I04-Macromolecular Crystallography
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Amelia K.
Gilio
,
Thomas W.
Thorpe
,
Alex
Heyam
,
Mark R.
Petchey
,
Balázs
Pogrányi
,
Scott P.
France
,
Roger M.
Howard
,
Michael J.
Karmilowicz
,
Russell
Lewis
,
Nicholas
Turner
,
Gideon
Grogan
Diamond Proposal Number(s):
[18598]
Open Access
Abstract: Imine reductases (IREDs) catalyze the asymmetric reduction of cyclic imines, but also in some cases the coupling of ketones and amines to form secondary amine products in an enzyme-catalyzed reductive amination (RedAm) reaction. Enzymatic RedAm reactions have typically used small hydrophobic amines, but many interesting pharmaceutical targets require that larger amines be used in these coupling reactions. Following the identification of IR77 from Ensifer adhaerens as a promising biocatalyst for the reductive amination of cyclohexanone with pyrrolidine, we have characterized the ability of this enzyme to catalyze couplings with larger bicyclic amines such as isoindoline and octahydrocyclopenta(c)pyrrole. By comparing the activity of IR77 with reductions using sodium cyanoborohydride in water, it was shown that, while the coupling of cyclohexanone and pyrrolidine involved at least some element of reductive amination, the amination with the larger amines likely occurred ex situ, with the imine recruited from solution for enzyme reduction. The structure of IR77 was determined, and using this as a basis, structure-guided mutagenesis, coupled with point mutations selecting improving amino acid sites suggested by other groups, permitted the identification of a mutant A208N with improved activity for amine product formation. Improvements in conversion were attributed to greater enzyme stability as revealed by X-ray crystallography and nano differential scanning fluorimetry. The mutant IR77-A208N was applied to the preparative scale amination of cyclohexanone at 50 mM concentration, with 1.2 equiv of three larger amines, in isolated yields of up to 93%.
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Jan 2023
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[9948]
Open Access
Abstract: Unspecific peroxygenases (UPOs) have emerged as valuable tools for the oxygenation of non-activated carbon atoms, as they exhibit high turnovers, good stability and depend only on hydrogen peroxide as the external oxidant for activity. However, the isolation of UPOs from their natural fungal sources remains a barrier to wider application. We have cloned the gene encoding an ‘artificial’ peroxygenase (artUPO), close in sequence to the ‘short’ UPO from Marasmius rotula (MroUPO), and expressed it in both the yeast Pichia pastoris and E. coli to compare the catalytic and structural characteristics of the enzymes produced in each system. Catalytic efficiency for the UPO substrate 5-nitro-1,3-benzodioxole (NBD) was largely the same for both enzymes, and the structures also revealed few differences apart from the expected glycosylation of the yeast enzyme. However, the glycosylated enzyme displayed greater stability, as determined by nano differential scanning fluorimetry (nano-DSF) measurements. Interestingly, while artUPO hydroxylated ethylbenzene derivatives to give the (R)- alcohols, also given by a variant of the ‘long’ UPO from Agrocybe aegerita (AaeUPO), it gave the opposite (S)-series of sulfoxide products from a range of sulfide substrates, broadening the scope for application of the enzymes. The structures of artUPO reveal substantial differences to that of AaeUPO, and provide a platform for investigating the distinctive activity of this and related ’short’ UPOs.
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Nov 2022
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I03-Macromolecular Crystallography
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Vanessa
Harawa
,
Thomas W.
Thorpe
,
James R.
Marshall
,
Jack J.
Sangster
,
Amelia K.
Gilio
,
Lucian
Pirvu
,
Rachel S.
Heath
,
Antonio
Angelastro
,
James D.
Finnigan
,
Simon J.
Charnock
,
Jordan W.
Nafie
,
Gideon
Grogan
,
Roger C.
Whitehead
,
Nicholas J.
Turner
Diamond Proposal Number(s):
[24948]
Open Access
Abstract: The development of efficient and sustainable methods for the synthesis of nitrogen heterocycles is an important goal for the chemical industry. In particular, substituted chiral piperidines are prominent targets due to their prevalence in medicinally relevant compounds and their precursors. A potential biocatalytic approach to the synthesis of this privileged scaffold would be the asymmetric dearomatization of readily assembled activated pyridines. However, nature is yet to yield a suitable biocatalyst specifically for this reaction. Here, by combining chemical synthesis and biocatalysis, we present a general chemo-enzymatic approach for the asymmetric dearomatization of activated pyridines for the preparation of substituted piperidines with precise stereochemistry. The key step involves a stereoselective one-pot amine oxidase/ene imine reductase cascade to convert N-substituted tetrahydropyridines to stereo-defined 3- and 3,4-substituted piperidines. This chemo-enzymatic approach has proved useful for key transformations in the syntheses of antipsychotic drugs Preclamol and OSU-6162, as well as for the preparation of two important intermediates in synthetic routes of the ovarian cancer monotherapeutic Niraparib.
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Nov 2022
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[24948]
Open Access
Abstract: Native Amine Dehydrogenases (nat-AmDHs) are NAD(P)H-enzymes performing reductive amination, mainly active towards small aliphatic aldehydes and cyclic ketones, due to active site volumes limited by the presence of several bulky amino acids. Herein, inspired by the diversity of residues at these positions among the family, we report the implementation of mutations F140A and W145A in Cfus AmDH and their transposition in nine other members. Moderate to high conversions were obtained with substrates not accepted by the native enzymes, notably n -alkylaldehydes (44.6% - 99.5% for hexanal to nonanal) and n -alkylketones (16.0% - 53.7% for hexan-2-one to nonan-2-one) with retention of excellent ( S )-enantioselectivity (>99% ee) . Complementary to the reported ( R )-selective AmDHs, the promising mutant Cfus AmDH-W145A was further characterized for its synthetic potential. Crystal structure resolution and molecular dynamics gave insights into the cofactor and substrate specificity and the whole structural dynamics, thus providing keys for mutagenesis work on this enzyme family.
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Aug 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[9948]
Open Access
Abstract: The asymmetric reduction of ketones to chiral hydroxyl compounds by alcohol dehydrogenases (ADHs) is an established strategy for the provision of valuable precursors for fine chemicals and pharmaceutics. However, most ADHs favor linear aliphatic and aromatic carbonyl compounds, and suitable biocatalysts with preference for cyclic ketones and diketones are still scarce. Among the few candidates, the alcohol dehydrogenase from Thauera aromatica (ThaADH) stands out with a high activity for the reduction of the cyclic α-diketone 1,2-cyclohexanedione to the corresponding α-hydroxy ketone. This study elucidates catalytic and structural features of the enzyme. ThaADH showed a remarkable thermal and pH stability as well as stability in the presence of polar solvents. A thorough description of the substrate scope combined with the resolution and description of the crystal structure, demonstrated a strong preference of ThaADH for cyclic α-substituted cyclohexanones, and indicated structural determinants responsible for the unique substrate acceptance.
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May 2022
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I03-Macromolecular Crystallography
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Thomas W.
Thorpe
,
James R.
Marshall
,
Vanessa
Harawa
,
Rebecca E.
Ruscoe
,
Anibal
Cuetos
,
James D.
Finnigan
,
Antonio
Angelastro
,
Rachel S.
Heath
,
Fabio
Parmeggiani
,
Simon J.
Charnock
,
Roger M.
Howard
,
Rajesh
Kumar
,
David S. B.
Daniels
,
Gideon
Grogan
,
Nicholas J.
Turner
Diamond Proposal Number(s):
[9948]
Abstract: Chiral amine diastereomers are ubiquitous in pharmaceuticals and agrochemicals1, yet their preparation often relies on low-efficiency multi-step synthesis2. These valuable compounds must be manufactured asymmetrically, as their biochemical properties can differ based on the chirality of the molecule. Herein we characterize a multifunctional biocatalyst for amine synthesis, which operates using a mechanism that is, to our knowledge, previously unreported. This enzyme (EneIRED), identified within a metagenomic imine reductase (IRED) collection3 and originating from an unclassified Pseudomonas species, possesses an unusual active site architecture that facilitates amine-activated conjugate alkene reduction followed by reductive amination. This enzyme can couple a broad selection of α,β-unsaturated carbonyls with amines for the efficient preparation of chiral amine diastereomers bearing up to three stereocentres. Mechanistic and structural studies have been carried out to delineate the order of individual steps catalysed by EneIRED, which have led to a proposal for the overall catalytic cycle. This work shows that the IRED family can serve as a platform for facilitating the discovery of further enzymatic activities for application in synthetic biology and organic synthesis.
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Apr 2022
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[24948]
Open Access
Abstract: Native Amine Dehydrogenases (nat-AmDHs) have recently emerged as a potentially valuable new reservoir of enzymes for the sustainable and selective synthesis of chiral amines, catalyzing the NAD(P)H-dependent ammoniation of carbonyl compounds with high activity and selectivity. MATOUAmDH2, recently identified from the Marine Atlas of Tara Oceans Unigenes (MATOUv1) database of eukaryotic genes, displays exceptional catalytic performance against its best identified substrate, isobutyraldehyde, as well as broader substrate scope than other nat-AmDHs. In the interests of providing a platform for the rational engineering of this and other nat-AmDHs, we have determined the structure of MATOUAmDH2 in complex with NADP + and also with the cofactor and cyclohexylamine. Monomers within the structure are representative of more open and closed conformations of the enzyme and illustrate the profound changes undergone by nat-AmDHs during the catalytic cycle. An alanine screen of active site residues revealed that M215A and L180A are more active than the wild-type enzyme for the amination of cyclohexanone with ammonia and methylamine respectively, the latter suggesting that AmDHs have the potential to be engineered for the improved production of secondary amines.
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Mar 2022
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[9948]
Open Access
Abstract: Imine reductases (IREDs) offer biocatalytic routes to chiral amines and have a natural preference for the NADPH cofactor. In previous work, we reported enzyme engineering of the ( R )-selective IRED from Myxococcus stipitatus (( R )-IRED- Ms _V8) yielding a NADH-dependent variant with high catalytic efficiency. However, no IRED with NADH specificity and ( S )-selectivity in asymmetric reductions has yet been reported. Herein, we applied semi-rational enzyme engineering to switch the selectivity of ( R )-IRED- Ms _V8. The quintuple variant A241V/H242Y/N243D/V244Y/A245L showed reverse stereopreference in the reduction of the cyclic imine 2-methylpyrroline compared to the wild-type and afforded the ( S )-amine product with >99% conversion and 91% enantiomeric excess. We also report the crystal-structures of the NADPH-dependent ( R )-IRED- Ms wild-type enzyme and the NADH-dependent ( R )-IRED- Ms _V8 variant and molecular dynamics (MD) simulations to rationalize the inverted stereoselectivity of the quintuple variant.
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Oct 2021
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[9948]
Open Access
Abstract: Cytochrome P450 CYP153AM.aq from Marinobacter aquaeolei serves as a model enzyme for the terminal (ω-) hydroxylation of medium- to long-chain fatty acids. We have engineered this enzyme using different mutagenesis approaches based on structure-sequence-alignments within the 3DM database and crystal structures of CYP153AM.aq and a homologue CYP153AP.sp. Applying these focused mutagenesis strategies and site-directed saturation mutagenesis, we created a variant that ω-hydroxylates octanoic acid. The M.aqRLT variant exhibited 151-fold improved catalytic efficiency and showed strongly improved substrate binding (25-fold reduced Km compared to the wild type). We then used molecular dynamics simulations to gain deeper insights into the dynamics of the protein. We found the tunnel modifications and the two loop regions showing greatly reduced flexibility in the engineered variant were the main features responsible for stabilizing the enzyme–substrate complex and enhancing the catalytic efficiency. Additionally, we showed that a previously known fatty acid anchor (Q129R) interacts significantly with the ligand to hold it in the reactive position, thereby boosting the activity of the variant M.aqRLT toward octanoic acid. The study demonstrates the significant effects of both substrate stabilization and the impact of enzyme flexibility on catalytic efficiency. These results could guide the future engineering of enzymes with deeply buried active sites to increase or even establish activities toward yet unknown types of substrates.
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Feb 2021
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[9948]
Abstract: The interconversion of non-activated alkenes and alcohols, catalyzed by (de)hydratases, has great potential in biotechnology for the generation of fine and bulk chemicals. LinD is a cofactor-independent enzyme that catalyzes the reversible (de)hydration of the tertiary alcohol (S)-linalool to the triene β-myrcene, and also its isomerization to the primary alcohol geraniol. Structure-informed mutagenesis of LinD, followed by activity studies, confirmed essential roles for residues C171, C180 and H129 in water activation for the hydration of β-myrcene to linalool. However, no evidence of covalent thioterpene intermediates was found using either X-ray crystallography, mass spectrometry, or QM/MM nudged elastic band simula-tions. Labelling and NMR experiments confirmed a role for residue D39 in (de)protonation of the linalool carbon C10 in the isomerization of linalool to geraniol and also the intermediacy of β-myrcene in this isomerization reaction. X-ray, molecular dynamics and activity studies also suggested a significant role in catalysis for a mobile methionine residue M125, which exists in substantially altered orientations in different mutant structures.
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Aug 2020
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