I03-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[20303]
Open Access
Abstract: Ene reductases (EREDs) catalyze asymmetric reduction with exquisite chemo-, stereo-, and regioselectivity. Recent discoveries led to unlocking other types of reactivities toward oxime reduction and reductive C–C bond formation. Exploring nontypical reactions can further expand the biocatalytic knowledgebase, and evidence alludes to yet another variant reaction where flavin mononucleotide (FMN)-bound ERs from the old yellow enzyme family (OYE) have unconventional activity with α,β-dicarbonyl substrates. In this study, we demonstrate the nonconventional stereoselective monoreduction of α,β-dicarbonyl to the corresponding chiral hydroxycarbonyl, which are valuable building blocks for asymmetric synthesis. We explored ten α,β-dicarbonyl aliphatic, cyclic, or aromatic compounds and tested their reduction with five OYEs and one nonflavin-dependent double bond reductase (DBR). Only GluER reduced aliphatic α,β-dicarbonyls, with up to 19% conversion of 2,3-hexanedione to 2-hydroxyhexan-3-one with an R-selectivity of 83% ee. The best substrate was the aromatic α,β-dicarbonyl 1-phenyl-1,2-propanedione, with 91% conversion to phenylacetylcarbinol using OYE3 with R-selectivity >99.9% ee. Michaelis–Menten kinetics for 1-phenyl-1,2-propanedione with OYE3 gave a turnover kcat of 0.71 ± 0.03 s–1 and a Km of 2.46 ± 0.25 mM. Twenty-four EREDs from multiple classes of OYEs and DBRs were further screened on 1-phenyl-1,2-propanedione, showing that class II OYEs (OYE3-like) have the best overall selectivity and conversion. EPR studies detected no radical signal, whereas NMR studies with deuterium labeling indicate proton incorporation at the benzylic carbonyl carbon from the solvent and not the FMN hydride. A crystal structure of OYE2 with 1.5 Å resolution was obtained, and docking studies showed a productive pose with the substrate.
|
Oct 2024
|
|
I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Diamond Proposal Number(s):
[28402, 12633]
Open Access
Abstract: Different strategies have previously been reported to convert cytochrome P450 monooxygenases to peroxygenases, allowing H2O2-driven oxyfunctionalization reactions. Comparison of the BM3 (CYP102A1) peroxygenase variant 21B3, obtained through enlargement of the active site by the F87A mutation followed by mutational stabilization towards H2O2, with the BM3_T268E variant, with an acid-base catalyst introduced, showed 21B3 to be the superior peroxygenase. A combination of these two strategies (21B3_T268E combinatorial mutant), however, resulted in reduced peroxygenase activity. The further introduction of the F(A)87V and A328F mutations (87–328 variants), previously reported to improve the regioselectivity of BM3 on n-alkanes, resulted in a loss of activity towards dodecanoic acid, a substrate commonly used to evaluate the activity of BM3. Although a reduction in activity was observed for styrene, the combinatorial mutants yielded higher enantioselectivities for R-styrene oxide. The activity towards α-olefins were, however, comparable between the different peroxygenase variants of BM3, but with absolute selectivity for epoxidation observed with the combinatorial mutants 21B3_87-328 and 21B3_T268E_87-382. Structural investigation of the active site architecture showed significant differences in the I-helix and heme accessibility. According to these results, future directed evolution studies will require selective pressure not only for H2O2, but also the specific substrate to be activated rather than surrogate substrates.
|
Mar 2024
|
|
I24-Microfocus Macromolecular Crystallography
|
Diamond Proposal Number(s):
[28402]
Open Access
Abstract: Cytochrome P450 monooxygenases (CYPs) are biocatalysts able to catalyze a variety of regio- and stereoselective oxyfunctionalization reactions using an iron(IV)-oxo porphyrin pi-cation radical prosthetic group, commonly referred to as compound I (Cpd I). The formation of Cpd I is, however, dependent on molecular oxygen and the sequential transfer of electrons from expensive nicotinamide cofactors via additional redox partner proteins. Recently, CYPs have been engineered to introduce or enhance peroxygenase activity, whereby Cpd I is formed from H2O2. Here we explore the potential of natural CYPs containing an aspartate instead of the conventional threonine on the distal side of the heme, for peroxygenase activity. Peroxygenase activity was demonstrated with three new CYPs, with SscaCYP from Streptomyces scabiei demonstrating the highest activity towards the hydroxylation of trans-β-methyl styrene and the sulfoxidation of thioanisole. The X-ray crystal structure of SscaCYP revealed two potential acid–base catalysts (D241 and E284) in close proximity to the axial water molecule for the heterolytic O–O cleavage during H2O2 activation for Cpd I formation. Both side chain groups are also located at H2O channels to the bulk solvent. Mutagenesis of either side chain increased the peroxygenase activity, whereas removal of both abolished the peroxygenase activity of SscaCYP. Spectral analysis confirmed the tight binding of the axial water molecule when both carboxylate groups are present. With only one carboxylate group present lower concentrations of H2O2 are also required for catalysis, suggesting H2O and H2O2 exchange as a rate limiting factor in the wild-type SscaCYP. The improved peroxygenase activity and increased substrate scope of the SscaCYP variants, have identified two “hot-spots” for future protein engineering with one an alternative site for the introduction of an acid–base catalyst for engineering peroxygenase activity in other CYPs.
|
Sep 2023
|
|
I03-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[20303]
Open Access
Abstract: Selective oxyfunctionalization of non-activated C–H bonds remains a major challenge in synthetic chemistry. The biocatalytic hydroxylation of non-activated C–H bonds by cytochrome P450 monooxygenases (CYPs), however, offers catalysis with high regio- and stereoselectivity using molecular oxygen. CYP153s are a class of CYPs known for their selective terminal hydroxylation of n-alkanes and microorganisms, such as the bacterium Alcanivorax dieselolei, have evolved extensive enzymatic pathways for the oxyfunctionalization of various lengths of n-alkanes, including a CYP153 to yield medium-chain 1-alkanols. In this study, we report the characterization of the terminal alkane hydroxylase from A. dieselolei (CYP153A71) for the oxyfunctionalization of medium-chain n-alkanes in comparison to the well-known CYP153A6 and CYP153A13. Although the expected 1-alkanols are produced, CYP153A71 readily converts the 1-alkanols to the corresponding aldehydes, fatty acids, as well as α,ω-diols. CYP153A71 is also shown to readily hydroxylate medium-chain fatty acids. The X-ray crystal structure of CYP153A71 bound to octanoic acid is solved, yielding an insight into not only the regioselectivity, but also the binding orientation of the substrate, which can be used in future studies to evolve CYP153A71 for improved oxidations beyond terminal n-alkane hydroxylation.
|
Oct 2022
|
|
I03-Macromolecular Crystallography
|
Radoslaw
Nowak
,
Anthony
Tumber
,
Eline
Hendrix
,
Mohammad Salik Zeya
Ansari
,
Manuela
Sabatino
,
Lorenzo
Antonini
,
Regina
Andrijes
,
Eidarus
Salah
,
Nicola
Mautone
,
Francesca Romana
Pellegrini
,
Klemensas
Simelis
,
Akane
Kawamura
,
Catrine
Johansson
,
Daniela
Passeri
,
Roberto
Pellicciari
,
Alessia
Ciogli
,
Donatella
Del Bufalo
,
Rino
Ragno
,
Mathew L.
Coleman
,
Daniela
Trisciuoglio
,
Antonello
Mai
,
Udo
Oppermann
,
Christopher J.
Schofield
,
Dante
Rotili
Diamond Proposal Number(s):
[10619]
Open Access
Abstract: MINA53 is a JmjC domain 2-oxoglutarate-dependent oxygenase that catalyzes ribosomal hydroxylation and is a target of the oncogenic transcription factor c-MYC. Despite its anticancer target potential, no small-molecule MINA53 inhibitors are reported. Using ribosomal substrate fragments, we developed mass spectrometry assays for MINA53 and the related oxygenase NO66. These assays enabled the identification of 2-(aryl)alkylthio-3,4-dihydro-4-oxoypyrimidine-5-carboxylic acids as potent MINA53 inhibitors, with selectivity over NO66 and other JmjC oxygenases. Crystallographic studies with the JmjC demethylase KDM5B revealed active site binding but without direct metal chelation; however, molecular modeling investigations indicated that the inhibitors bind to MINA53 by directly interacting with the iron cofactor. The MINA53 inhibitors manifest evidence for target engagement and selectivity for MINA53 over KDM4–6. The MINA53 inhibitors show antiproliferative activity with solid cancer lines and sensitize cancer cells to conventional chemotherapy, suggesting that further work investigating their potential in combination therapies is warranted.
|
Nov 2021
|
|
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[20303]
Open Access
Abstract: CYP505A30 is a fungal, self-sufficient cytochrome P450 monooxygenase that can selectively oxyfunctionalise n-alkanes, fatty alcohols, and fatty acids. From alkanes, it produces a mixture of non-vicinal diols by two sequential hydroxylation reactions. Here we report the structure of the haem domain of CYP505A30, the first structure for a member of the CYP505 family, with dodecanoic acid bound within the active site. Overall, a high structural similarity to the related bacterial CYP102A1 was observed, despite low sequence identity (<40 %). Comparison of the active sites, however, showed a high degree of conservation with only two amino acid differences close to the haem. Stabilisation of the acid substrate in CYP505A30 also occurs, as in CYP102A1, via an arginine residue. However, compared to R47, which is situated in the β1 region of CYP102A1, R358 is located in the β3 region of CYP505A30. We furthermore created mutants to test if it is possible to rationally transfer the knowledge on active site mutations in CYP102A1 to change the regioselectivity of CYP505A30. The introduction of F93V, I334F mutations resulted in increased ω-1 (C2) regioselectivity, similar to CYP102A1 328-87, of more than 80 % for n-octane and 90 % for n-decane. Changing residues to resemble the 102A1 wildtype increased the regioselectivity towards ω-2 (C3) to over 60 % for both substrates. The knowledge gained from this study unlocks a more selective production of symmetrical non-vicinal diols from n-alkanes.
|
Oct 2021
|
|
I04-1-Macromolecular Crystallography (fixed wavelength)
|
Diamond Proposal Number(s):
[15292]
Abstract: We report the crystal structure of the copper-containing nitrite reductase (NirK) from the Gram-negative bacterium Sinorhizobium meliloti 2011 (Sm), together with complex structural alignment and docking studies with both non-cognate and the physiologically-related pseudoazurins, SmPaz1 and SmPaz2, respectively. S. meliloti is a rhizobacterium used for the formulation of Medicago sativa bionoculants, and SmNirK plays a key role in this symbiosis through the denitrification pathway. The structure of SmNirK, solved at a resolution of 2.5 å, showed a striking resemblance with the overall structure of the well-known class I NirKs composed of two Greek key β-barrel domains. The activity of SmNirK is ~12% of the activity reported for classical NirKs, which could be attributed to several factors such as subtle structural differences in the secondary proton channel, solvent accessibility of the substrate channel, and that the denitrifying activity has to be finely regulated within the endosymbiont. In vitro kinetics performed in homogenous and heterogeneous media showed that both SmPaz1 and SmPaz2, which are coded in different regions of the genome, donate electrons to SmNirK with similar performance. Even though the energetics of the interprotein electron transfer (ET) process is not favorable with either electron donors, adduct formation mediated by conserved residues allows minimizing the distance between the copper centers involved in the interprotein ET process.
|
Sep 2021
|
|
I04-Macromolecular Crystallography
|
Minkyung
Baek
,
Frank
Dimaio
,
Ivan
Anishchenko
,
Justas
Dauparas
,
Sergey
Ovchinnikov
,
Gyu Rie
Lee
,
Jue
Wang
,
Qian
Cong
,
Lisa N.
Kinch
,
R. Dustin
Schaeffer
,
Claudia
Millán
,
Hahnbeom
Park
,
Carson
Adams
,
Caleb R.
Glassman
,
Andy
Degiovanni
,
Jose H.
Pereira
,
Andria V.
Rodrigues
,
Alberdina A.
Van Dijk
,
Ana C.
Ebrecht
,
Diederik J.
Opperman
,
Theo
Sagmeister
,
Christoph
Buhlheller
,
Tea
Pavkov-Keller
,
Manoj K.
Rathinaswamy
,
Udit
Dalwadi
,
Calvin K.
Yip
,
John E.
Burke
,
K. Christopher
Garcia
,
Nick V.
Grishin
,
Paul D.
Adams
,
Randy J.
Read
,
David
Baker
Diamond Proposal Number(s):
[20303]
Abstract: DeepMind presented remarkably accurate predictions at the recent CASP14 protein structure prediction assessment conference. We explored network architectures incorporating related ideas and obtained the best performance with a three-track network in which information at the 1D sequence level, the 2D distance map level, and the 3D coordinate level is successively transformed and integrated. The three-track network produces structure predictions with accuracies approaching those of DeepMind in CASP14, enables the rapid solution of challenging X-ray crystallography and cryo-EM structure modeling problems, and provides insights into the functions of proteins of currently unknown structure. The network also enables rapid generation of accurate protein-protein complex models from sequence information alone, short circuiting traditional approaches which require modeling of individual subunits followed by docking. We make the method available to the scientific community to speed biological research.
|
Jul 2021
|
|
I03-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[12633]
Abstract: We report an engineered panel of ene-reductases (ERs) from Thermus scotoductus SA-01 (TsER) that combines control over facial selectivity in the reduction of electron deficient Cdouble bondC double bonds with thermostability (up to 70 °C), organic solvent tolerance (up to 40 % v/v) and a broad substrate scope (23 compounds, three new to literature). Substrate acceptance and facial selectivity of 3-methylcyclohexenone was rationalized by crystallisation of TsER C25D/I67T and in silico docking. The TsER variant panel shows excellent enantiomeric excess (ee) and yields during bi-phasic preparative scale synthesis, with isolated yield of up to 93 % for 2R,5S-dihydrocarvone (3.6 g). Turnover frequencies (TOF) of approximately 40 000 h−1 were achieved, which are comparable to rates in hetero- and homogeneous metal catalysed hydrogenations. Preliminary batch reactions also demonstrated the reusability of the reaction system by consecutively removing the organic phase (n-pentane) for product removal and replacing with fresh substrate. Four consecutive batches yielded ca. 27 g L−1 R-levodione from a 45 mL aqueous reaction, containing less than 17 mg (10 μM) enzyme and the reaction only stopping because of acidification. The TsER variant panel provides a robust, highly active and stereocomplementary base for further exploitation as a tool in preparative organic synthesis.
|
Feb 2021
|
|
I04-1-Macromolecular Crystallography (fixed wavelength)
|
Diamond Proposal Number(s):
[18938]
Open Access
Abstract: Baeyer-Villiger monooxygenases (BVMOs) are flavin-dependent enzymes that primarily convert ketones to esters, but can also catalyze heteroatom oxidation. Several structural studies have highlighted the importance of the ‘control loop’ in BVMOs, which adopts different conformations during catalysis. Central to the ‘control loop’ is a conserved tryptophan that has been implicated in NADP(H) binding. BVMOAFL210 from Aspergillus flavus, however, contains a threonine in the equivalent position. Here, we report the structure of BVMOAFL210 in complex with NADP+ in both the ‘open’ and ‘closed’ conformations. In neither conformation does Thr513 contact the NADP+. Although mutagenesis of Thr513 did not significantly alter the substrate scope, changes in peroxyflavin stability and reaction rates were observed. Mutation of this position also brought about changes in the regio- and enantioselectivity of the enzyme. Moreover, lower rates of overoxidation during sulfoxidation of thioanisole were also observed.
|
Mar 2020
|
|
