Ph.D. Molecular Biology
1993, University of Pennsylvania
School of Medicine
Research Focus Areas
Protein Structure and Function
Acetyl-CoA, a key metabolite at the interface of a number of metabolic pathways, is generated from the breakdown of carbohydrates, lipids, and amino acids, and from the activation of acetate. Acetate kinase (ACK) catalyzes the reversible phosphorylation of acetate by ATP yielding acetyl phosphate and ADP. ACK is a key enzyme in bacterial metabolism and physiology, and its primary role in bacteria is to function with phosphotransacetylase (PTA: acetyl phosphate + HSCoA ↔ acetyl-CoA + Pi) to form a pathway for the interconversion of acetate and acetyl-CoA. Under high acetate conditions, the pathway can operate to activate acetate to acetyl-CoA for use as a carbon and/or energy source. The ACK-PTA pathway is completely reversible and thus also plays a vital catabolic role. Under conditions that result in mixed acid fermentation, acetyl-CoA cannot enter the TCA cycle and excess acetyl-CoA is used to produce acetate and ATP via the ACK-PTA pathway. Under aerobic conditions, when the carbon flux into cells exceeds the amphibolic capacity of the central metabolic pathways, cells again use the ACK-PTA pathway to excrete acetate and generate ATP.
ACK was previously thought to be found strictly in prokaryotes; however, our laboratory has identified genes encoding ACK in a number of eukaryotic microbes. In the alga Chlamydomonas reinhardtii and oomycete Phytophthora spp., PTA appears to be the partner enzyme for ACK, as is usually the case in bacteria. However, PTA has not been identified in other eukaryotic microbes.
ACK partners with the bacterial enzyme XFP in Cryptococcus neoformans and other fungi.
We have identified xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (XFP) as a putative partner enzyme in all fungi that have ACK. XFP, also previously thought to be a strictly bacterial enzyme, catalyzes the formation of acetyl phosphate from either xylulose 5-phosphate or fructose 6-phosphate and inorganic phosphate. One fungus with the ACK-XFP pathway is Cryptococcus neoformans, a basidiomycete fungal pathogen of humans that has diverged considerably from other model fungi such as Neurospora crassa, Aspergillus nidulans, Saccharomyces cerevisiae, and the common human fungal pathogen Candida albicans. C. neoformans is an invasive opportunistic pathogen of the central nervous system and the most frequent cause of fungal meningitis worldwide. Although exposure to C. neoformans is common, an increased rate of infection occurs in individuals with impaired cell-mediated immunity, particularly those with AIDS and recipients of immunosuppressive therapy. Recombinant C. neoformans ACK has been produced and purified to electrophoretic homogeneity and biochemical and kinetic characterization is nearly complete. Unlike prokaryotic ACKs, which show a preference for the two-carbon substrate acetate but can also utilize the three-carbon substrate propionate very well, C. neoformans ACK can only utilize acetate and has very limited activity with propionate. Activity in the acetyl phosphate forming direction is substantially lower (100-fold or more) than that observed for prokaryotic ACKs. This suggests that the physiological function of C. neoformans ACK is to catalyze the reaction in the opposite direction (acetyl phosphate + ADP → acetate + ATP) for energy generation.
Preliminary gene knockout experiments in a haploid C. neoformans strain suggest that ACK may be essential. C. neoformans is also one of several fungi that have two putative genes encoding XFP. RNA interference of XFP2, but not XFP1, resulted in a severe growth defect. We are currently confirming the essentiality of these genes.
Entamoeba histolytica PPi-dependent ACK may have a novel role.
Entamoeba histolytica, which infects about 50 million people worldwide, is the third leading cause of morbidity and mortality due to parasitic disease in humans (after malaria and schistosomiasis) and is estimated to be responsible for 50,000 - 100,000 deaths every year. Infection can lead to amoebiasis, resulting from trophozoites invading the intestinal wall and spreading from the intestine via the bloodstream to the liver, lung, and brain. Amoebiasis is predominantly seen in developing countries where the high incidence of infection is the result of fecal contamination of the food and water supply, factors that cannot be easily resolved because of the limited financial resources of these countries. E. histolytica is listed by NIAID as a category B priority pathogen with Bioterrorism Potential due to its low infectious dose and potential for dissemination through compromised food and water supplies.
Recombinant E. histolytica ACK has been produced and purified to electrophoretic homogeneity and biochemical and kinetic characterization is complete. The kinetic characterization has shown that this enzyme utilizes inorganic pyrophosphate (PPi) as the sole phosphoryl donor but cannot utilize nucleotide triphosphates (i.e., ATP), as is the case with all other characterized ACKs. The Entamoeba enzyme also has a much broader acyl substrate range than other ACKs, utilizing fatty acids as long as hexanoate. The Entamoeba ACK not only is unique enzymatically, but may have a novel physiological role. Genes encoding PTA, XFP, or any other known enzyme that produces acetyl phosphate as a product are absent.
ACK as a possible drug target.
ACK is present in a number of significant human pathogens including the eukaryotic microbes C. neoformans, E. histolytica, Aspergillus fumigatus, Histoplasma capsulatum, and Coccidioides species as well as a number of bacterial pathogens listed under the NIAID A, B, and C categories of priority pathogens for biodefense such as Salmonella species, Staphylococcus aureus, Listeria monocytogenes, Mycobacterium tuberculosis, Yersinia species, Bacillus anthracis, and Clostridium botulinum. Thus, the presence of ACK in these pathogens and its absence in humans, animals, and plants suggests ACK may provide a novel target for chemotherapeutic agents.
National Institutes of Health
The Presence of a Bacterial Metabolic Pathway in Eukaryotic Fungi
National Science Foundation
Biochemistry and Physiology of Novel Acetate Kinases from Eukaryotic Microbes
Biochemistry of Metabolism
General Biochemistry Lab II
Biochemical Basis of Disease
Microbial Gene Regulation
M. Fowler, C. Ingram-Smith, and K.S. Smith. 2011. Direct detection of the acetate-forming activity of the enzyme acetate kinase. JOVE, in press.
R. Barber, L. Zhang, M. Harnack, M. Olson, R. Kaul, C. Ingram-Smith, K. Smith. 2011. Complete genome sequence of Methanosaeta concilii, a specialist in aceticlastic methanogenesis. Journal of Bacteriology 193:3668-9.
Y. Meng, C. Ingram-Smith, L. Cooper, and K. S. Smith. 2010. Characterization of an archaeal medium-chain acyl-CoA synthetase from Methanosarcina acetivorans. Journal of Bacteriology 192:5982-90.
M. B. Shah, C. Ingram-Smith, L. Cooper, J. Qu, Y. Meng, K. S. Smith, and A. M. Gulick. 2009. The 2.1 Å crystal structure of an acyl-CoA synthetase from Methanosarcina acetivorans reveals an alternate acyl-binding pocket for small branched acyl substrates. Proteins 77:685-698.
J. W. Chambers, M. T. Morris, K. S. Smith, and J. C. Morris 2008. Residues in an ATP binding domain influence sugar binding in a trypanosome hexokinase. Biochemical and Biophysical Research Communications 365:420-5.
K. S. Smith and C. Ingram-Smith. 2007 Methanosaeta, the abandoned aceticlastic methanogen. Trends in Microbiology 15:150-5.
M. T. Morris, C. DeBruin, Z. Yang, J. W. Chambers, K. S. Smith, and J. C. Morris. 2006. Activity of a second Trypanosoma brucei hexokinase is controlled by an 18 amino acid C-terminal tail. Eukaryotic Cell. 5: 2014-23.
C. Ingram-Smith and K. S. Smith. 2006. AMP-forming acetyl-CoA synthetases in Archaea show unexpected diversity in substrate utilization. Archaea 2: 2: 95-107.
C. Ingram-Smith, B. I. Woods, and K. S. Smith. 2006. Characterization of the acyl substrate binding pocket of acetyl-CoA synthetase. Biochemistry 45: 11482-11490.
C. Ingram-Smith, S. R. Martin, and K. S. Smith. 2006. Acetate kinase: not just a bacterial enzyme. Trends in Microbiology 14: 249-253.