Crystal Structure of Bacterial Morphinone Reductase and Properties of the C191A Mutant Enzyme

Barna, T.; Messiha, Hanan L.; Petosa, Carlo; Bruce, Neil C.; Scrutton, Nigel S.; Moody, P.C.E.

doi:10.1074/jbc.m202846200

Cited by 74 publications

(113 citation statements)

References 44 publications

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“…3b). This structural correspondence is also found for nearly all structurally characterized members of the OYE family (data not shown) with the exception of His-188, which is replaced by asparagine in several enzymes (19,20,(25)(26)(27)(28). The corresponding atoms Asn N␦2 and His N␦1 that bind the carbonyl oxygen overlay perfectly, however, indicating a similar effect on substrate activation and positioning.…”

Section: Resultsmentioning

confidence: 77%

Crystal structure of 12-oxophytodienoate reductase 3 from tomato: Self-inhibition by dimerization

Breithaupt

Kurzbauer

Lilie

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

118

109

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12-Oxophytodienoate reductase (OPR) 3, a homologue of old yellow enzyme (OYE), catalyzes the reduction of 9S,13S-12-oxophytodienoate to the corresponding cyclopentanone, which is subsequently converted to the plant hormone jasmonic acid (JA). JA and JA derivatives, as well as 12-oxophytodienoate and related cyclopentenones, are known to regulate gene expression in plant development and defense. Together with other oxygenated fatty acid derivatives, they form the oxylipin signature in plants, which resembles the pool of prostaglandins in animals. Here, we report the crystal structure of OPR3 from tomato and of two OPR3 mutants. Although the catalytic residues of OPR3 and related OYEs are highly conserved, several characteristic differences can be discerned in the substrate-binding regions, explaining the remarkable substrate stereoselectivity of OPR isozymes. Interestingly, OPR3 crystallized as an extraordinary self-inhibited dimer. Mutagenesis studies and biochemical analysis confirmed a weak dimerization of OPR3 in vitro, which correlated with a loss of enzymatic activity. Based on structural data of OPR3, a putative mechanism for a strong and reversible dimerization of OPR3 in vivo that involves phosphorylation of OPR3 is suggested. This mechanism could contribute to the shaping of the oxylipin signature, which is critical for fine-tuning gene expression in plants.flavoprotein ͉ jasmonic acid biosynthesis ͉ plant defense ͉ oxylipin signature ͉ 12-oxophytodienoic acid F lavoproteins catalyze a wide variety of essential biochemical reactions, including electron transfer, dehydrogenation, and hydroxylation reactions. Old yellow enzyme (OYE), the first flavin-dependent enzyme identified (1), and homologues of OYE from bacteria, yeast, and plants are able to reduce the CAC double bond of ␣,␤-unsaturated carbonyl compounds, an activity that is rather uncommon for flavoenzymes (2, 3). This reaction has been shown to proceed by a ping-pong bi-bi mechanism, during which the FMN cofactor is reduced by NAD(P)H before the substrate is bound and reduced by hydride transfer to the substrate's C ␤ (4). Despite extensive efforts, the physiological substrate has been revealed only for one member of the OYE family, the plant enzyme 12-oxophytodienoate reductase (OPR) 3, which catalyzes one step in the biosynthesis of the plant hormone jasmonic acid (JA) (5, 6). JA and JA derivatives act as signaling compounds in the defense response against herbivores and pathogens and are involved in the regulation of various developmental processes, such as fruit ripening, pollen maturation, and senescence (7,8). JA is synthesized from ␣-linolenic acid, which is oxidized and cyclized, resulting in the cyclopentenone 9S,13S-12-oxophytodienoate (9S,13S-OPDA). OPR3 reduces 9S,13S-OPDA to the corresponding cyclopentanone, which is converted to JA by repeated ␤-oxidation ( Fig. 1) (9). Several OPR isozymes have been identified in plants, including 3 isoforms in Lycopersicon esculentum, 5 in Arabidopsis thaliana, and 13 in Oryza sativa (10, 11). O...

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Section: Resultsmentioning

confidence: 77%

Crystal structure of 12-oxophytodienoate reductase 3 from tomato: Self-inhibition by dimerization

Breithaupt

Kurzbauer

Lilie

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

118

109

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“…PETN reductase was prepared from Escherichia coli JM109/pONR1 and purified as described previously (32) but also incorporating a final chromatographic step using Q-Sepharose (28). MR was purified from a recombinant strain of E. coli transformed with plasmid pMORB2, which expresses the enzyme from the cloned mor B gene as described previously (33), but also incorporating a final chromatographic step using Q-Sepharose (29). NADPH and NADH were from Sigma.…”

Section: Methodsmentioning

confidence: 99%

“…In the oxidative half-reaction of MR, absorption changes at 464 nm were reported upon flavin oxidation by 2-cyclohexenone as described previously (29). Transients were biphasic, with the fast phase (k obs1 ; 90% of the total absorption change) reporting upon flavin oxidation.…”

Section: Solvent Isotopementioning

confidence: 99%

“…MR catalyzes the NADH-dependent saturation of the carbon-carbon bond of both morphinone and codeinone to produce hydromorphone (a powerful analgesic) and hydrocodone (an antitussive), which are valuable semi-synthetic opiate drugs (34,35). The half-reactions of MR and PETN reductase have been investigated using stopped-flow methods (29,36,37), enabling elucidation of the kinetic mechanisms for each half-reaction. We demonstrate in this report that hydride transfer from nicotinamide coenzyme to flavin in both enzymes occurs by quantum tunneling but that the nature of the tunneling reaction is different.…”

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confidence: 99%

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H-tunneling in the Multiple H-transfers of the Catalytic Cycle of Morphinone Reductase and in the Reductive Half-reaction of the Homologous Pentaerythritol Tetranitrate Reductase

Basran

Harris

Sutcliffe

et al. 2003

Journal of Biological Chemistry

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100

180

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The mechanism of flavin reduction in morphinone reductase (MR) and pentaerythritol tetranitrate (PETN) reductase, and flavin oxidation in MR, has been studied by stopped-flow and steady-state kinetic methods. The temperature dependence of the primary kinetic isotope effect for flavin reduction in MR and PETN reductase by nicotinamide coenzyme indicates that quantum mechanical tunneling plays a major role in hydride transfer. In PETN reductase, the kinetic isotope effect (KIE) is essentially independent of temperature in the experimentally accessible range, contrasting with strongly temperaturedependent reaction rates, consistent with a tunneling mechanism from the vibrational ground state of the reactive C-H/D bond. In MR, both the reaction rates and the KIE are dependent on temperature, and analysis using the Eyring equation suggests that hydride transfer has a major tunneling component, which, unlike PETN reductase, is gated by thermally induced vibrations in the protein. The oxidative half-reaction of MR is fully rate-limiting in steady-state turnover with the substrate 2-cyclohexenone and NADH at saturating concentrations. The KIE for hydride transfer from reduced flavin to the ␣/␤ unsaturated bond of 2-cyclohexenone is independent of temperature, contrasting with strongly temperaturedependent reaction rates, again consistent with groundstate tunneling. A large solvent isotope effect (SIE) accompanies the oxidative half-reaction, which is also independent of temperature in the experimentally accessible range. Double isotope effects indicate that hydride transfer from the flavin N5 atom to 2-cyclohexenone, and the protonation of 2-cyclohexenone, are concerted and both the temperature-independent KIE and SIE suggest that this reaction also proceeds by ground-state quantum tunneling. Our results demonstrate the importance of quantum tunneling in the reduction of flavins by nicotinamide coenzymes. This is the first observation of (i) three H-nuclei in an enzymic reaction being transferred by tunneling and (ii) the utilization of both passive and active dynamics within the same native enzyme.Enzymes are phenomenal catalysts that can achieve rate enhancements of up to ϳ21 orders of magnitude over the uncatalyzed reaction rate (1). Our quest to understand the physical basis of this catalytic power is challenging and has involved sustained and intensive research efforts by many workers in the physical and life sciences (for recent reviews see Refs. 2-5). Recent years have witnessed new activity in this area and extended our theoretical understanding beyond the shortcomings of transition state theory (6) to include roles for protein "motion" (7, 8), low barrier hydrogen bonds (e.g. Refs. 9 -11), active site preorganization (e.g. reviewed in Refs. 4 and 12), and quantum mechanical tunneling (for recent reviews see Refs. 13-15). New theoretical frameworks incorporating quantum mechanical tunneling and protein motion are emerging to address the catalytic potency of enzymes. These invoke motion in the protein and/or subs...

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“…Enoate reductases are members of the "old yellow enzyme" (OYE) family (Barna et al 2002) and contain a noncovalently bound cofactor flavin mononucleotide (FMN).…”

Section: Introductionmentioning

confidence: 99%

An enoate reductase Achr-OYE4 from Achromobacter sp. JA81: characterization and application in asymmetric bioreduction of C=C bonds

Wang

Pei

2013

Appl Microbiol Biotechnol

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A putative enoate reductase, Achr-OYE4, was mined from the genome of Achromobacter sp. JA81, expressed in Escherichia coli, and was characterized. Sequence analysis and spectral properties indicated that Achr-OYE4 is a typical flavin mononucleotide-dependent protein; it preferred NADH over NADPH as a cofactor. The heterologously expressed protein displayed good activity and excellent stereoselectivity toward some activated alkenes in the presence of NADH, NADPH, or their recycling systems. The glucose dehydrogenase-based recycling system yielded the best results in most cases, with a product yield of up to 99 % and enantiopurity of >99 % ee. Achr-OYE4 is an important addition to the asymmetric reduction reservoir as an "old yellow enzyme" from Achromobacter.

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Crystal Structure of Bacterial Morphinone Reductase and Properties of the C191A Mutant Enzyme

Cited by 74 publications

References 44 publications

Crystal structure of 12-oxophytodienoate reductase 3 from tomato: Self-inhibition by dimerization

Crystal structure of 12-oxophytodienoate reductase 3 from tomato: Self-inhibition by dimerization

H-tunneling in the Multiple H-transfers of the Catalytic Cycle of Morphinone Reductase and in the Reductive Half-reaction of the Homologous Pentaerythritol Tetranitrate Reductase

An enoate reductase Achr-OYE4 from Achromobacter sp. JA81: characterization and application in asymmetric bioreduction of C=C bonds

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