Promiscuous esterases counterintuitively are less flexible than specific ones

Abstract

34Understanding mechanisms of promiscuity is increasingly important from a fundamental and 35 application point of view. As to enzyme structural dynamics, more promiscuous enzymes 36 generally have been recognized to also be more flexible. However, examples for the opposite 37 received much less attention. Here, we exploit comprehensive experimental information on 38 the substrate promiscuity of 147 esterases tested against 96 esters together with 39 computationally efficient rigidity analyses to understand the molecular origin of the observed 40 promiscuity range. Unexpectedly, our data reveal that promiscuous esterases are significantly 41 less flexible than specific ones, are significantly more thermostable, and have a significantly 42 increased specific activity. These results may be reconciled with a model according to which 43 structural flexibility in the case of specific esterases serves for conformational proofreading. 44 Our results signify that esterase sequence space can be screened by rigidity analyses for 45 promiscuous esterases as starting points for further exploration in biotechnology and synthetic 46 chemistry. 47 48 3 1. Introduction 49 Enzymes involved in primary metabolism typically exquisitely discriminate against other 50metabolites. Yet, evolution of specificity is only pushed by nature to the point at which 51 'unauthorized' reactions do not impair the fitness of the organism (1). As a result, the universe 52 of promiscuous activities available in nature has been suggested to be enormous (2,3). 53 Understanding mechanisms of promiscuity has thus become increasingly important for the 54 fundamental understanding of molecular recognition and how enzyme function has evolved 55 over time(4) but also to optimize enzyme engineering applications (5). A particular challenge 56 in the latter case is the ability to discover a suitable enzyme with 'sufficient' promiscuous 57 activity to serve as a starting point for further exploration (1). 58Enzyme structural dynamics, besides its role in catalysis (6, 7) and allosteric regulation (8-59 11), has been recognized as likely the single most important mechanism by which promiscuity 60 can be achieved (5). Prominent examples are human cytochrome P450 (CYP) enzymes, for 61 which crystallographic studies and molecular simulations demonstrated that more 62 promiscuous CYPs show larger structural plasticity and mobility (12-14), or TEM-1 -63 lactamase and a resurrected progenitor, for which molecular simulations show that the pocket 64 of the ancestral, and more promiscuous, enzyme fluctuates to a greater extent (15). However, 65 examples for the opposite, i.e., conformational changes selected in evolution such that they 66 enhance specificity in molecular recognition (16), have received much less attention in the 67 context of enzyme promiscuity. 68A clear limitation for scrutinizing the link between enzyme structural dynamics and substrate 69 promiscuity is the general lack of large-scale data on one enzyme (super)family tested against 70 a multitude of ...

Can constraint network analysis guide the identification phase of KnowVolution? A case study on improved thermostability of an endo-β-glucanase

“…For identifying structural weak spots on EGLII, thermal unfolding simulations were carried out by CNA (section Constraint Network Analysis ) on structural ensembles of wildtype EGLII generated by MD simulations (section Generation of structural ensembles ), as done previously [21] , [31] , [34] , [43] . By visual inspection of the unfolding trajectory, four major phase transitions, T1 – T4, were identified ( Fig.…”

“…As done previously [43] , structural ensembles of wildtype EGLII (PDB ID 5L9C) were generated by all-atom MD simulations of in total 5 μs simulation time. For details on starting structure preparation, parametrization, equilibration, and production runs, see SI Method M1.…”

“…As done previously [43] , the thermal unfolding simulations of wildtype EGLII was performed with the Constraint Network Analysis (CNA) software package (version 3.0) [21] , [22] , [23] , [24] . For details on thermal unfolding simulations, see SI Method M2.…”

“…In the present study, we applied the neighbor stability map rc ij ,neighbor to investigate short-range rigid contacts. For this, as done previously [31] , [33] , [43] , rc ij was filtered such that only rigid contacts between two residues that are at most 5 Å apart from each other were considered.…”

General theory of specific binding: insights from a genetic-mechano-chemical protein model

“…This can be illustrated by two case studies that relate con icting accounts of the role of exibility in enzyme promiscuity: Flexibility and promiscuity are correlated in a group of 57 human cytochrome P450 (CYT) enzymes, 94 while the opposite trend is observed in a group of 147 esterases. 95 In the case of CYT, we know that the binding pocket is quite small (Δ𝜃 0 < 0), so we can infer that the proteins fall to the right of the optimal line (bottom purple arrow, Fig. 5D).…”