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Reaction pathway engineering converts a radical hydroxylase into a halogenase

Nature Chemical Biology volume 18, pages 171–179 (2022)Cite this article

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Abstract

FeII/α-ketoglutarate (FeII/αKG)-dependent enzymes offer a promising biocatalytic platform for halogenation chemistry owing to their ability to functionalize unactivated C–H bonds. However, relatively few radical halogenases have been identified to date, limiting their synthetic utility. Here, we report a strategy to expand the palette of enzymatic halogenation by engineering a reaction pathway rather than substrate selectivity. This approach could allow us to tap the broader class of FeII/αKG-dependent hydroxylases as catalysts by their conversion to halogenases. Toward this goal, we discovered active halogenases from a DNA shuffle library generated from a halogenase–hydroxylase pair using a high-throughput in vivo fluorescent screen coupled to an alkyne-producing biosynthetic pathway. Insights from sequencing halogenation-active variants along with the crystal structure of the hydroxylase enabled engineering of a hydroxylase to perform halogenation with comparable activity and higher selectivity than the wild-type halogenase, showcasing the potential of harnessing hydroxylases for biocatalytic halogenation.

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Fig. 1: Identification of a closely related halogenase and hydroxylase pair.
Fig. 2: Crystal structure of Hydrox.
Fig. 3: Strategy for screening library variants.
Fig. 4: Screen identifies key residues.
Fig. 5: Engineering halogenase activity using insights from the screen and structure.

Data availability

Accession codes for proteins in this study are as follows: Hal, WP_122981682; Hydrox, WP_107105619. The PDB accession code for the structure of the hydroxylase from Streptomyces sp. MBT76 (Hydrox, WP_107105619) is 7JSD. The PDB accession code for BesD is 6NIE. NMR data and shuffle sequencing data are also provided. Any additional datasets from the supplemental information generated during and/or analyzed during the current study are available from the corresponding author on request. Source data are provided with this paper.

Code availability

The code used to analyze the shuffle data is included in the Supplementary Information.

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Acknowledgements

This work was supported by DOE/LBNL DEAC02-05CH11231, FWP CH030201. M.E.N. acknowledges the support of a National Science Foundation Graduate Research Fellowship. E.N.K. acknowledges the support of a National Institutes of Health NRSA Training Grant (1 T32 GMO66698). J.A.M. acknowledges the support of a UC Berkeley Chancellor’s Fellowship, Howard Hughes Medical Institute Gilliam Fellowship and National Institutes of Health NRSA Training Grant (1 T32 GMO66698). X-ray data were collected at the Advanced Light Source Beamline 8.3.1, which is operated by the University of California Office of the President, Multicampus Research Programs and Initiatives (MR-15- 328599), the National Institutes of Health (R01 GM124149 and P30 GM124169), Plexxikon and the Integrated Diffraction Analysis Technologies program of the US Department of Energy Office of Biological and Environmental Research. The Advanced Light Source is a national user facility operated by Lawrence Berkeley National Laboratory on behalf of the US Department of Energy under contract number DEAC02-05CH11231, Office of Basic Energy Sciences. The funds for the 900 MHz NMR spectrometer housed in the QB3 Institute in Stanley Hall at University of California, Berkeley, were kindly provided by the NIH (GM68933).

Author information

Authors and Affiliations

  1. Department of Chemical & Biomolecular Engineering, University of California, Berkeley, Berkeley, CA, USA

    Monica E. Neugebauer, Jorge A. Marchand, Douglas C. Millar & Michelle C. Y. Chang

  2. Department of Chemistry, University of California, Berkeley, Berkeley, CA, USA

    Elijah N. Kissman & Michelle C. Y. Chang

  3. QB3 Institute, University of California, Berkeley, Berkeley, CA, USA

    Jeffrey G. Pelton

  4. Department of Molecular & Cell Biology, University of California, Berkeley, Berkeley, CA, USA

    Nicholas A. Sambold & Michelle C. Y. Chang

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  1. Monica E. Neugebauer
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Contributions

M.E.N. performed enzyme characterization experiments, library generation, screen development, library analysis and assisted with protein crystallography. E.N.K. performed protein crystallography and enzyme characterization experiments. J.A.M. and D.C.M. contributed to screen development. J.G.P. performed NMR experiments. N.A.S. assisted with enzyme characterization experiments. M.C.Y.C. designed experiments and administered the project. M.E.N. and M.C.Y.C. wrote the paper with contributions from all authors.

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Correspondence to Michelle C. Y. Chang.

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Extended data

Extended Data Fig. 1 Divergent mechanisms of FeII/αKG-dependent halogenases and hydroxylases.

FeII/αKG-dependent halogenases and hydroxylases are mechanistically related. Upon oxygen binding, both enzymes decarboxylate αKG to generate a FeIV-oxo intermediate for abstracting a hydrogen atom from the substrate. However, differences in the primary coordination sphere of iron contribute to the subsequent reaction outcome. In hydroxylases, iron is coordinated by Asp or Glu, and hydroxyl rebound with the substrate radical leads to hydroxylation. In contrast, halogenases lack Asp or Glu in the primary coordination sphere and instead have Gly or Ala, leaving space for chloride to coordinate to Fe. Although rebound of either the halogen or the hydroxyl is possible, halogenases have evolved to favor halogen rebound.

Extended Data Fig. 2 Sequence alignment of BesD, Hal, and Hydrox.

(A) Sequence identity matrix for the halogenases from S. cattleya (BesD) and A. teichomyceticus (Hal, WP_122981682), and the hydroxylase from S. sp. MBT76 (Hydrox, WP_107105619). (B) Sequence alignment of BesD, Hal, and Hydrox highlighting the HXG and HXD motifs.

Extended Data Fig. 3 Steady-state kinetic analysis of Hal and Hydrox.

The amino acid lysine was used as a substrate. Data are mean ± sd (n = 3 technical replicates). Table contains kcat, KM, and kcat/KM calculated by non-linear curve fitting to the Michaelis-Menten equation. Data in the table (kinetic parameters) are mean ± s.e. Error in kcat/KM is obtained by propagation from the individual kinetic terms.

Source data

Extended Data Fig. 4 Results of screening the shuffle library with CuAAC.

Cells expressing BesB, BesC, and a member of the shuffle library are grown in M9 media supplemented with 0.5 mM lysine for two days. Controls are present on each plate as follows: Hydrox (A1-4), Hydrox D144G (A5-8), Hal (A9-12). After two days of expression, cells were pelleted by centrifugation and the supernatant was transferred to a fresh plate and mixed 1:1 with CuAAC solution to yield the final reaction mixture (CalFluor 488 azide (1 μM), copper (II) sulfate (0.5 mM), BTTAA (0.1 mM), and sodium ascorbate (5 mM)). Following incubation in the dark for 15 min, the fluorescence was measured using a SynergyMx Microplate Reader (BioTek) at room temperature (λex = 485 nm, λem = 528 nm). Values for each well are reported relative to the average of wells A1-4 (Hydrox) on each plate.

Source data

Extended Data Fig. 5 Purification and LC/MS analysis of Hal G144D I151N N225V.

(A) SDS-PAGE gel of purified Hal G144D I151N N225V. Protein purification was performed once. (B) Extracted ion chromatograms of LC/MC analysis of chlorolysine (m/z = 181.0738) and hydroxylysine (m/z = 163.1077) produced by Hal, Hydrox, and Hal G144D I151N N225V. Reactions contained Fe, lysine, NaCl, αKG, and purified enzyme variants and proceeded for 25 min before quenching in methanol containing 1% (v/v) formic acid to precipitate protein prior to LC/MS analysis. The Hal triple mutant produces no chlorolysine as expected given the G144D mutation likely abolishes chloride binding. A 3-fold increase in hydroxylysine production is observed in Hal G144D I151N N225V the compared to wild-type Hal, which remains 1.5-fold lower than wild-type Hydrox.

Source data

Extended Data Fig. 6 Purification and LC/MS analysis of HalI151 variants.

(A) SDS-PAGE gel of purified Hal and Hydrox variants. Lane 8, Hal I151V; 9, Hal I151A; 10, Hal I151N. Protein purification was performed once. (B) LC/MS analysis of chlorolysine (red, m/z = 181.0738) and hydroxylysine (black, m/z = 163.1077) produced by Hal, Hal I151V, Hal I151A, Hal I151N, and Hydrox. Reactions containing Fe, lysine, NaCl, αKG, and purified enzyme variants proceeded for 25 min before quenching in methanol containing 1% (v/v) formic acid to precipitate protein prior to LC/MS analysis. Mean and standard error are shown for n = 3 technical replicates. Selectivity values are reported in Supplementary Table 3.

Source data

Extended Data Fig. 7 Designing a chimera based on the shuffle library.

To design an enzyme which captures the key sequence motifs required for halogenation, a chimeric protein was engineered based on the shuffle results. (A) Conservation of halogenase residues that were conserved in library members capable of performing halogenation. Residues that were conserved in 100% of the halogenating hits were grafted into the sequence of the hydroxylase. All the residues that differ between the Chi-14 and Hydrox are listed below the sequence map. Key primary and secondary sphere residues are highlighted in green. (B) Sequence alignment of Hydrox (blue), Hal (red), and the engineered Chi-14.

Extended Data Fig. 8 Steady-state kinetic analysis of Hydrox triple mutant and Chi-14.

The amino acid lysine was used as a substrate. Data are mean ± sd (n = 3 technical replicates). Table contains kcat, KM, and kcat/KM calculated by non-linear curve fitting to the Michaelis-Menten equation. Data in the table (kinetic parameters) are mean ± s.e. Error in kcat/KM is obtained by propagation from the individual kinetic terms.

Source data

Extended Data Fig. 9 Total turnover number (TTN) and succinate coupling ratios for Hal, Hydrox, Hydrox D144G N151I V225N, and Chi-14.

Reactions (50 µL) contained l-lysine (4-7 mM), sodium αKG (10 mM), sodium ascorbate (5 mM), (NH4)2Fe(SO4)2 · 6H2O (1 mM), and sodium chloride (10 mM) in 100 mM MOPS buffer (pH 7.5). Reactions were initiated by addition of purified Hal or Hydrox variants (12.5 or 25 µM final concentration) and allowed to proceed for 24 hours at room temperature before quenching in 2.5 vol of methanol with 1% (v/v) formic acid. Following centrifugation at 20,000 × g for 20 min (at 4 °C) to remove precipitated protein, lysine and succinate concentrations were analyzed by LC/MS. (A) Standard curve for lysine quantification by LC/MS. Mean and standard error are shown for n = 3 technical replicates. (B) Lysine turnover for Hydrox, Hal, Hydrox D144G N151I V225N, and Chi-14. Mean and standard error are shown for n = 3 technical replicates. (C) Standard curve for succinate quantification by LC/MS. Mean and standard error are shown for n = 3 technical replicates. (D) Succinate turnover for Hydrox, Hal, Hydrox D144G N151I V225N, and Chi-14. Mean and standard error are shown for n = 3 technical replicates.

Source data

Extended Data Fig. 10 Purification and LC/MS analysis of Hydrox and Chi-14 variants.

(A) SDS-PAGE gel of purified Hydrox D144G N151I V225N (triple mutant, HydroxTM) and Chi-14 variants. Lane 11, Chi-14 L150F; 12, Chi-14 ST218-219GAV; 13, Chi-14 I222M; 14, Chi-14 M225V T226S; 15, Chi-14 A228S; 16, Chi-14 K230E E234G D236V; 17, HydroxTM F150L; 18, HydroxTM GAV218/219 to ST; 19, HydroxTM M223I; 20, HydroxTM V226M S227T; 21, HydroxTM S229A; 22, HydroxTM E231K G235E V237D. Proteins were purified once. (B) LC/MS analysis of chlorolysine (red, m/z = 181.0738) and hydroxylysine (black, m/z = 163.1077) produced by Hydrox and Chi-14 variants. The variants 11-22 correspond to the purified proteins in (A). An asterisk and the corresponding dotted line provide points of comparison for Chi-14 mutants that diminish activity relative to Chi-14 or Hydrox D144G N151I V225N (HydroxTM) mutants that increase activity relative to the triple mutant, Hydrox D144G N151I V225N. Reactions containing Fe, lysine, NaCl, αKG, and purified enzyme variants proceeded for 25 min before quenching in methanol containing 1% (v/v) formic acid to precipitate protein prior to LC/MS analysis. Mean and standard error are shown for n = 3 technical replicates. Selectivity values are reported in Supplementary Table 3.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–11, Tables 1–6, Notes 1–3.

Reporting Summary

Supplementary Data 1

NMR analysis of 4-hydroxylysine produced by Hydrox.

Supplementary Data 2

NMR analysis of 2-amino-4-hydroxy-ε-lactam produced by PfHalA.

Supplementary Data 3

NMR analysis of 2-amino-4-hydroxy-ε-lactam produced by Chi-14.

Supplementary Data 4

Raw sequencing data used to generate Fig. 4.

Source data

Source Data Fig. 1

LC–MS data.

Source Data Fig. 3

Fluorescence plate reader data.

Source Data Fig. 4

Residue conservation scatter data.

Source Data Fig. 5

LC–MS data, integrated EIC.

Source Data Extended Data Fig. 3

Michaelis–Menten kinetics data.

Source Data Extended Data Fig. 4

Fluorescence plate reader data, CuAAC.

Source Data Extended Data Fig. 5

LC–MS data.

Source Data Extended Data Fig. 6

LC–MS data, integrated EIC.

Source Data Extended Data Fig. 8

Michaelis–Menten kinetics data.

Source Data Extended Data Fig. 9

Succinate coupling data.

Source Data Extended Data Fig. 10

LC–MS data, integrated EIC.

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Neugebauer, M.E., Kissman, E.N., Marchand, J.A. et al. Reaction pathway engineering converts a radical hydroxylase into a halogenase. Nat Chem Biol 18, 171–179 (2022). https://doi.org/10.1038/s41589-021-00944-x

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  • DOI: https://doi.org/10.1038/s41589-021-00944-x

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