Single-mutation fitness landscapes for an enzyme on multiple substrates reveal specificity is globally encoded.


Our lack of total understanding of the intricacies of how enzymes behave has constrained our ability to robustly engineer substrate specificity. Furthermore, the mechanisms of natural evolution leading to improved or novel substrate specificities are not wholly defined. Here we generate near-comprehensive single-mutation fitness landscapes comprising >96.3% of all possible single nonsynonymous mutations for hydrolysis activity of an amidase expressed in E. coli with three different substrates. For all three selections, we find that the distribution of beneficial mutations can be described as exponential, supporting a current hypothesis for adaptive molecular evolution. Beneficial mutations in one selection have essentially no correlation with fitness for other selections and are dispersed throughout the protein sequence and structure. Our results further demonstrate the dependence of local fitness landscapes on substrate identity and provide an example of globally distributed sequence-specificity determinants for an enzyme.

Submission Details

ID: aX45V7uD

Submitter: Shu-Ching Ou

Submission Date: Jan. 31, 2019, 5:38 p.m.

Version: 1

Publication Details
Wrenbeck EE;Azouz LR;Whitehead TA,Nat Commun (2017) Single-mutation fitness landscapes for an enzyme on multiple substrates reveal specificity is globally encoded. PMID:28585537
Additional Information

NS = Nonsense mutations, nd = not determined. For wild-type, the measured Km are 4.7±0.5 mM (ACT), 52.7±8.3 mM (PR), 297.2±54.8 mM (IB). Measured kcat are 59.0±2.0 s-1 (ACT), 144.7±9.9 s-1 (PR), 13.3±1.1 s-1 (IB).

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)

Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
2UXY 2007-04-02T00:00:00+0000 1.25 Aliphatic amidase

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
91.2 Aliphatic amidase A5W2C0 AMIE_PSEP1
99.1 Aliphatic amidase A6V262 AMIE_PSEA7
99.7 Aliphatic amidase Q02QK0 AMIE_PSEAB
99.7 Aliphatic amidase B7V2X1 AMIE_PSEA8
100.0 Aliphatic amidase P11436 AMIE_PSEAE