatic properties.The NC-loop of FClip1shared some common features with those of other α/β hydrolases,such as participating the formation of substrate pocket and the entrance of substrate channel.There are also obvious differences, such as diverse length, sequence, conformation, andfunction. This suggested that NC-loop might play an important role in the divergent evolutionof α/β hydrolase superfamily. In addition, the sequence and length of NC-loop of FClip1weremore similar with perhydrolases, and FClip1displayed a low activity of perhydrolysis (3mU/mg). It indicated that FClip1might be the evolutionary intermediate between lipases andperhydrolases.