Reaction pathway engineering converts a radical hydroxylase into a halogenase

Neugebauer, Monica E; Kissman, Elijah N; Marchand, Jorge A.; Pelton, Jeffrey G.; Sambold, Nicholas A; Millar, Douglas C.; Chang, Michelle C Y

doi:10.1038/s41589-021-00944-x

Cited by 43 publications

(62 citation statements)

References 59 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This study also explores the function of H-bonding in the active sites of halogenases, and their implications on catalysis. Our results serve as a warning to others attempting to engineer non-heme iron enzymes for non-native functions, [38][39][40] as placement of redox-active residues can result in catalytically non-functional enzymes.…”

Section: Discussionmentioning

confidence: 74%

Role of a secondary coordination sphere residue in halogenation catalysis of non-heme iron enzymes

Wilson

Chatterjee

Smithwick

et al. 2022

Preprint

View full text Add to dashboard Cite

Chemo- and regio-selective catalysis of C(sp3)-H halogenation reaction is a formidable goal in chemical synthesis. 2-oxo-glutarate (2OG) dependent non-heme iron halogenases catalyze selective chlorination/bromination of C-H bonds and exhibit high sequence and structural similarities with non-heme iron hydroxylases. How the secondary coordination sphere (SCS) of these two enzyme systems differentiate and determine their reactivity is not understood. In this work, we show that tyrosine placement in the SCS of non-heme iron halogenases have a huge impact on their structure, function, and reactivity. We discover that a tyrosine mutant (F121Y) in SyrB2 halogenase undergoes post-translational oxidation to dihydroxyphenylalanine (DOPA) physiologically. A combination of spectroscopic, mass-spectrometric, and biochemical studies show that the DOPA modification in SyrB2 renders the enzyme non-functional. Further bioinformatics analysis suggests that halogenases, unlike hydroxylases, have a conserved placement of phenylalanine at position 121 to preclude such unproductive oxidation. Overall, this study demonstrates the importance of the SCS in controlling the structure and enzymatic activity of non-heme iron halogenases. Our results will have significant implications towards the design of small-molecule and protein-based halogenation catalysts.

show abstract

Section: Discussionmentioning

confidence: 74%

Role of a secondary coordination sphere residue in halogenation catalysis of non-heme iron enzymes

Wilson

Chatterjee

Smithwick

et al. 2022

Preprint

View full text Add to dashboard Cite

show abstract

“…(A) GFP (green) and Dronpa2 (gold) protein environments suppress excited-state isomerization of the chromophore to different degrees compared to that in vacuum (gray), rendering GFP less photoisomerizable than Dronpa2 (Figure D). (B) Y(M210)F mutant (purple) of Rhodobacter sphaeroides photosynthetic reaction center reveals that tyrosine at M210, which stabilizes the first intermediate, is in part responsible for the unidirectional excited-state electron transfer of wild type (orange). , (C) Wild-type Fe(II)/2-oxoglutarate (2OG)-dependent halogenases (orange) chlorinate their substrates, but their intrinsic hydroxylating power can be unleashed upon mutation (purple). ,, The default (blue) and the side pathways (red for all and green for panel A) are shown on the right and left for each panel, respectively. Energies are not drawn to scale.…”

Section: Resultsmentioning

confidence: 99%

“…The availability of different reaction pathways and the potential for pathway selection, existing for numerous ground-state and excited-state enzymes, − are primarily inscribed in the PES(s) of the chromophore/substrate and constrain how the PES(s) can be tuned in response to an environment, illustrated by diverse examples in Figure . On the basis of directed evolution studies on enzymes, new chemistries are not created out of nowhere but rather the substrates are found to already exhibit low reactivities toward the said chemistries. − Therefore, to rationally design enzymes that are superior at catalyzing a reaction, it is important to sample a wide range of perturbations to substrates (or chromophores capable of structural change) and the environment’s steric or electrostatic influences on the energetics of nonproductive yet competitive pathways rather than only those that exhibit more desirable phenotypes . Only when those less desirable cases are understood can we mechanistically deduce why the more productive pathway is not taken, guiding future design efforts to optimize the desired function.…”

Section: Discussionmentioning

confidence: 99%

“…62,63 (C) Wild-type Fe(II)/2-oxoglutarate (2OG)-dependent halogenases (orange) chlorinate their substrates, but their intrinsic hydroxylating power can be unleashed upon mutation (purple). 60,64,65 The default (blue) and the side pathways (red for all and green for panel A) are shown on the right and left for each panel, respectively. Energies are not drawn to scale.…”

Section: Predictive Model For Steric and Electrostaticmentioning

confidence: 99%

See 1 more Smart Citation

Energetic Basis and Design of Enzyme Function Demonstrated Using GFP, an Excited-State Enzyme

et al. 2022

View full text Add to dashboard Cite

The past decades have witnessed an explosion of de novo protein designs with a remarkable range of scaffolds. It remains challenging, however, to design catalytic functions that are competitive with naturally occurring counterparts as well as biomimetic or nonbiological catalysts. Although directed evolution often offers efficient solutions, the fitness landscape remains opaque. Green fluorescent protein (GFP), which has revolutionized biological imaging and assays, is one of the most redesigned proteins. While not an enzyme in the conventional sense, GFPs feature competing excited-state decay pathways with the same steric and electrostatic origins as conventional ground-state catalysts, and they exert exquisite control over multiple reaction outcomes through the same principles. Thus, GFP is an “excited-state enzyme”. Herein we show that rationally designed mutants and hybrids that contain environmental mutations and substituted chromophores provide the basis for a quantitative model and prediction that describes the influence of sterics and electrostatics on excited-state catalysis of GFPs. As both perturbations can selectively bias photoisomerization pathways, GFPs with fluorescence quantum yields (FQYs) and photoswitching characteristics tailored for specific applications could be predicted and then demonstrated. The underlying energetic landscape, readily accessible via spectroscopy for GFPs, offers an important missing link in the design of protein function that is generalizable to catalyst design.

show abstract

“…To probe the significance of Met246 on regioselectivity in HalB, we generated a M246 NNK library to determine whether mutagenesis could perturb the regioselectivity of HalB for modifying C 5 and result in chlorination of C 4 as well. To assay the variants, we employed a fluorogenic screen 69 based on the role of 4-Cl-lysine as an intermediate in the biosynthesis of propargylglycine (Pra), a terminal-alkyne containing amino acid ( Fig. 4A ) 58 .…”

Section: Resultsmentioning

confidence: 99%

Regioselective control of biocatalytic C–H activation and halogenation

Kissman¹,

Neugebauer²,

Sumida³

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

Biocatalytic C–H activation has the potential to merge enzymatic and synthetic strategies for bond formation. FeII/αKG-dependent halogenases are particularly distinguished for their ability both to control selective C-H activation as well as to direct group transfer of a bound anion along a reaction axis separate from oxygen rebound, enabling the development of new transformations. In this context, we elucidate the basis for selectivity of enzymes that perform selective halogenation to yield 4-Cl-lysine (BesD), 5-Cl-lysine (HalB), and 4-Cl-ornithine (HalD), allowing us to probe how regioselectivity and chain length selectivity are achieved. We now report the crystal structure of the HalB and HalD, revealing the key role of the substrate-lid in positioning the substrate for C4 vs C5 chlorination and recognition of lysine vs ornithine. Targeted engineering of the substrate-binding lid further demonstrates that these selectivities can be altered or switched, showcasing the potential to develop halogenases for biocatalytic applications.

show abstract

Reaction pathway engineering converts a radical hydroxylase into a halogenase

Cited by 43 publications

References 59 publications

Role of a secondary coordination sphere residue in halogenation catalysis of non-heme iron enzymes

Role of a secondary coordination sphere residue in halogenation catalysis of non-heme iron enzymes

Energetic Basis and Design of Enzyme Function Demonstrated Using GFP, an Excited-State Enzyme

Regioselective control of biocatalytic C–H activation and halogenation

Contact Info

Product

Resources

About