Advance Biochemistry

1. Advance Biochemistry

2. Introduction • Goals • To cover aspects of biochemistry unique and important to plants • Sometimes will involve bacterial biochemistry • See some of the many biochemical pathways critical to plants (Structures will be shown!) • Hear about techniques important in plant biochemistry • Molecular biology, mass spectrometry etc. • Major emphasis on regulation • Grading • Three one hour exams (in class, Tuesdays) (90 points each) • 30 points for homework assignments • Total points at the end used to determine grade

3. Textbook and reading • Biochemistry &Molecular Biology of Plants, ASPB • Plant Biochemistry and Molecular Biology by Hans-Walter Heldt • Readings from original literature (PDFs supplied for UW-Madison licensed materials)

4. Overall plan • Cell and Cellular Constituents Cell structure and functions Water and solutions Carbohydrates Fatty acids and lipids Amino acids and protein Enzymes Vitamins and minerals • Metabolism • Strategy for Processing of Nutrients in Plants • Applied Biochemistry

5. Overall plan • Photosynthesis • Carbon metabolism, Electron transport • Nitrogen, reduction and metabolism • Carbon end products • Cell constituents • Membranes, Cell walls • Cellular metabolism • Ion pumps • Protein turnover • Symbiotic nitrogen fixation

6. Regulation of Metabolism • Plant cells do a wide range of biochemistry • Regulation of metabolism • Stoichiometric requirements (e.g. amino acids) • Avoid waste (energy that is needed when it is needed) • Directionality of metabolism • Most reactions are reversible • The cytoplasm as a soup, how does anything get done?

7. http://www.sigmaaldrich.com/img/assets/ 4202/sigma_metabolic_path-new.pdf

9. Plant and animal biochemistry sometimes differ

10. Methods of regulation • Properties of enzymes • Compartmentation • Gene expression

11. Methods of regulation • Properties of enzymes • Affinity for substrate, inherent catalytic capacity • Feedback regulation/feedforward/loopgain • Allosteric effects, competitive versus non-competitive inhibition • Fructose 2,6-bisphosphate as an example • Redox control of enzymes (vicinal cysteines can become cystine) • pH and Mg regulation • Especially chloroplast enzymes

12. Methods of regulation • Properties of enzymes (Post-translational regulation) • Phosphorylation • Protein kinases and phosphatases • Turns enzymes on or off, can affect sensitivity to effectors (SPS) • Fatty acids • Palmitic acid in a regulatory way, myristic acid is non-regulatory • Prenylation • Fanesylation (3 isoprenoids, 15 C) CaaX C-terminus • Geranylgeranylation (20 carbons) CaaL C-terminus • Fatty acids and prenylation anchors proteins to membranes or to other proteins

13. Anchoring proteins to membranes Buchannan et al. (ASPB book) Fig. 1.10 page 9

14. Methods of regulation • Cellular compartmentation • Hallmark of eukaryotic cells • Oxygen reactions mostly in mitochondria and chloroplasts • Chloroplasts – more generally plastids – are what make plants unique • Cell walls, vacuoles also distinctive but not unique • Plastids are biochemical powerhouses • I hope this course will leave you with an appreciation for the unique biochemistry of plants, and where in the cell it happens

15. The family of plastids Buchannan et al. Fig. 1.44

16. Endosymbiosis • Well accepted that chloroplasts and mitochondria were once free living bacteria • Their metabolism is bacterial (e.g. photosynthesis) • Retain some DNA (circular chromosome) • Protein synthesis sensitive to chloramphenicol • Cytosolic P synthesis sensitive to cycloheximide • Most genes transferred from symbiont to nucleus • Requires protein tageting

17. Phylogenetic location of chloroplasts and mitochondria

18. DNA for chloroplast proteins can be in the nucleus or chloroplast genome Buchannan et al. Fig. 4.4

19. Import of proteins into chloroplasts Buchannan et al. Fig. 4.6

20. Biochemistry inside plastids • Photosynthesis – reduction of C, N, and S • Amino acids, essential amino acid synthesis restricted to plastids • Phenylpropanoid amino acids and secondary compounds start in the plastids (shikimic acid pathway) • Site of action of several herbicides, including glyphosate • Branched-chain amino acids • Sulfur amino acids • Fatty acids – all fatty acids in plants made in plastids

21. Biochemistry inside plastids • Carotenoids – source of vitamin A • Thiamin and pyridoxal, B vitamins • Ascorbic acid – vitamin C • Tocopherol – vitamin E • Phylloquinone (an electron accepttor in PS I – vitamin K)

22. Photorespiration is highly compartmentalized Buchannan et al. Fig. 1.40

23. Methods of regulation • Gene expression • Normally slow relative to metabolic control that will be discussed most of the time in this course • Allows metabolism to be changed in response to environmental factors • Transcriptional control most common • Sometimes variation in transcription rate not reflected in enzyme amount • Translational control also found • No change in mRNA levels but changes in protein amounts

24. Gene structure relevant to metabolic regulation

25. Promoters

26. Exploring metabolism by genetic methods • Antisense – what happens when the amount of an enzyme is reduced • not clear how antisense works • Knockouts • Often more clear-cut since all of the enzyme is gone • Use of t-DNA, Salk lines • Overexpression • Use an unregulated version of the protein or express on a strong promoter • Sometimes leads to cosuppression • RNA interference • 21 to 26 mers seem very effective in regulating translation

27. What do we expect for the reaction of metabolism to changes in amount of an enzyme? • Is subtracting 50% likely to give exactly the opposite result as adding 50%? • Are there threshholds? • Are there optimal amounts? • Are there compensatory pathways? • Are there compensatory regulatory mechanisms? • Kacser H, Porteous JW. Control of metabolism: what do we have to measure. Trends Biochem.Sci. 1987;12:5-14. • Koshland DE. Switches, thresholds and ultrasensitivity. Trends Biochem.Sci. 1987;12:225-9.