Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QT, UK1
Author for correspondence: Neil C. Bruce. Tel: +44 1223 334168. Fax: +44 1223 334162. e-mail: n.bruce{at}biotech.cam.ac.uk
Keywords: /ß-unsaturated carbonyl compounds, Old Yellow Enzyme, oxidative stress, xenobiotic compounds
a Present address: MRC Centre for Protein Engineering, Hills Road, Cambridge CB2 2QH, UK.
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Overview |
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Old Yellow Enzyme |
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The observed similarity between the sequences of the gene encoding an OYE (Saito et al., 1991 ) and a gene from the bile-acid-inducible operon in Eubacterium sp. suggested that OYE could also be involved in sterol metabolism. This would involve reduction of a 2-cyclohexenone functional group, and it was demonstrated that 2-cyclohexenone is a very good oxidant of OYE (Stott et al., 1993
), giving a turnover number in excess of that seen with quinones such as menadione. However, no evidence has been found for sterol metabolism by OYE in vivo.
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An Old Yellow Enzyme family of flavoproteins |
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Several bacterial enzymes with amino acid sequence homology to OYE have now been reported. Multiple independent studies of the bacterial degradation of nitrate ester explosives have identified very similar OYE-related proteins. The enzymes catalyse the nicotinamide-cofactor-dependent reductive cleavage of nitrate esters to give the alcohol and nitrite. Pentaerythritol tetranitrate (PETN) reductase from Enterobacter cloacae PB2 (Binks et al., 1996 ) sequentially removes two of the four nitro groups of PETN, and two of the three groups of nitroglycerine (GTN). The crystal structure of this enzyme has now been elucidated (Barna et al., 2001
). Similar enzymes are responsible for nitrate ester degradation by Agrobacterium radiobacter (Snape et al., 1997
) nitrate ester reductase and by strains of Pseudomonas fluorescens and Pseudomonas putida (Blehert et al., 1999
) xenobiotic reductases. OYE also catalyses the cleavage of nitrate esters, albeit at a slower rate than these bacterial enzymes, probably via a radical-based mechanism (Meah et al., 2001
).
Research into the degradation of morphine alkaloids by a strain of P. putida led to the identification of a two-stage transformation sequence from morphine via morphinone to hydromorphone (Hailes & Bruce, 1993 ). The second enzyme in the pathway, morphinone reductase, is homologous to OYE (French & Bruce, 1994
). This enzyme is responsible for the reduction of an
/ß-unsaturated carbonyl functionality analogous to that of known OYE substrates. Another similar enzyme has been found to be cold-shock-induced in Pseudomonas syringae, a plant pathogen (Rohde et al., 1999
).
A close relative of PETN reductase, N-ethylmaleimide reductase (Miura et al., 1997 ), has been identified in Escherichia coli. N-Ethylmaleimide is a variation on the
/ß-unsaturated carbonyl functional group, and is also a substrate for OYE (Vaz et al., 1995
). Original characterization of the enzyme (Mizugaki et al., 1979
) described it as a cis-enoyl-CoA reductase, involved in the ß-oxidation of fatty acids such as linoleic acid.
Plant homologues of OYE were first identified during various screens for genes induced under different circumstances. OYE-related messenger RNAs are produced during cytokinin induction in Chenopodium rubrum (Peters et al., 1996 ), sulphate starvation in Catharanthus roseus (GenBank accession no. AF005237) and dehydration stress in Vigna unguiculata (Iuchi et al., 1996
). Three Arabidopsis OYE homologues have been identified as 12-oxophytodienoate reductases (Schaller & Weiler, 1997
), which can catalyse the reduction of an
/ß-unsaturated carbonyl intermediate in the synthesis of jasmonic acid, a wound-response hormone. Only one of the paralogues appears to be involved in the wound-response pathway in vivo (Schaller et al., 2000
). Interestingly, other lipids with
/ß-unsaturated carbonyl functionality appear to be produced in response to physical wounding and P. syringae infection, being implicated both in signalling and as causative agents of tissue damage (Vollenweider et al., 2000
). The smaller, volatile,
/ß-unsaturated carbonyl compound trans-2-hexenal is also involved in response to bacterial pathogens (Farmer, 2001
). A tomato orthologue of 12-oxophytodienoate reductase has been compared to OYE in some detail (Strassner et al., 1999
), and the structure of this enzyme has very recently been determined (Breithaupt et al., 2001
).
A series of more distantly related proteins is present in the sequence databases. A barrel domain related to OYE forms part of trimethylamine dehydrogenase (Lim et al., 1986 ), and is used as a module in several other multi-domain proteins (Scrutton, 1994
), such as E. coli 2,4-dienoyl-CoA-reductase (He et al., 1997
) and enoate reductases of clostridia (Rohdich et al., 2001
). Whilst some of the putative active site residues differ in these distant relatives of OYE, there is a strong sequence conservation in a core region of around 40 amino acids. The second domain in trimethylamine dehydrogenase is also redox-active, being related to glutathione reductase and dihydrolipoamide dehydrogenase. There is clear evidence for intra-protein electron transfer from the OYE-related domain to an ironsulphur cluster located in the second domain (Falzon & Davidson, 1996
). Thus rather than having a common binding site for both the reducing and oxidizing substrates (as in OYE), these enzymes would appear to have separate sites, permitting optimization of each site for the half-reaction it catalyses. However, a two-domain 2-aminobenzoate monooxygenase/reductase with an OYE-related domain as the C-terminus has recently been reported (Schuhle et al., 2001
), in which each domain would appear to act independently, albeit to catalyse consecutive steps in a metabolic pathway.
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Structure and mechanism of OYE family members |
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OYE is a single-domain protein of around 45 kDa, with an /ß barrel fold (Fox & Karplus, 1994
), as shown in Fig. 4
. There is a ß-hairpin structure covering the base of the barrel, whilst the FMN is bound at the top of the barrel, with the si-face of the flavin accessible to the solvent, where it forms the bottom of the active site. From representation of the active site of OYE in complex with p-hydroxybenzaldehyde in Fig. 5
, it can be seen that the phenolic ring (white bonds) undergoes a stacking interaction with the flavin (grey bonds). The phenol oxygen forms two hydrogen bonds to His-191 and Asn-194, displacing a chloride ion bound in the empty oxidized enzyme structure. Binding studies with a variety of substituted phenols suggest that it is the phenolate anion that is a ligand for the enzyme (Abramovitz & Massey, 1976
; Brown et al., 1998
). The crystal structure of OYE soaked with the non-substrate analogue of NADPH, (c-THN)TPN, suggests a novel cofactor binding arrangement where only the nicotinamide moeity of the cofactor is bound tightly, whilst the adenine phosphate is not fixed. This fits the observations that the
-anomer of NADPH reacts as well or even slightly better than the usual ß-anomer, and that 2'-phospho-5'-AMP is not a competitive inhibitor. However, it makes the observed selectivity for NADPH over NADH hard to explain.
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In the three enzymes for which crystal structures are available, the FMN cofactor is bound in a very similar manner (Barna et al., 2001 ; Breithaupt et al., 2001
; Fox & Karplus, 1994
). Where the side-chains of residues contact the cofactor, these residues are conserved across the family. The residues lying immediately above the plane of the flavin ring, whether involved in catalysis as described above, or forming the hydrophobic substrate-binding site, are also highly conserved. Asn-194 in OYE is replaced by histidine in PETN reductase and 12-oxophytodienoate reductase; however, this does not significantly alter the position of ligand binding in the structures. This substitution might account for some difference in reactivity and ligand binding within the OYE family (Strassner et al., 1999
). The major structural differences between the enzymes occur in the loop regions at the top of the barrel (Fig. 6
). Such differences are likely to be more significant when considering the binding of larger ligands, such as the steroids and 12-oxophytodienoate.
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Towards an appreciation of the true physiological roles of the enzymes |
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The high degree of conservation of regions of primary and tertiary structure across the family, at least within the close family of single-domain yeast, plant and Gram-negative bacterial enzymes, would suggest that the enzymes are orthologous. If this is the case, the conserved functional role is still to be discovered. The suggestion of a role in the general detoxification of a broad spectrum of electrophilic molecules is attractive; however, it is not clear why a general purpose enzyme should resist evolutionary change. Small /ß-unsaturated carbonyl compounds are substrates for all family members; however, activity towards these compounds varies considerably [e.g. 2-cyclohexenone: OYE has a Km=<10 µM, and kcat=4·2 s-1 (Brown et al., 1998
); morphinone reductase has an apparent Km=5·5 mM, and an apparent kcat=0·67 s-1 (French et al., 1998
)].
Plant homologues of OYE are increasingly implicated in the metabolism of larger lipid molecules with /ß-unsaturated carbonyl functionality (Schaller & Weiler, 1997
; Strassner et al., 1999
). These compounds are made during insect attack (Farmer, 2001
), and, perhaps more interestingly, during bacterial pathogenesis (Vollenweider et al., 2000
). The presence of an extremely similar enzyme in both the plant pathogen P. syringae (Rohde et al., 1999
) and the host plants raises the possibility of horizontal transfer of a detoxification enzyme from host to enemy.
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Biotechnological applications of the enzymes |
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Whilst the OYE-related nitrate ester reductases were isolated on the basis of their ability to liberate nitrite from the high explosives PETN and GTN (Fig. 3a), it has become apparent that they might also share an ability to reduce trinitrotoluene (TNT; Fig. 3b
), which is a significant environmental pollutant. Enterobacter cloacae PB2 is able to grow very slowly on TNT as a sole nitrogen source (French et al., 1998
), and incubation of E. cloacae PETN reductase with NADPH and TNT resulted in the conversion of TNT to a mixture of reduced products. Many flavin-containing enzymes act as nitroreductases, and hydroxylamino- and amino-dinitrotoluenes are produced from TNT by PETN reductase. However, additional strongly coloured products are observed with PETN reductase (French et al., 1998
) and the P. fluorescens nitrate ester reductase (Blehert et al., 1999
). These coloured products arise from hydride addition to the aromatic ring, yielding hydride and dihydrideMeisenheimer complexes (Vorbeck et al., 1998
). Thus at least some nitrate ester reductases appear to reduce both the nitro groups and the aromatic ring of TNT. Nitrite is formed by the action of these nitrate ester reductases on TNT (French et al., 1998
), but the nature of other end products and the mechanism of their formation is as yet unknown. Phytoremediation, the use of plants to remediate contaminated land, is the subject of considerable research at present. Plants are fundamentally self-sustaining, and transpire large volumes of groundwater. Phytoremediation is therefore an attractive option for the remediation of large expanses of land and groundwater containing moderate concentrations of explosives. The success of such an approach relies upon the ability of the plant to metabolize the target compounds. Genetic modification of plants could permit the combination of the metabolic abilities of soil microbes with the high biomass and deep root systems of plants. PETN reductase has been introduced into tobacco plants (French et al., 1999
), and the resulting transgenic lines show considerably increased tolerance to GTN and TNT in growth media.
Morphine and its derivatives provide some of the most potent analgesic compounds in clinical use today. Small changes in the chemical structure of the morphine alkaloids can significantly alter the pharmaceutical properties of the drug. The semi-synthetic opiates are prepared by chemical modifications from the naturally occurring alkaloids morphine, codeine and thebaine isolated from the opium poppy. These processes are often difficult to achieve due to the complexity of the molecules and the abundance of functional groups. There has therefore been considerable interest in the use of biocatalysts to produce semi-synthetic derivatives (Rathbone et al., 2001 ). As shown in Fig. 3(c)
, morphinone reductase catalyses the reduction of morphinone and codeinone to the potent analgesics hydromorphone and hydrocodone, respectively (French & Bruce, 1994
). Coexpression of morphinone reductase with a morphine dehydrogenase in E. coli resulted in efficient biocatalysts that transformed morphine and codeine to hydromorphone and hydrocodone, with high yields (Boonstra et al., 2001
).
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Conclusions |
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