Office: 843-792-0845
Lab: 843-792-0875
Fax: 843-792-8565
Email: krupenko@musc.edu
BSB-512B

Research Interests

My research interests lay in the area of protein structure and function, enzyme mechanisms and enzyme regulation. At present my lab is focused on studies of one of the major enzymes of folate metabolism. Folate coenzymes carry out one-carbon transfers and play an essential role in several major cellular processes, including nucleic acid biosynthesis, mitochondrial and choloroplast protein biosynthesis, amino acid metabolism, methyl group biogenesis, vitamin metabolism and DNA/protein methylation. Higher animals are unable to synthesize folate and thus depend on the diet to provide the folate necessary for their metabolic function. Folate deficiency causes a number of severe disorders, increases the risk of vascular disease and also causes uracil misincorporation into DNA and chromosome breakage that is implicated in progression of cancer. Numerous enzymes are involved in folate metabolism interconverting the coenzymes by transferring one-carbon units. The multifunctional multidomain enzyme, 10-formyltetrahydrofolate dehydrogenase (FDH) regulates 10-formyltetrahydrofolate and tetrahydrofolate pools converting 10-formyltetrahydrofolate to tetrahydrofolate. The enzyme is a natural fusion of two unrelated proteins and has two catalytic centers, which work in concert to create enzyme activity. The mainstream project in my lab is directed to explore FDH structure and mechanism. This includes: characterization of the folate binding site of FDH and identification of amino acid residues important for folate binding and catalysis; study of the mechanisms of FDH activation; study of FDH oligomerization; resolution of the crystal structure of FDH and its domains expressed as separate proteins; study of the role of the flexibility of FDH domains in the protein structure and enzyme regulation.  FDH substrate, 10-formyltetrahydrofolate, is also a substrate for two reactions in the de novo purine biosynthesis pathway. This allowed us to hypothesize that FDH may regualte purine biosynthesis by controlling the level of 10-formyltetrahydrofolate, thus influencing cellular metabolism. We have recently discovered that overexpression of FDH suppresses growth of several types of cancer cells. We have further observed that this enzyme being abundant in normal tissues is absent in most cancers. This suggests that in order to proliferate, cancer cells must "turn off" the enzyme and, in opposite, high enzyme levels will inhibit cancer cell proliferation. We propose that this enzyme suppresses cancer cells through inhibition of biosynthesis of purines, DNA/RNA precursors, thus inhibiting nucleic acid biosynthesis and cell growth/proliferation. This can also influence DNA repair and other purine-dependent processes that might contribute in the FDH suppressor effects and cancer cell apoptosis. The enzyme might also be relevant to individual proclivity of patients to carcinogenesis: its lower levels in certain types of tissues might indicate preexisting conditions for tumor development or early stages of initiated oncogenesis. Therefore, we have recently started a new project to investigate the role of FDH as a tumor suppressor, to explore the mechanisms of inhibitory effects of FDH on cancer cells and to elucidate the role of the enzyme in carcinogenesis. These studies apply cell culture models, human tumor xenografts in nude mice and apoptosis-related techniques to address the above questions. FDH will be further evaluated as a potential target for gene therapy.

Selected Publications

Click here to view list of publications on the National Library of Medicine's PubMed online database.

• Chumanevich, A.A., Krupenko S.A., and Davies, C.  (2004) The crystal structure of the hydrolase domain of 10-       formyltetrahydrofolate dehydrogenase: mechanism of hydrolysis and its interplay with the dehydrogenase domain.        J. Biol. Chem. (in press, e-pub ahead of print Jan. 16, 2004)

• Oleinik, N.V., and Krupenko, S.A. (2003) Ectopic expression of FDH in A549 cells induces G1 cell cycle arrest and apoptosis. Mol. Cancer Res. 1, 577-588

• Reuland, S.N., Vlasov, A.P., and Krupenko, S.A. (2003) Disruption of a calmodulin central helix-like region of FDH impairs its dehydrogenase activityby uncoupling the functional domains. J. Biol. Chem., 278, 22894-22900

• Krupenko, S.A., and Oleinik, N.V. (2002) 10-Formyltetrahydrofolate dehydrogenase, one of the major folate enzymes, is down-regulated in tumor tissues and possesses suppressor effect on cancer cells. Cell Growth Diff. 13, 227-236

• Chumanevich, A.A.,  Davies, C., and Krupenko, S.A. (2002) Crystallization and preliminary X-ray diffraction analysis of recombinant hydrolase domain of 10-formyltetrahydrofolate dehydrogenase. Acta Cryst.  D58, 1841-1842

• Krupenko, S.A., Vlasov, A.P., and Wagner, C. (2001) On the role of conserved histidine 106 in 10-formyltetrahydrofolate dehydrogenase catalysis: connection between hydrolase and dehydrogenase mechanisms. J. Biol. Chem., 276(26): 24030-24037.
• Krupenko, S.A., and Wagner, C. (1999) Aspartate 142 is involved in both hydrolase and dehydrogenase catalytic centers of 10-formyltetrahydrofolate dehydrogenase. J. Biol. Chem. 274:35777-35784.
• Krupenko, S.A., and Wagner, C. (1998) Overexpression of functional hydrolase domain of rat liver 10-formyltetrahydrofolate dehydrogenase in Escherichia coli. Protein Expression and Purification 14:146-152.
• Krupenko, S.A., Wagner, C., and Cook, R.J. (1997) Expression, purification and properties of the aldehyde dehydrogenase homologous carboxyl-terminal domain of rat 10-formyltetrahydrofolate dehydrogenase. J. Biol. Chem. 272:10266-10272.
• Krupenko, S.A., Wagner, C., and Cook, R.J. (1997) Domain structure of rat 10-formyltetrahydrofolate dehydrogenase. J. Biol. Chem. 272:10273-10278.
• Krupenko, S.A., Wagner, C., and Cook, R.J. (1995) Cysteine 707 is involved in the dehydrogenase active site of rat 10-formyltetrahydrofolate dehydrogenase. J. Biol. Chem. 270:519-522.
• Krupenko, S.A., Wagner, C., and Cook, R.J. (1995) Recombinant 10-formyltetrahydrofolate dehydrogenase catalyses both dehydrogenase and hydrolase reactions utilizing the synthetic substrate 10-formyl-5,8-dideazafolate. Biochem. J. 306:651-655.
• Krupenko, S.A., Horstman, D.A., Wagner, C., and Cook, R.J. (1995) Baculovirus expression and purification of rat 10-formyltetrahydrofolate dehydrogenase. Protein Expression and Purification 6:457-464.