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!