Professor Emeritus, PH.D. Washington University, 1965
Plant Biology Program

Investigations in our lab focus on two topics: The Structural Organization of Metabolism & From Activity to ORF

 The Structural Organization of Metabolism. The cell is a very crowded place. This suggests that  pathway metabolism does not proceed by way of diffusion of intermediates from one “soluble” enzyme to the next. Rather, it has been postulated that sequential pathway enzymes interact, often transiently and, as a consequence, that intermediates are channeled from one enzyme to the next without equilibrating with molecules in the cytosol. While the evidence for this hypothesis is strong, most studies have been with in vitro systems that lack the structural organization presumed to play an important role in channeling. Elegant electron microscope studies of whole cells have demonstrated the close proximity of sequential enzymes, but data from these studies do not result in quantitative estimates of the fractional contribution of channeled flux to total flux through the pathway. We have developed methods that allow us to make quantitative estimates of the channeled flux in in vivo systems and are currently applying these methods to studying channeling in glycolysis and the oxidative limb of the pentose phosphate pathway.

 From Activity to ORF. Although vast databases of DNA sequences are now available for many organisms, there are still many known protein activities that are not associated with DNA sequences.  Identifying the connections between function and gene sequence is a fundamental and necessary step for genomics to become maximally useful in applications that are both practical and of fundamental scientific significance. A typical approach to this problem has been to grow an organism under two conditions, compare the intensity of spots on a 2D gel and identify the gene for the proteins in spots showing changes in intesity. At the end of the experiment, the investigator knows the protein and its gene, but not the reaction catalyzed by the protein. We have chosen to start with known activities not associated with an ORF. By correlating the specific activity  of samples resulting from partial purification of the protein with the quantity of the protein, we can identify proteins that might have the catalytic activity of interest. The ORFs for the limited number of proteins can be determined by mass spectrometry. These ORFs can be checked for the activity of interest by over-expressing them in an appropriate system and assaying the activity.

Selected Papers

Grayson, M. A., Walters, J. J., Hughes^, M. M., Gross, M. L .Bashkin, J, Kohl, D. H.  (2005) In vivo quantitative glucose metabolite analysis in yeast using ¹³C labeled sugars to determine enzyme channeling  In Press J. Am. Soc Mass Spectrometry

Kohl, D. H.,J-a. Koo, J. C. Lee, G. Shearer (2005) Quantitative Estimation of Channeling From Early Glycolytic Intermediates to CO₂ in Intact Escherichia coli (2004) FEBS Journal : 272 (13), 3260-3269

Debnam, P.M., G.Shearer, L. Blackwood, and D.H. Kohl (1997) Evidence for channelling of intermediates in the oxidative pentose phosphate pathwaiy by soybean and pea nodule extracts, yeast extracts, and purified yeast enzymes.  Eur. J. Biochem. 246: 283-290.

FROM ACTIVITY TO ORF

Chen, CN, G. Shearer, M. Svrakic, L. Holden, J. Dover, M. Johnston, L. Porubleva, P. Chitnis, P and D. H. Kohl (2003) Associating protein activities with their genes: rapid identification of a gene encoding a methylglyoxal reductase in the yeast Saccharomyces cerevisiae. Yeast 20: 545-554.

PROLINE CATABOLISM IN N₂-FIXING SYSTEMS

Curtis, J, G Shearer, D. H. Kohl,  (2004) Bacteroid Proline Catabolism Affects N₂ Fixation Rate of Drought-Stressed Soybeans Plant Physiol. 136: 3313-3318

Kohl, D.H., Straub P.F., and Shearer, G. Does proline play a special role in bacteroid metabolism? Plant Cell and Environment 17:1257-12628, (1994).

Kohl, D.H., Schubert, K.R., Carter, M.B., Hagedorn, C.H., and Shearer, G. Proline metabolism in N2-fixing root nodules: Energy transfer and regulation of purine synthesis. Proc. Natl. Acad. Sci. USA 85:2036-2040, (1988)

N CYCLE PROCESSES: N₂ FIXATION AND DENITRIFICATION

Shearer, G. and D.H. Kohl. 1986.  N2-fixation in field settings:  Estimations based on natural 15N abundance.  Aust. J. Plant Physiol 13: 699-757.

Shearer, G. and D.H. Kohl. 1992.  Information derived from variation in the natural abundance of 15N in complex biological systems.  In Isotopes in Organic Chemistry: Heavy Atom Isotope Effects.  Eds., E. Buncel and W.H. Saunders, Jr., Elsevier, Amsterdam.    pp. 133-190.

Shearer, G., J.R. Jones and D.H. Kohl. 1992.  The consequences of the isotope effect on proline dehydrogenation rates estimated by the tritium loss method.  Anal. Biochem. 203: 191-200.

Kohl, D.H. and G. Shearer.  1989. Ecosystem estimates of N2-fixation and investigations of nodule metabolism using variations in the natural abundance of 15N.  In: Advances in Legume Biology. Eds, C.H. Stirton and J. L. Zarucchi.  Publ.  Monographs in Systematic Botany. pp 579-594

Shearer, G., D.H. Kohl, R.A. Virginia, B.A. Bryan, J.L. Skeeters, E.T. Nilsen, M.R. Sharifi and P.W. Rundel. l983. Estimates of N2-fixation from variation in the natural abundance of 15N in Sonoran Desert ecosystems.  Oecologia (Berl.) 56:365-373.

Vitousek, P., G. Shearer, D.H. Kohl. 1989.  Floral 15N natural abundance in Hawaiian rain forest:  Patterns and possible mechanisms.  Oecologia  (Berl.): 78: 383-388.

Shearer, G. and D.H. Kohl.  1988.  Nitrogen isotopic fractionation and 18O exchange in relation to the mechanism of denitrification of nitrite by Pseudomonas stutzeri.  J. Biol. Chem.  263: 13231-13245.

Kohl, D.H., G. Shearer and J.E. Harper.  1979.  The natural abundance of 15N in nodulating and non nodulating isolines of soybeans.  In Proceedings of the Third International Conference on Stable Isotopes.  Eds. S. Klein and P. Klein.  Academic Press.  N.Y.  pp. 317-325

Shearer, G., D.H. Kohl and S.H. Chien.  1978.  15N abundance in a wide variety of soils.  Soil Sci. Soc. Amer. J. 42: 889-902.

Feigin, A., D.H. Kohl, G. Shearer and B. Commoner.  1974.  Variation in the natural nitrogen-15 abundance in nitrate mineralized during incubation of several Illinois soils.  Soil Sci. Soc. of Amer. Proc. 38: 90-95.

Kohl, D.H. and G. Shearer.  1981.  The use of soils lightly enriched in 15N to screen N2-fixing activity in plants.  Plant Soil 60: 487-489.

N CYCLE PROCESSES: SOURCES OF NITRATE N TO SURFACE WATERS

Kohl, D.H., G. Shearer and B. Commoner.  1971.  Fertilizer nitrogen:  Contribution to nitrate in surface water in a corn belt watershed.  Science 174: 1331-1334.

Kohl, D.H., G. Shearer and B. Commoner.  1972. A reply to use of variations in natural nitrogen isotope abundance for environmental studies:  A questionable approach.  Science 177: 453-456.

PHOTOSYNTHESIS

Kohl, D.H. 1972.  Photosynthesis.  In Schwarz, H.M., D.C. Borg, and J.R. Bolton (eds) Biological Application of Electron Spin Resonance, pp. 213-264.  John Wiley, New York

Kohl, D.H. and P.M. Wood. 1969.  On the molecular identity of ESR Signal II observed in photosynthetic systems:  The effect of heptane extraction and reconstitution with plastoquinone and deuterated plastoquinone.  Plant Physiology 44: 1439-1445.

Kohl, D.H., J. Townsend, B. Commoner, H.L. Crespi, R.C. Dougherty and J.J. Katz.  1965.  Effects of isotopic substitution on electron spin resonance signals in photosynthetic organisms.  Nature 206, 1105-1110.

Commoner, B., D.H. Kohl and J. Townsend.  1963.  Fast kinetics of unpaired electrons in photosynthetic systems.  Proc. Natl. Acad. Sci. U.S. 50, 638-644.

Kohl, D.H. 1965.  Studies of photosynthesis in intact cells by electron spin resonance.  Dissertation, Washington University, St. Louis, Missouri.

MISCELLANEOUS

Kohl, D. H. (2001) GM Food - Another view. The Nation. Ap 16: 7. Letters to Editor and Reply July 16, 2001

Kohl, D.H. 1973.  Food:  Meat and the environment.  Environment 15 (5):3.

Kohl, D.H.  1975.  The environmental movement:  What might it be?  Natural Resources Journal 15: 337-351.

Kohl, D.H.  1976.  The IQ game:  Bait and switch--A review essay.  School Review 84: 572–604.