PDBsum entry 1c00

Hydrolase

PDB id: 1c00

Contents

Protein chain

483 a.a.

Metals

_CA

Waters ×203

References listed in PDB file

Key reference

Title

A structural view of evolutionary divergence.

Authors

B.Spiller, A.Gershenson, F.H.Arnold, R.C.Stevens.

Ref.

Proc Natl Acad Sci U S A, 1999, 96, 12305-12310. [DOI no: 10.1073/pnas.96.22.12305]

PubMed id

10535917

Abstract

Two directed evolution experiments on p-nitrobenzyl esterase yielded one enzyme with a 100-fold increased activity in aqueous-organic solvents and another with a 17 degrees C increase in thermostability. Structures of the wild type and its organophilic and thermophilic counterparts are presented at resolutions of 1.5 A, 1.6 A, and 2.0 A, respectively. These structures identify groups of interacting mutations and demonstrate how directed evolution can traverse complex fitness landscapes. Early-generation mutations stabilize flexible loops not visible in the wild-type structure and set the stage for further beneficial mutations in later generations. The mutations exert their influence on the esterase structure over large distances, in a manner that would be difficult to predict. The loops with the largest structural changes generally are not the sites of mutations. Similarly, none of the seven amino acid substitutions in the organophile are in the active site, even though the enzyme experiences significant changes in the organization of this site. In addition to reduction of surface loop flexibility, thermostability in the evolved esterase results from altered core packing, helix stabilization, and the acquisition of surface salt bridges, in agreement with other comparative studies of mesophilic and thermophilic enzymes. Crystallographic analysis of the wild type and its evolved counterparts reveals networks of mutations that collectively reorganize the active site. Interestingly, the changes that led to diversity within the alpha/beta hydrolase enzyme family and the reorganization seen in this study result from main-chain movements.

Figure 1.
Fig. 1. MOLSCRIPT diagrams (15, 16) of pNB esterases looking into the active site cavity, showing loops that are not visible in the electron density as dashed lines and loops that reorganize most significantly in gold. The catalytic triad is shown in red and mutations are shown in blue. (A) The WT structure with secondary structural elements labeled. (B) The 5-6c8 structure. (C) The 8g8 structure, rotated slightly from the others to clarify the location of the mutations. (D) A wall-eyed stereo overlay of the C[ ]positions of the three structures, oriented as in A and B. WT is shown in blue, organophile 5-6c8 in green, and thermophile 8g8 in purple.

Figure 3.
Fig. 3. Comparison between the thermophile 8g8 (purple) and WT (blue). (A) A superposition of the 315-324 and 265-275 loops. The His-322 Tyr mutation introduces a direct interaction between the loops. The 315-324 loop is pulled closer to the active site to accommodate the smaller substrate, allowing a 3.5-Å H bond between Tyr-322 and the main-chain N of Ile-270. Additionally, the new orientation of 315-324 allows an H bond between the side chains of Ser-323 and Thr-326, stabilizing helix 12 and the 315-324 loop. (B) A superposition of WT and 8g8 shows the effect of the Met-358 Val mutation. In the absence of the His-322 Arg mutation, the large reorganization of 265-275 is not seen. Leu-362 and Ile-270 move to fill the cavity created by the Met-358 Val mutation.