Structure of 6-Oxo Camphor Hydrolase H122A Mutant Bound to Its Natural Product, (2S,4S)-α-Campholinic Acid

Leonard, Philip; Grogan, Gideon

doi:10.1074/jbc.m403514200

Cited by 27 publications

(18 citation statements)

References 29 publications

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“…A range of other aldehyde substrates for BAL have recently been identified (Sanchez-Gonzalez & Rosazza, 2003;Demir et al, 2003) and there is an increasing number of reports of the optimization of its activity for possible application of the enzyme in industrial processes (Stilger et al, 2006). As part of our continuing studies into the structural enzymology of unusual C-C bond lyases using X-ray crystallography (Leonard & Grogan, 2004;Leonard et al, 2006), we were interested in the molecular structural determinants of both catalysis and enantioselectivity in the reaction catalysed by BAL and we therefore crystallized the protein. Soon after our acquisition of a data set to a maximum resolution of 1.65 Å , the X-ray structure of BAL at a resolution of 2.6 Å was published by Schulz and coworkers (Mosbacher et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

Structure of the ThDP-dependent enzyme benzaldehyde lyase refined to 1.65 Å resolution

et al. 2007

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Benzaldehyde lyase (BAL; EC 4.1.2.38) is a thiamine diphosphate (ThDP) dependent enzyme that catalyses the enantioselective carboligation of two molecules of benzaldehyde to form (R)-benzoin. BAL has hence aroused interest for its potential in the industrial synthesis of optically active benzoins and derivatives. The structure of BAL was previously solved to a resolution of 2.6 Å using MAD experiments on a selenomethionine derivative [Mosbacher et al. (2005), FEBS J. 272, 6067-6076]. In this communication of parallel studies, BAL was crystallized in an alternative space group (P2 1 2 1 2 1) and its structure refined to a resolution of 1.65 Å , allowing detailed observation of the water structure, active-site interactions with ThDP and also the electron density for the co-solvent 2-methyl-2,4-pentanediol (MPD) at hydrophobic patches of the enzyme surface.

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Section: Introductionmentioning

confidence: 99%

Structure of the ThDP-dependent enzyme benzaldehyde lyase refined to 1.65 Å resolution

et al. 2007

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“…Nearly 62% of the residues responsible for dimer formation are conserved in BoxC orthologs, suggesting that the dimeric structure of this novel group of ring-cleaving enzymes will be consistent. In the broader context of the crotonase superfamily, the dimeric form is rare, with most members adopting trimer, tetramer, or hexamer (dimers of trimers) forms (32)(33)(34)(35)(36)(37)(38)(39)(40) and, in one case, proposed to form a trimer of dimers (41). Only the carboxyltransferase subunits for the acetyl-CoA carboxylase from Saccharomyces cerevisiae (42) and the ␣-subunit of glutaconyl-CoA decarboxylase (GCD␣) from A. fermentans (43), which are subunits of larger multifunctional enzymes, are reported to be dimers.…”

Section: Overall Structurementioning

confidence: 99%

Structural and Biophysical Characterization of BoxC from Burkholderia xenovorans LB400

Bains

León

Boulanger

2009

Journal of Biological Chemistry

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The mineralization of aromatic compounds by microorganisms relies on a structurally and functionally diverse group of ring-cleaving enzymes. The recently discovered benzoate oxidation pathway in Burkholderia xenovorans LB400 encodes a novel such ring-cleaving enzyme, termed BoxC, that catalyzes the conversion of 2,3-dihydro-2,3-dihydroxybenzoyl-CoA to 3,4-dehydroadipyl-CoA without the requirement for molecular oxygen. Sequence analysis indicates that BoxC is a highly divergent member of the crotonase superfamily and nearly double the size of the average superfamily member. The structure of BoxC determined to 1.5 Å resolution reveals an intriguing structural demarcation. A highly divergent region in the C terminus probably serves as a structural scaffold for the conserved N terminus that encompasses the active site and, in conjunction with a conserved C-terminal helix, mediates dimer formation. Isothermal titration calorimetry and molecular docking simulations contribute to a detailed view of the active site, resulting in a compelling mechanistic model where a pair of conserved glutamate residues (Glu 146 and Glu 168 ) work in tandem to deprotonate the dihydroxylated ring substrate, leading to cleavage. A final deformylation step incorporating a water molecule and Cys 111 as a general base completes the formation of 3,4-dehydroadipyl-CoA product. Overall, this study establishes the basis for BoxC as one of the most divergent members of the crotonase superfamily and provides the first structural insight into the mechanism of this novel class of ring-cleaving enzymes.Aromatic compounds comprise approximately one-quarter of the earth's biomass (1) and are the second most abundant natural product next to carbohydrates. The majority of aromatic compounds in the environment are in the form of the organic polymer lignin that plays a structural role in cross-linking cell wall polysaccharides in plants. Despite the inherent thermostability of the aromatic ring, these naturally occurring compounds are efficiently mineralized by various microorganisms. Human-made aromatic compounds, such as those used in industrial processes, however, are often recalcitrant to microbial degradation due to their chemical complexity, decreased bioavailability, and increased thermostability. Moreover, bacteria have only been exposed to these compounds for a relatively short period of time. As a result, these compounds persist in the environment, where they can increase to toxic levels and cause irreversible damage to the biosphere.The common structural blueprint shared by natural and human-made aromatic compounds is the resonance-stabilized planar ring system. Microorganisms overcome the stability of these aromatic structures by employing specific ring-cleaving enzymes that form part of complex catabolic pathways. Until recently, two general classes of microbial processes were characterized that catalyze the degradation of aromatic compounds. These classifications, termed the aerobic and anaerobic pathways, were based primarily on the mode of ...

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“…When the binding of the carbonyl group of the sixmembered ring in the oxyanion hole was retained throughout the simulation, the geometries on the 5-membered ring are such that the attack by a water activated by histidine 145 would not be as readily undergone as for the (1R,6S)-enantiomer, as the now distorted geometry of any putative Asp154-His145 dyad will make this event less likely. Hence, although the absolute configuration of the favoured enantiomer in these resolutions is not known, a knowledge of the structure of the active site in complex with the natural product [4] in conjunction with the modelling studies are suggestive of the (1R,6S)-enantiomer as being that transformed most rapidly.…”

Section: Resultsmentioning

confidence: 96%

“…We have also obtained an X-ray crystal structure of both native OCH [3] and a low activity mutant, in which one of the natural products of reaction was observed. [4] The latter structure has helped to shed light on the molecular determinants of both mechanism and enantiotopic selectivity in reactions catalysed by OCH. At this stage, we have proposed a mechanism for the activity of OCH that is initiated by general base activation of a water molecule by histidine 145.…”

Section: Introductionmentioning

confidence: 98%

“…An additional measure of prochiral control is afforded by the nascent cyclopentane ring of the product forming a stacking interaction with phenyalanine 82. [4] With a view to examining the wider substrate specificity of OCH, we decided to synthesise a series of asymmetric, heteroannular bicyclic b-diketones that would test the ability of the enzyme to catalyse the resolution of racemic substrates. As the substrate specificity of OCH had proven to be quite narrow, accepting only mono-or bicyclic diketones that were non-enolisable, either through restrictions imposed by Bredts rule (in the case of bicycloA C H T U N G T R E N N U N G [2.2.2]octane-2,6-dione) or by alkylation at the carbon atom between the carbonyl groups (as in the series of 1-alkylbicyclo- [2] it was decided to synthesise compounds based around bicyclo A general route to this class of bicyclic b-diketones was recently published.…”

Section: Introductionmentioning

confidence: 99%

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On the Resolution of Chiral Substrates by a retro‐Claisenase Enzyme: Biotransformations of Heteroannular Bicyclic β‐Diketones by 6‐Oxocamphor Hydrolase

et al. 2007

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The enzyme 6-oxocamphor hydrolase (OCH) from Rhodococcus sp. NCIMB 9784 catalyses the cleavage of a carbon-carbon bond between two carbonyl groups in both mono-and bicyclic non-enolisable b-diketone substrates. In this mode OCH has been shown to effect the desymmetrisation of both bridged symmetrical bicyclic [2.2.1] and [2.2.2] systems and a series of 1-alkylbicycloA C H T U N G T R E N N U N G [3.3.0]octane-2,8-diones, yielding chiral substituted cyclopentanone and cyclohexanone products in high optical purity. In the present study, OCH has been challenged with a series of heteroannular substrates including 1-methylbicycloA C H T U N G T R E N N U N G [4.3.0]nonane-2,9-dione (7a-methylhexahydroindene-1,7-dione) in an effort to assess the competence of the enzyme for kinetic resolutions of asymmetric, racemic substrates. OCH was shown to catalyse the resolution of 1-methylbicyclo-A C H T U N G T R E N N U N G [4.3.0]nonane-2,9-dione with an E value of 2.9. The effect of increasing the length of the alkyl chain in the 1-position, or enlarging one of the rings, was to increase the enantioselectivity of the enzyme to 5.7 and 3.1 for the substrates 1-allylbicyclo-(9a-methyloctahydrobenzocycloheptene-1,9-dione) was not a substrate for OCH. These experiments constitute the first description of the resolution behaviour of such a retro-Claisenase enzyme, and suggest a maximum steric limit for substrate recognition by OCH.

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Structure of 6-Oxo Camphor Hydrolase H122A Mutant Bound to Its Natural Product, (2S,4S)-α-Campholinic Acid

Cited by 27 publications

References 29 publications

Structure of the ThDP-dependent enzyme benzaldehyde lyase refined to 1.65 Å resolution

Structure of the ThDP-dependent enzyme benzaldehyde lyase refined to 1.65 Å resolution

Structural and Biophysical Characterization of BoxC from Burkholderia xenovorans LB400

On the Resolution of Chiral Substrates by a retro‐Claisenase Enzyme: Biotransformations of Heteroannular Bicyclic β‐Diketones by 6‐Oxocamphor Hydrolase

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