I04-Macromolecular Crystallography
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Mary
Ortmayer
,
Karl
Fisher
,
Jaswir
Basran
,
Emmanuel M.
Wolde-michael
,
Derren J.
Heyes
,
Colin
Levy
,
Sarah
Lovelock
,
J. L. Ross
Anderson
,
Emma L.
Raven
,
Sam
Hay
,
Stephen E. J.
Rigby
,
Anthony P.
Green
Diamond Proposal Number(s):
[12788]
Abstract: Nature employs a limited number of genetically encoded axial ligands to control diverse heme enzyme activities. Deciphering the functional significance of these ligands requires a quantitative understanding of how their electron donating capabilities modulate the structures and reactivities of the iconic ferryl intermediates compounds I and II. However, probing these relationships experimentally has proven challenging as ligand substitutions accessible via conventional mutagenesis do not allow fine tuning of electron donation and typically abolish catalytic function. Here we exploit engineered translation components to replace the histidine ligand of cytochrome c peroxidase (CcP) by a less electron donating Nδ-methyl histidine (Me-His) with little effect on enzyme structure. The rate of formation (k1) and the reactivity (k2) of compound I are unaffected by ligand substitution. In contrast, proton coupled electron transfer to compound II (k3) is 10-fold slower in CcP Me-His, providing a direct link between electron do-nation and compound II reactivity which can be explained by weaker electron donation from the Me-His ligand (‘the push’) affording an electron deficient ferryl-oxygen with reduced proton affinity (‘the pull’). The deleterious effects of the Me-His ligand can be fully compensated by introducing a W51F mutation, designed to increase ‘the pull’ by removing a hydrogen bond to the ferryl-oxygen. Analogous substitutions in ascorbate peroxidase (APX) lead to similar activity trends to those observed in CcP, suggesting a common mechanistic strategy is employed by enzymes using distinct electron transfer pathways. Our study highlights how non-canonical active site substitutions can be used to directly probe and deconstruct highly evolved bioinorganic mechanisms.
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Jan 2020
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[8997, 12788, 17773]
Open Access
Abstract: The UbiX-UbiD enzymes are widespread in microbes, acting in concert to decarboxylate alpha-beta unsaturated carboxylic acids using a highly modified flavin cofactor, prenylated FMN (prFMN). UbiX serves as the flavin prenyltransferase, extending the isoalloxazine ring system with a fourth non-aromatic ring, derived from sequential linkage between a dimethylallyl moiety and the FMN N5 and C6. Using structure determination and solution studies of both dimethylallyl monophosphate (DMAP) and dimethyallyl pyrophosphate (DMAPP) dependent UbiX enzymes, we reveal the first step, N5-C1’ bond formation, is contingent on the presence of a dimethylallyl substrate moiety. Hence, an SN1 mechanism similar to other prenyltransferases is proposed. Selected variants of the (pyro)phosphate binding site are unable to catalyse subsequent Friedel-Crafts alkylation of the flavin C6, but can be rescued by addition of (pyro)phosphate. Thus, retention of the (pyro)phosphate leaving group is required for C6-C3’ bond formation, resembling pyrophosphate initiated class I terpene cyclase reaction chemistry.
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May 2019
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Tobias
Hedison
,
Rajesh
Shenoy
,
Andreea Iulia
Iorgu
,
Derren
Heyes
,
Karl
Fisher
,
Gareth
Wright
,
Sam
Hay
,
Robert Roy
Eady
,
Svetlana
Antonyuk
,
S. Samar
Hasnain
,
Nigel S.
Scrutton
Diamond Proposal Number(s):
[11740]
Abstract: It is generally assumed that tethering enhances rates of electron harvesting and delivery to active sites in multi-domain enzymes by proximity and sampling mechanisms. Here, we explore this idea in a tethered 3-domain, trimeric copper-containing nitrite reductase. By reverse engineering, we find that tethering does not enhance the rate of electron delivery from its pendant cytochrome c to the catalytic copper-containing core. Using a linker that harbors a gatekeeper tyrosine in a nitrite access channel, the tethered haem domain enables catalysis by other mechanisms. Tethering communicates the redox state of the haem to the distant T2Cu center that helps initiate substrate binding for catalysis. It also tunes copper reduction potentials, suppresses reductive enzyme inactivation, enhances enzyme affinity for substrate and promotes inter-copper electron transfer. Tethering has multiple unanticipated beneficial roles, the combination of which fine-tunes function beyond simplistic mechanisms expected from proximity and restrictive sampling models.
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May 2019
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[12788, 17773]
Abstract: The biological production of FDCA is of considerable value as a potential replacement for petrochemical derived monomers such as terephthalate, used in polyethylene terephthalate (PET) plastics. HmfF belongs to an uncharacterised branch of the prenylated flavin (prFMN) dependent UbiD-family of reversible (de)carboxylases, and is proposed to convert 2,5-furan-dicarboxylic acid (FDCA) to furoic acid in vivo. We present detailed characterisation of HmfF and demonstrate that HmfF can catalyse furoic acid carboxylation at elevated CO2 levels in vitro. We report the crystal structure of a thermophilic HmfF from Pelotomaculum thermopropionicum, revealing the active site located above the prFMN cofactor contains a furoic acid/FDCA binding site composed of residues H296-R304-R331 specific to the HmfF branch of UbiD enzymes. Variants of the latter are compromised in activity, while H296N alters the substrate preference to pyrrol compounds. Solution studies and crystal structure determination of an engineered dimeric form of the enzyme revealed an unexpected key role for a UbiD-family wide conserved Leu residue in activity. The structural insights into substrate and cofactor binding provide a template for further exploitation of HmfF in the production of FDCA plastic precursors, and improve our understanding of catalysis by members of the UbiD enzyme family.
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Feb 2019
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8997]
Abstract: The UbiD family of reversible decarboxylases act on aromatic, heteroaromatic, and unsaturated aliphatic acids and utilize a prenylated flavin mononucleotide (prFMN) as cofactor, bound adjacent to a conserved Glu-ArgGlu/Asp ionic network in the enzyme's active site. It is proposed that UbiD activation requires oxidative maturation of the cofactor, for which two distinct isomers, prFMNketimine and prFMNiminium have been observed. It also has been suggested that only the prFMNiminium form is relevant to catalysis, which requires transient cycloaddition between substrate and cofactor. Using Aspergillus niger Fdc1 as a model system, we reveal isomerization of prFMNiminium to prFMNketimine is a light-dependent process that is largely independent of the Glu277-Arg173-Glu282 network and accompanied by irreversible loss of activity. On the other hand, efficient catalysis was highly dependent on an intact Glu-Ar-Glu network, as only Glu to Asp substitutions retain activity. Surprisingly, oxidative maturation to form the prFMNiminium species is severely affected only for the R173A variant. In summary, the unusual irreversible isomerization of prFMN is light dependent and likely proceeds via high-energy intermediates, but is independent of the Glu-Arg-Glu network. Our results from mutagenesis, crystallographic, spectroscopic and kinetic experiments indicate a clear role for the Glu-Arg-Glu network in both catalysis and oxidative maturation.
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Dec 2017
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I03-Macromolecular Crystallography
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Stefan
Payer
,
Stephen A.
Marshall
,
Natalie
Bärland
,
Xiang
Sheng
,
Tamara
Reiter
,
Andela
Dordic
,
Georg
Steinkellner
,
Christiane
Wuensch
,
Susann
Kaltwasser
,
Karl
Fisher
,
Stephen E. J.
Rigby
,
Peter
Macheroux
,
Janet
Vonck
,
Karl
Gruber
,
Kurt
Faber
,
Fahmi
Himo
,
David
Leys
,
Tea
Pavkov-keller
,
Silvia M.
Glueck
Diamond Proposal Number(s):
[12788]
Abstract: The utilization of CO2 as carbon source for organic syntheses meets the urgent demand of more sustainability in chemical production. Here we report on the enzyme-catalyzed para-carboxylation of catechols, employing 3,4-dihydroxybenzoic acid decarboxylases (AroY) that belong to the UbiD enzyme family. Crystal structures and accompanying solution data confirm that AroY utilizes the recently discovered prenylated FMN (prFMN) cofactor, and requires oxidative maturation to form the catalytically competent prFMNiminium species. This study reports on the in vitro reconstitution and activation of a prFMN-dependent enzyme that is capable of directly carboxylating aromatic catechol substrates under ambient conditions. A reaction mechanism for the reversible decarboxylation involving a monocovalent quinoid-cofactor intermediate is proposed, which is distinct from the prFMN associated 1,3 dipolar cycloaddition mechanism in related enzymes.
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Aug 2017
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[8997, 12788]
Abstract: The activity of the reversible decarboxylase enzyme Fdc1 is dependent on prenylated FMN (prFMN), a recently discovered cofactor. The oxidized prFMN supports a 1,3-dipolar cycloaddition mechanism that underpins reversible decarboxylation. Fdc1 is a distinct member of the UbiD family of enzymes, with the canonical UbiD catalyzing the (de)carboxylation of para-hydroxybenzoic acid-type substrates. Here we show that the Escherichia coli UbiD enzyme, which is implicated in ubiquinone biosynthesis, cannot be isolated in an active holo-enzyme form, despite the fact active holo-Fdc1 is readily obtained. Formation of holo-UbiD requires reconstitution in vitro of the apo-UbiD with reduced prFMN. Furthermore, while the Fdc1 apo-enzyme can be readily reconstituted and activated, in vitro oxidation to the mature prFMN cofactor stalls at formation of a radical prFMN species in holo-UbiD. Further oxidative maturation in vitro occurs only at alkaline pH, suggesting a proton-coupled electron transfer precedes formation of the fully oxidized prFMN. Crystal structures of holo-UbiD reveal a relatively open active site, potentially occluded from solvent through domain motion. The presence of a prFMN sulfite-adduct in one of the UbiD crystal structures confirms oxidative maturation does occur at ambient pH on a slow time scale. Activity could not be detected for a range of putative para-hydroxybenzoic acid substrates tested. However, the lack of an obvious hydrophobic binding pocket for the octaprenyl-tail of the proposed ubiquinone precursor substrate does suggest UbiD might act on a non-prenylated precursor. Our data reveals unexpected variation occurs in domain mobility, prFMN binding and maturation by the UbiD enzyme family.
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Jan 2017
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Mary
Ortmayer
,
Pierre
Lafite
,
Binuraj R. K.
Menon
,
Tewes
Tralau
,
Karl
Fisher
,
Lukas
Denkhaus
,
Nigel S.
Scrutton
,
Stephen E. J.
Rigby
,
Andrew W.
Munro
,
Sam
Hay
,
David
Leys
Diamond Proposal Number(s):
[8997]
Abstract: The universal Per-ARNT-Sim (PAS) domain functions as a signal transduction module involved in sensing diverse stimuli such as small molecules, light, redox state and gases. The highly evolvable PAS scaffold can bind a broad range of ligands, including haem, flavins and metal ions. However, although these ligands can support catalytic activity, to our knowledge no enzymatic PAS domain has been found. Here we report characterization of the first PAS enzyme: a haem-dependent oxidative N-demethylase. Unrelated to other amine oxidases, this enzyme contains haem, flavin mononucleotide, 2Fe-2S and tetrahydrofolic acid cofactors, and specifically catalyses the NADPH-dependent oxidation of dimethylamine. The structure of the ? subunit reveals that it is a haem-binding PAS domain, similar in structure to PAS gas sensors. The dimethylamine substrate forms part of a highly polarized oxygen-binding site, and directly assists oxygen activation by acting as both an electron and proton donor. Our data reveal that the ubiquitous PAS domain can make the transition from sensor to enzyme, suggesting that the PAS scaffold can support the development of artificial enzymes.
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Nov 2016
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[7146, 8997]
Open Access
Abstract: O-Demethylation by acetogenic or organohalide-respiring bacteria leads to the formation of methyltetrahydrofolate from aromatic methyl ethers. O-Demethylases, which are cobalamin-dependent, three-component enzyme systems, catalyse methyl-group transfers from aromatic methyl ethers to tetrahydrofolate via methylcobalamin intermediates. In this study, crystal structures of the tetrahydrofolate-binding methyltransferase module from a Desulfitobacterium hafniense DCB-2 O-demethylase were determined both in complex with tetrahydrofolate and the product methyltetrahydrofolate. While these structures are similar to previously determined methyltransferase structures, the position of key active-site residues is subtly altered. A strictly conserved Asn is displaced to establish a putative proton-transfer network between the substrate N5 and solvent. It is proposed that this supports the efficient catalysis of methyltetrahydrofolate formation, which is necessary for efficient O-demethylation.
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Sep 2015
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8997, 12788]
Open Access
Abstract: Queuosine (Q) is a hypermodified RNA base that replaces guanine in the wobble positions of 5prime-GUN-3prime tRNA molecules. Q is exclusively made by bacteria, and the corresponding queuine base is a micronutrient salvaged by eukaryotic species. The final step in Q biosynthesis is the reduction of the epoxide precursor, epoxyqueuosine, to yield the Q cyclopentene ring. The epoxyqueuosine reductase responsible, QueG, shares distant homology with the cobalamin-dependent reductive dehalogenase (RdhA), however the role played by cobalamin in QueG catalysis has remained elusive. We report the solution and structural characterization of Streptococcus thermophilus QueG, revealing the enzyme harbours a redox chain consisting of two [4Fe-4S] clusters and a cob(II)alamin in the base-off form, similar to RdhAs. In contrast to the shared redox chain architecture, the QueG active site shares little homology with RdhA, with the notable exception of a conserved Tyr that is proposed to function as a proton donor during reductive dehalogenation. Docking of an epoxyqueuosine substrate suggests the QueG active site places the substrate cyclopentane moiety in close proximity of the cobalt. Both the Tyr and a conserved Asp are implicated as proton donors to the epoxide leaving group. This suggests that, in contrast to the unusual carbon-halogen bond chemistry catalyzed by RdhAs, QueG acts via Co-C bond formation. Our study establishes the common features of Class III cobalamin-dependent enzymes, and reveal an unexpected diversity in the reductive chemistry catalyzed by these enzymes.
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Sep 2015
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