Download PCB 5530 Take-home exam 2008

PCB 5530 Take-home exam 2008
Background
Proline accumulation. Besides being a protein amino acid, proline is an osmoprotectant or
compatible solute that accumulates in response to osmotic stress (drought and salinity) in many
bacteria and plants (1,2). The proline biosynthesis and degradation pathways and their regulation
have accordingly been well studied in bacteria and plants and the synthesis pathway is a target
for metabolic engineering of stress resistance in both groups of organisms (3-5).
1. Verbruggen N, Hermans C (2008) Proline accumulation in plants: a review. Amino Acids 2008 Apr 1
[Epub ahead of print]
2. Wood JM, Bremer E, Csonka LN, Kraemer R, Poolman B, van der Heide T, Smith LT (2001)
Osmosensing and osmoregulatory compatible solute accumulation by bacteria. Comp Biochem
Physiol A Mol Integr Physiol 130:437-60
3. Fujita T, Maggio A, Garcia-Rios M, Stauffacher C, Bressan RA, Csonka LN (2003) Identification of
regions of the tomato gamma-glutamyl kinase that are involved in allosteric regulation by proline. J
Biol Chem 278:14203-10
4. Dandekar AM, Uratsu SL (1988) A single base pair change in proline biosynthesis genes causes
osmotic stress tolerance. J Bacteriol 170:5943-5.
5. Adams E, Frank L (1980) Metabolism of proline and the hydroxyprolines. Annu Rev Biochem
49:1005-61.
Questions
1. Draw a scheme showing the chemical structures and enzymes of the proline biosynthesis pathways in plants and bacteria, and indicate the names of the E. coli genes. Indicate also the compartments in which the plant enzymes are located. Describe in 100 words or less the main ways
in which the pathway is regulated in E. coli and in plants, giving complete literature references.
2. One of various attempts to engineer greater proline accumulation in plants is:
Hong Z, Lakkineni K, Zhang Z, Verma DP (2000) Removal of feedback inhibition of delta(1)-pyrroline5-carboxylate synthetase results in increased proline accumulation and protection of plants from osmotic
stress. Plant Physiol 122:1129-36.
a. In one sentence, state what metabolic engineering/MCA principle(s) this study illustrates.
b. Assuming all proline is in the cytoplasm, convert the data in Fig. 2 to units of millimolar proline in the cytoplasm, making a reasonable and clearly stated assumption about the relationship
between fresh weight and cytoplasmic volume. Comment briefly on whether the data recalculated in this way support the idea that the wild-type transgene product is severely feedbackinhibited in salt-stressed plants.
c. Using reasonable and clearly stated assumptions about the total reduced (amino) nitrogen content (protein + non-protein) of leaves, estimate the percentage of total reduced nitrogen that is
accounted for by proline in vector-alone control plants without NaCl stress, and in F129A line 3
plus NaCl stress. What does the latter value suggest about prospects for engineering a further
increase in proline accumulation?
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3. The YggS protein of bacteria (the E. coli YggS sequence is below) is also present in plants and
other eukaryotes. YggS has no known function although its sequence shows clearly that it is an
enzyme belonging to a well-known class.
>E_coli_YggS
MNDIAHNLAQVRDKISAAATRCGRSPEEITLLAVSKTKPASAIAEAIDAGQRQFGENYVQ
EGVDKIRHFQELGVTGLEWHFIGPLQSNKSRLVAEHFDWCHTIDRLRIATRLNDQRPAEL
PPLNVLIQINISDENSKSGIQLAELDELAAAVAELPRLRLRGLMAIPAPESEYVRQFEVA
RQMAVAFAGLKTRYPHIDTLSLGMSDDMEAAIAAGSTMVRIGTAIFGARDYSKK
a. Indicate the class of enzyme to which YggS belongs, the cofactor that is specific to this class,
the typical substrates, and the main types of reaction mediated by this class of enzymes.
b. Use SEED to search for associations (clustering on the chromosome, co-occurrence in genomes) between YggS and known metabolic enzymes (i.e. those with EC numbers). Create a subsystem; name it YggS_XX where XX are the initials of your name. [Your subsystem is part of
your answer and will count towards the grade for this homework – so be sure to ‘publish’ it]. For
the strongest associations, summarize your findings in 100 words or less, indicating the major
bacterial groups in which the associations are seen. List the organisms in which YggS has been
shown to be essential or non-essential.
c. Give the AGI codes(s) for the yggS gene(s) in Arabidopsis. Use three named prediction algorithms and alignment with the E. coli and yeast YggS proteins to predict the subcellular location.
Integrate all this evidence to make a one-sentence conclusion about localization of the protein(s).
State in one sentence how the predicted localization(s) relate to localization of ProC in plants.
d. Using the Golm Transcriptome DB Multiple Expression Query tool, plot the profiles of
Arabidopsis yggS gene expression with respect to development and to stress in the root and
shoot. Summarize in one sentence the main inference(s) that you can draw from these data about
the biochemical function of the yggS gene(s).
e. Using the Golm Transcriptome DB Transcript Co-response tool, compare the expression
patterns of the Arabidopsis yggS gene(s) with those of the metabolic enzyme(s) with which you
found associations in 3b above. Present the results as Tables showing all correlations among
these genes for developmental, and stress aboveground and belowground datasets. Summarize
any inferences about biochemical function of the yggS gene(s) in one sentence.
f. Using the Golm Transcriptome DB Transcript Co-Response tool, check for classes of genes
(Mapman ‘Bins’) that are especially highly represented among the top 100 genes in the developmental dataset that are positively or negatively correlated with each Arabidopsis yggS gene.
Summarize any inferences about biochemical function of the yggS gene(s).
g. Use all the information that you gleaned in a-f above to formulate one specific hypothesis
about the reaction mediated by YggS, and draw the structures of the substrate(s) and product(s).
Provide a brief (100 words or less) rationale for your hypothesis. Then explain in 150 words or
less how the hypothesis fits the observations, and how it does not.
h. State in 100 words or less how you would test this hypothesis experimentally via a genetic or a
biochemical approach. The experimental tests can be made in any suitable organism; use
Arabidopsis only if it offers a unique advantage for the tests you propose.
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