CYP74C3 and CYP74A1, plant cytochrome P450 enzymes whose activity is regulated by ...

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

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.

Source: Biochem Soc Trans. (2006) vol. 34, p. 1223-1227

Preparative enzymatic solid phase synthesis of cis(+)-12-oxo-phytodienoic acid – physical interaction of AOS and AOC is not necessary

The pathway of jasmonic acid (JA) biosynthesis was established in the 1980s by Vick and Zimmerman but, until now, the preparative biosynthesis of the jasmonic acid precursors 12-oxo-phytodienoic acid (OPDA) and 3-oxo-2-[2′-pentenyl]-cyclopentan-1-octanoic acid (OPC-8:0) in their endogenous and biologically relevant cis(+)-configuration was only possible in small amounts and had to put up with high costs. This was mainly due to the lack of high amounts of pure and enzymatically active allene oxide cyclase (AOC), which is a key enzyme in the biosynthesis of jasmonates in that it releases, in a coupled reaction with allene oxide synthase (AOS), the first cyclic and biological active metabolite – OPDA. We describe here the expression and purification of AOS and AOC and their subsequent coupling to solid matrices to produce an enantioselective, reusable bioreactor for octadecanoid production. With the method described here it is possible to produce optically pure enantiomers of octadecanoids in high amounts in a cost- and time-efficient manner. Furthermore, it could be demonstrated that a physical interaction of AOS and AOC, hitherto postulated to be required for substrate channeling from AOS to AOC, is not necessary for the in vitro cyclization of the unstable epoxide generated by the AOS reaction.

Keywords: Allene oxide cyclase; Allene oxide synthase; Jasmonates; Jasmonic acid; 12-Oxo-phytodienoic acid; Octadecanoid pathway; Oxylipins; Plant hormones

Source: Phytochemistry (2007) vol. 68, Issue 2, p. 229-236

Evidence for an Ionic Intermediate in the Transformation of Fatty Acid ...

Evidence for an Ionic Intermediate in the Transformation of Fatty Acid Hydroperoxide by a Catalase-related Allene Oxide Synthase from the Cyanobacterium Acaryochloris marina

Allene oxides are reactive epoxides biosynthesized from fatty acid hydroperoxides by specialized cytochrome P450s or by catalase-related hemoproteins. Here we cloned, expressed, and characterized a gene encoding a lipoxygenase-catalase/peroxidase fusion protein from Acaryochloris marina. We identified novel allene oxide synthase (AOS) activity and a by-product that provides evidence of the reaction mechanism. The fatty acids 18.4ω3 and 18.3ω3 are oxygenated to the 12R-hydroperoxide by the lipoxygenase domain and converted to the corresponding 12R,13-epoxy allene oxide by the catalase-related domain. Linoleic acid is oxygenated to its 9R-hydroperoxide and then, surprisingly, converted ~70% to an epoxyalcohol identified spectroscopically and by chemical synthesis as 9R,10S-epoxy-13S-hydroxyoctadeca-11E-enoic acid and only ~30% to the 9R,10-epoxy allene oxide. Experiments using oxygen-18-labeled 9R-hydroperoxide substrate and enzyme incubations conducted in H218O indicated that ~72% of the oxygen in the epoxyalcohol 13S-hydroxyl arises from water, a finding that points to an ionic intermediate (epoxy allylic carbocation) during catalysis. AOS and epoxyalcohol synthase activities are mechanistically related, with a reacting intermediate undergoing a net hydrogen abstraction or hydroxylation, respectively. The existence of epoxy allylic carbocations in fatty acid transformations is widely implicated although for AOS reactions, without direct experimental support. Our findings place together in strong association the reactions of allene oxide synthesis and an ionic reaction intermediate in the AOS-catalyzed transformation.

Source: J. Biol. Chem. (2009) vol. 284, p. 22087-22098

Tomato CYP74C3 is a Multifunctional Enzyme not only Synthesizing Allene Oxide but also Catalyzing its Hydrolysis and Cyclization

The mechanism of the recombinant tomato allene oxide synthase (LeAOS3, CYP74C3) was studied. Incubations of linoleic acid (9S)-hydroperoxide with dilute suspensions of LeAOS3 (10-20 s, 0 °C) yield mostly the expected allene oxide (12Z)-9,10-epoxy-10,12-octadecadienoic acid (9,10-EOD), which was detected as its methanol-trapping product. In contrast, the relative yield of 9,10-EOD progressively decreased when the incubations were performed with fourfold, tenfold, or 80-fold larger amounts of LeAOS3, while -ketol and the cyclopentenone rac-cis-10-oxo-11-phytoenoic acid (10-oxo-PEA) became the predominant products. Both the -ketol and 10-oxo-PEA were also produced when LeAOS3 was exposed to preformed 9,10-EOD, which was generated by maize allene oxide synthase (CYP74A). LeAOS3 also converted linoleic acid (13S)-hydroperoxide into the corresponding allene oxide, but with about tenfold lower yield of cyclopentenone. The results indicate that in contrast to the ordinary allene oxide synthases (CYP74A subfamily), LeAOS3 (CYP74C subfamily) is a multifunctional enzyme, catalyzing not only the synthesis, but also the hydrolysis and cyclization of allene oxide.

Keywords: allene oxide synthase • enzyme catalysis • metabolism • oxylipins • tomato

Source: ChemBioChem (2008) Vol. 9, Issue 15, p. 2498 - 2505

Determinants governing the CYP74 catalysis: Conversion of allene oxide synthase into hydroperoxide lyase by site-directed mutagenesis

Bioinformatics analyses enabled us to identify the hypothetical determinants of catalysis by CYP74 family enzymes. To examine their recognition, two mutant forms F295I and S297A of tomato allene oxide synthase LeAOS3 (CYP74C3) were prepared by site-directed mutagenesis. Both mutations dramatically altered the enzyme catalysis. Both mutant forms possessed the activity of hydroperoxide lyase, while the allene oxide synthase activity was either not detectable (F295I) or significantly reduced (S297A) compared to the wild-type LeAOS3. Thus, both sites 295 and 297 localized within the “I-helix central domain” (“oxygen binding domain”) are the primary determinants of CYP74 type of catalysis.

Keywords: Cytochrome P450; CYP74 family; Allene oxide synthase; Hydroperoxide lyase; Site-directed mutagenesis

Source: FEBS Letters (2008) Vol. 582, Issues 23-24, Pages 3423-3428

My Article on BKCS 2009

A Substrate Serves as a Hydrogen Atom Donor in the Enzyme-Initiated Catalytic Mechanism of Dual Positional Specific Maize Lipoxygenase-1

The maize lipoxgyenase-1 is a non-traditional dual positional specific enzyme and the reaction proceeds via enzyme-initiated catalysis. Bioinformatic analysis indicated that the maize lipoxygenase-1 is structurally more similar to soybean LOX1 than pea LOXN2 in that it has an additional external loop (residues 318-351) in the carboxy-terminal catalytic domain. We analyzed the dependence of product distribution on concentration of linoleic acid and monitored the formation of hydroperoxyoctadecadienoic acid as a function of enzyme concentration. Product distribution was strongly influenced by substrate concentration, such that kinetically-controlled regioisomers were enriched and thermodynamically-controlled regioisomers were depleted at high substrate concentration. Kinetic studies indicated that the formation of hydroperoxyoctadecadienoic acid saturated rapidly in an enzyme concentration-dependent manner, which implied that reactivation by reoxidation of inactive Fe(II) failed to occur. Our results support the previously proposed enzyme-initiated catalytic mechanism of the maize lipoxgyenase-1 and reveals that a substrate molecule serves as a hydrogen atom donor in its enzyme-initiated catalysis.

Bulletin of the Korean Chemical Society. (2009) vol 30, pages 719-723

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Biosynthesis and Metabolism of Jasmonates

Jasmonates are derived from oxygenated fatty acids via the octadecanoid pathway and characterized by a pentacyclic ring structure. They have regulatory function as signaling molecules in plant development and adaptation to environmental stress. Until recently, it was the cyclopentanone jasmonic acid (JA) that attracted most attention as a plant growth regulator. It becomes increasingly clear, however, that biological activity is not limited to JA but extends to, and may even differ between its many metabolites and conjugates as well as its cyclopentenone precursors. The enzymes of jasmonate biosynthesis and metabolism may thus have a regulatory function in controlling the activity and relative levels of different signaling molecules. Such a function is supported by both the characterization of loss of function mutants in Arabidopsis, and the biochemical characterization of the enzymes themselves.

Source: Journal of Plant Growth Regulation (2005) vol. 23, p. 179-199

Jasmonate-Responsive Gene Expression

Jasmonic acid (JA) and its volatile methyl ester (MeJA) belong to a family of lipid-derived signalling molecules that affect many aspects of plant life, including defence against certain pathogens and insects and some developmental processes. JA signal transduction leads to modulation of the expression of primary response genes, the products of which lead to the expression of secondary response genes. The ORCA3 transcription factor from Catharanthus roseus is a good candidate for a terminal component of the JA signal transduction pathway. To our knowledge, not a single component of the primary JA signal transduction pathway has been characterized to date in Arabidopsis. Many transcriptional components of secondary JA response pathways have been described in this model plant species, and are reviewed here. Our review advocates a strong adherence to signal transduction terminology as employed in the animal research field and in molecular biology textbooks, to simplify and correct current models about JA signal transduction leading to gene expression.

Source: Journal of Plant Growth Regulation (2005) Vol. 23, p. 200-210

12 Steps to Living Well

Kevin Bungert is the owner, head coach and motivation behind Living Well Systems, Inc., a personal motivation and coaching company located in East Northport, New York, USA. Over the years he has developed 12 steps that he use in his coaching business to help people improve the problem areas of their lives. These 12 ideas are:

1. Define Your Purpose: What is it that you really want to accomplish in life, in your business, with regards to your health, with regards to your family and friends?

2. Be Honest With Yourself: Stop blaming someone else for your circumstances, your choices equals your life! Be honest with yourself! Because when you are, than you take back total control of your life.

3. Define Your Short: Everybody has goals, whether you write them down or not, you have goals. For example, without writing it down every month, you want to pay your mortgage, right? You want to eat healthy, right? You want to be in a loving relationship, right? You want some fun in your life, right?

4. Stay Positive and Believe In Yourself: Just believe in yourself and listen to the ACHIEVERS, not the whiners!

5. Learn New Information: The reason we must learn new information is because the knowl-edge we have right now has only gotten us to the level of production that we are at right now. We want to learn how to do more business!

6. Make Different Choices: Your business can change dramatically if you choose to make it happen!

7. Take Action: We must take action! That's where the great stuff is!

8. Reprogram Your Negative: Have you ever called yourself FAT, LAZY, STUPID, SILLY, UNORGANIZED, OLD, DISGUSTING, OR A LOSER? Well, that's negative self talk! And we have ALL done it, to some degree or other. The sad part is, it's so damaging. We call ourselves names all the time, that if other people said it, we would be really hurt! Well as adults, it is up to us to STOP OUR NEGATIVE SELF TALK AND RE-PLACE IT WITH MORE HELPFUL THOUGHTS!

9. Make New Associations: It means meeting new people who can help you achieve your goals. But the rub is, they don't come and knock on your door! We have to step out of our comfort zones and go seek out their help. Are you going to ask for help to make your life better?

10. Refine - Continually: Step #10, basically means put all 9 earlier steps into action and then take a look. If you want to make a change in a certain area of your life that is caus-ing you pain, begin to put these ideas into force in your life and keep try-ing! NOTHING CHANGES OVERNIGHT, PROGRESS TAKES TIME! You job is to NOT GIVE UP! EVER!

11. Help Others: Success in life is in what you do for others.

12. Don’t Be Afraid: We are all afraid that we are not good enough ins some way… We have to accept that some things are going to scare us… and we have to move forward anyway. START TODAY! DON'T BE AFRAID!

Food processing a tool to pesticide residue dissipation

Food safety is an area of growing worldwide concern on account of its direct bearing on human health. The presence of harmful pesticide residues in food has caused a great concern among the consumers. Hence, world over to tackle food safety issues, organic farming is being propagated. However, due to several reasons, diffusion and acceptance of this approach in developing countries has been very slow. Therefore, it is important in the transient phase that some pragmatic solution should be developed to tackle this situation of food safety. Food processing treatments such as washing, peeling, canning or cooking lead to a significant reduction of pesticide residues. In this background this paper reviews the common food processing operations along with the degree of residue removal in each process. The processes reviewed include: baking, bread making, dairy product manufacture, drying, thermal processing, fermentation, freezing, infusion, juicing, malting, milling, parboiling, peeling, peeling and cooking, storage, storage and milling, washing, washing and cooking, washing and drying, washing and peeling, washing peeling and juicing and wine making. Extensive literature review demonstrates that in most cases processing leads to large reductions in residue levels in the prepared food, particularly through washing, peeling and cooking operations.

Source: Food Research International (2009) vol. 42, p. 26-40

Sleep! Sleep! Sleep!

The following can interfere with sleep:
• Caffeine - coffee, sodas, tea, chocolate
• Tobacco
• A room that is too hot or too cold
• Light
• Noise
• An uncomfortable bed
• Using alcohol before bedtime
• Being hungry
• Eating a large meal close to bedtime
• A snoring bed partner
• A pet in the bedroom
• TV in the bedroom
• Getting too stirred up before bedtime can make it hard to go to sleep

Some tips that promote sleep:
• Make 8 hours of sleep a regular habit. Sleeping less during the week and trying to catch up on the weekend doesn’t work.
• Try to go to bed at the same time every night.
• If you have a clock that is always lit up, turn it so you can’t see the time.
• Exercise every day.
• Turn off your TV and computer an hour or two before bedtime.
• If you nap, keep it short and early in the day.
• Try reading before bedtime, but use a low-watt bulb.
• Do not eat a few hours before bedtime but don’t go to bed hungry. If you eat something, choose food that is light and nutritious. Avoid spicy or greasy food.
• Take a hot bath before retiring.
• If you feel you need to worry, tell yourself that you will only worry in the daytime. Make your bedroom a worryfree zone. Learn relaxation techniques to reduce stress and worrying.
• Listen to relaxation tapes before retiring.
• Do not lay awake in bed for more than 20 to 30 minutes. Get up, do something boring for a little while, and then go back to bed.
• Your bed is for sleep and sex. If you are not doing either of these, stay out of bed.

Cell signaling under salt, water and cold stresses

Low temperature, drought, and high salinity are common stress conditions that adversely affect plant growth and crop production. The cellular and molecular responses of plants to environmental stress have been studied intensively (Thomashow, 1999; Hasegawa et al., 2000). Understanding the mechanisms by which plants perceive environmental signals and transmit the signals to cellular machinery to activateadaptive responses is of fundamental importance to biology. Knowledge about stress signal transduction is also vital for continued development of rational breeding and transgenic strategies to improve stress tolerance in crops. In this review, we first consider common characteristics of stress signal transduction in plants, and then examine some recent studies on the functional analysis of signaling components. Finally, we attempt to put these components and pathways into signal transduction networks that are grouped into three generalized signaling types.

Source: The Plant Cell (2002) vol. 14, p. S165-S183