Advance%20Biochemistry

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Advance Biochemistry
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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

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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)

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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

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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

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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?

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http//www.sigmaaldrich.com/img/assets/ 4202/sigma
_metabolic_path-new.pdf
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Plant and animal biochemistry sometimes differ
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Methods of regulation

1. Properties of enzymes
2. Compartmentation
3. Gene expression

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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

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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

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

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The family of plastids
Buchannan et al. Fig. 1.44
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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

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Phylogenetic location of chloroplasts and
mitochondria
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DNA for chloroplast proteins can be in the
nucleus or chloroplast genome
Buchannan et al. Fig. 4.4
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Import of proteins into chloroplasts
Buchannan et al. Fig. 4.6
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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

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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)

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Photorespiration is highly compartmentalized
Buchannan et al. Fig. 1.40
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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

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Gene structure relevant to metabolic regulation
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Promoters
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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

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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.
  1987125-14.
• Koshland DE. Switches, thresholds and
  ultrasensitivity. Trends Biochem.Sci.
  198712225-9.