Computational stabilization of a non-heme iron enzyme enables efficient evolution of new function

Abstract

Directed evolution has emerged as a powerful tool for engineering new biocatalysts. However, introducing new catalytic residues can be destabilizing, and it is generally beneficial to start with a stable enzyme parent. Here we show that the deep learning-based tool ProteinMPNN can be used to redesign Fe(II)/αKG superfamily enzymes for greater stability, solubility, and expression while retaining both native activity and industrially-relevant non-native functions. For the Fe(II)/αKG enzyme tP4H, we performed site-saturation mutagenesis with both the wild-type and stabilized design variant and screened for activity increases in a non-native C-H hydroxylation reaction. We observed substantially larger increases in non-native activity for variants obtained from the stabilized scaffold compared to those from the wild-type enzyme. ProteinMPNN is user-friendly and widely-accessible, and straightforward structural criteria were sufficient to obtain stabilized, catalytically-functional variants of the Fe(II)/αKG enzymes tP4H and GriE. Our work suggests that stabilization by computational sequence redesign could be routinely implemented as a first step in directed evolution campaigns for novel biocatalysts.

Figure 1.

Initial whole-cell reaction screen data with a panel of Fe(II)/αKG amino acid hydroxylases and free acid substrate analogues. Reactions were performed in whole cell from 50 mL expression cultures where whole cell volume was 1/20^th the expression volume. Reactions were carried out in MOPS (pH 7.0, 50 mM) with 20 mM substrate, 60 mM αKG (as disodium salt), 1 mM ferrous ammonium sulfate, and 1 mM L-ascorbic acid.

Figure 2.

Validation of tP4H activity with free acid 1. (A) Reaction of tP4H with substrate 1 to form trans-4-hydroxycyclohexane carboxylic acid 4. (B) Yield of 4 after reaction of 1 with tP4H variants, as well as negative control reaction with Fe(II) and bovine serum albumin (BSA). The Fe 1 mM control was run in the absence of added enzyme. Purified enzyme concentration was varied between 10–40 μM with 20 mM 1, 40 mM αKG, 1 mM ferrous ammonium sulfate, and 1 mM L-ascorbic acid in MES buffer (50 mM, pH 6.8). Reactions were carried out for 24 hours at 25 °C and quantified with analytical LC-MS. (C) Structural model of the tP4H showing key active site residues. Fe(II), αKG, and L-Pipecolic acid were modeled in Chimera.

Figure 3.

Stability and activity of wild-type tP4H and ProteinMPNN design R2_11. (A) tP4H structure (Alphafold2 model, Figure S9) color-coded to show sites fixed in the design process (blue, Supplementary spreadsheet – ProteinMPNN sequences_metrics) and sites mutated in the ProteinMPNN R2_11 redesign (orange-red). Sites colored black were neither fixed nor redesigned in the R2_11 variant. Side chains for first shell active site residues (Table S4) are shown in blue. (B) Temperature-dependent CD spectroscopy of wild-type tP4H and R2_11. (C) Activity-stability analysis of wild-type tP4H. (D) Activity-stability analysis of R2_11. Relative activity was determined using PBP assay described in Supplementary Information.

Figure 4.

(A) Reaction scheme for C-H hydroxylation of substrate 1 with tP4H, R2_11, and associated variants. (B) Directed evolution of wild-type tP4H. (C) Directed evolution of stabilized variant R2_11. Reactions were carried out for 24 hr at 25 °C using purified enzyme (10–20 μM) in MES buffer (50 mM, pH 6.8), with 20 mM cyclohexane carboxylic acid 1, 40 mM αKG, 1 mM ferrous ammonium sulfate, and 1 mM ascorbic acid. Concentration of 4 in quenched reaction samples was quantified by analytical LC-MS analysis.

Figure 5

A) Temperature dependent CD of the R2_11 and tP4H triple mutants. B) TTN for formation of 4 (d.r. 4:1, Figure S4) with the R2_11 and tP4H triple mutants at two different temperatures. Reactions were carried out for 6 hrs. at 25 °C and 35 °C using purified enzyme (15 μM) in MES buffer (50 mM, pH 6.8), with 20 mM cyclohexane carboxylic acid 1, 40 mM αKG, 1 mM ferrous ammonium sulfate, and 1 mM ascorbic acid.