CYP74C3 and CYP74A1, plant cytochrome P450 enzymes whose activity is regulated by detergent micelle association, and proposed new rules for the classification of CYP74 enzymes

2006 ◽  
Vol 34 (6) ◽  
pp. 1223-1227 ◽  
Author(s):  
R.K. Hughes ◽  
E.J. Belfield ◽  
R. Casey
Keyword(s):  
Cytochrome P450 ◽  
Major Effect ◽  
Hydroperoxide Lyase ◽  
Allene Oxide ◽  
Cytochrome P450 Enzymes ◽  
Oligomeric State ◽  
Product Specificity ◽  
Detergent Micelle ◽  
Sequence Relatedness ◽  
Monomeric Proteins

CYP74C3 (cytochrome P450 subfamily 74C3), an HPL (hydroperoxide lyase) from Medicago truncatula (barrel medic), and CYP74A1, an AOS (allene oxide synthase) from Arabidopsis thaliana, are key membrane-associated P450 enzymes in plant oxylipin metabolism. Both recombinant detergent-free enzymes are monomeric proteins with dual specificity and very low enzyme activity that can be massively activated with detergent. This effect is a result of the formation of a complex between the protein monomer and a single detergent micelle and, in the case of CYP74A1, has a major effect on the substrate specificity of the enzyme. Association with a detergent micelle without an effect on protein oligomeric state represents a new mechanism of activation for membrane-associated P450 enzymes. This may represent a second unifying feature of CYP74 enzymes, in addition to their known differences in reaction mechanism, which separates them functionally from more classical P450 enzymes. Highly concentrated and monodispersed samples of detergent-free CYP74C3 and CYP74A1 proteins should be suitable for structural resolution. On the basis of recent evidence for incorrect assignment of CYP74 function, using the current rules for CYP74 classification based on sequence relatedness, we propose an alternative based on substrate and product specificity for debate and discussion.

scholarly journals ALLENE OXIDE SYNTHASE and HYDROPEROXIDE LYASE, Two Non-Canonical Cytochrome P450s in Arabidopsis thaliana and Their Different Roles in Plant Defense

2019 ◽  
Vol 20 (12) ◽  
pp. 3064 ◽  
Author(s):  
Sachin Rustgi ◽  
Armin Springer ◽  
ChulHee Kang ◽  
Diter von Wettstein ◽  
Christiane Reinbothe ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Plant Defense ◽  
Metabolic Regulation ◽  
Insect Pests ◽  
Green Leaf Volatiles ◽  
Hydroperoxide Lyase ◽  
Allene Oxide ◽  
Allene Oxide Synthase ◽  
Cytochrome P450 Enzymes ◽  
Oxide Synthase

The channeling of metabolites is an essential step of metabolic regulation in all living organisms. Multifunctional enzymes with defined domains for metabolite compartmentalization are rare, but in many cases, larger assemblies forming multimeric protein complexes operate in defined metabolic shunts. In Arabidopsis thaliana, a multimeric complex was discovered that contains a 13-lipoxygenase and allene oxide synthase (AOS) as well as allene oxide cyclase. All three plant enzymes are localized in chloroplasts, contributing to the biosynthesis of jasmonic acid (JA). JA and its derivatives act as ubiquitous plant defense regulators in responses to both biotic and abiotic stresses. AOS belongs to the superfamily of cytochrome P450 enzymes and is named CYP74A. Another CYP450 in chloroplasts, hydroperoxide lyase (HPL, CYP74B), competes with AOS for the common substrate. The products of the HPL reaction are green leaf volatiles that are involved in the deterrence of insect pests. Both enzymes represent non-canonical CYP450 family members, as they do not depend on O2 and NADPH-dependent CYP450 reductase activities. AOS and HPL activities are crucial for plants to respond to different biotic foes. In this mini-review, we aim to summarize how plants make use of the LOX2–AOS–AOC2 complex in chloroplasts to boost JA biosynthesis over volatile production and how this situation may change in plant communities during mass ingestion by insect pests.

scholarly journals Characterization of Medicago truncatula (barrel medic) hydroperoxide lyase (CYP74C3), a water-soluble detergent-free cytochrome P450 monomer whose biological activity is defined by monomer–micelle association

2006 ◽  
Vol 395 (3) ◽  
pp. 641-652 ◽  
Author(s):  
Richard K. Hughes ◽  
Eric J. Belfield ◽  
Mylrajan Muthusamay ◽  
Anuja Khan ◽  
Arthur Rowe ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Cytochrome P450 ◽  
Medicago Truncatula ◽  
Water Soluble ◽  
Hydroperoxide Lyase ◽  
Cytochrome P450 Enzyme ◽  
Cytochrome P450 Enzymes ◽  
P450 Enzyme ◽  
Detergent Micelles

We describe the detailed biochemical characterization of CYP74C3 (cytochrome P450 subfamily 74C3), a recombinant plant cytochrome P450 enzyme with HPL (hydroperoxide lyase) activity from Medicago truncatula (barrel medic). Steady-state kinetic parameters, substrate and product specificities, RZ (Reinheitszahl or purity index), molar absorption coefficient, haem content, and new ligands for an HPL are reported. We show on the basis of gel filtration, sedimentation velocity (sedimentation coefficient distribution) and sedimentation equilibrium (molecular mass) analyses that CYP74C3 has low enzyme activity as a detergent-free, water-soluble, monomer. The enzyme activity can be completely restored by re-activation with detergent micelles, but not detergent monomers. Corresponding changes in the spin state equilibrium, and probably co-ordination of the haem iron, are novel for cytochrome P450 enzymes and suggest that detergent micelles have a subtle effect on protein conformation, rather than substrate presentation, which is sufficient to improve substrate binding and catalytic-centre activity by an order of magnitude. The kcat/Km of up to 1.6×108 M−1·s−1 is among the highest recorded, which is remarkable for an enzyme whose reaction mechanism involves the scission of a C–C bond. We carried out both kinetic and biophysical studies to demonstrate that this effect is a result of the formation of a complex between a protein monomer and a single detergent micelle. Association with a detergent micelle rather than oligomeric state represents a new mechanism of activation for membrane-associated cytochrome P450 enzymes. Highly concentrated and monodispersed samples of detergent-free CYP74C3 protein may be well suited for the purposes of crystallization and structural resolution of the first plant cytochrome P450 enzyme.

scholarly journals Substrate channeling in oxylipin biosynthesis through a protein complex in the plastid envelope of Arabidopsis thaliana

2018 ◽  
Author(s):  
Stephan Pollmann ◽  
Armin Springer ◽  
Sachin Rustgi ◽  
Diter von Wettstein ◽  
ChulHee Kang ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Insect Pests ◽  
Fine Tuning ◽  
Hydroperoxide Lyase ◽  
Allene Oxide ◽  
Cytochrome P450 Enzymes ◽  
Defense Compounds ◽  
Plastid Envelope ◽  
Volatile Aldehydes ◽  
Abiotic Stimuli

ABSTRACTOxygenated membrane fatty acid derivatives dubbed oxylipins play important roles in the plant’s defense against biotic and abiotic cues. Plants challenged by insect pests, for example, synthesize a blend of different defense compounds that, amongst others, comprise volatile aldehydes and jasmonic acid (JA). Because all oxylipins are derived from the same pathway, we asked how their synthesis might be regulated and focused on two closely related, atypical cytochrome P450 enzymes designated CYP74A and CYP74B, i.e., allene oxide synthase (AOS) and hydroperoxide lyase (HPL). Both enzymes compete for the same substrate but give rise to different products. While the final product of the AOS branch is JA, those of the HPL branch comprise volatile aldehydes and alcohols. AOS and HPL are plastid envelope enzymes in Arabidopsis thaliana but accumulate at different locations. Biochemical experiments identified AOS as constituent of complexes also containing lipoxygenase 2 (LOX2) and allene oxide cyclase (AOC), which catalyze consecutive steps in JA precursor biosynthesis, while excluding the concurrent HPL reaction. Based on published X-ray data, the structure of this complex could be modelled and amino acids involved in catalysis and subunit interactions identified. Genetic studies identified the microRNA 319 (miR319)-regulated clade of TCP (TEOSINTE BRANCHED/CYCLOIDEA/PCF) transcription factor genes and CORONATINE INSENSITIVE 1 (COI1) to control JA production through the AOS-LOX2-AOC2 complex. Together, our results define a molecular branch point in oxylipin biosynthesis that allows fine-tuning the plant’s defense machinery in response to biotic and abiotic stimuli.

Sign in / Sign up

Export Citation Format

Share Document