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Jan Zarzycki
PostDoc (fourth year) Department of Biochemistry, Plant Biology, Michigan State University, East Lansing, MI Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA Michigan State University DOE-Plant Research Laboratory 612 Wilson Road, Room 222 East Lansing, MI 48824 Phone: (517) 432-4349 E-mail: jzarz@msu.edu, jzarzycki@lbl.gov 
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A bicarbonate fixing photorespiratory bypass
As in plants, autotrophic CO2 fixation in cyanobacteria occurs primarily by the Calvin-Benson cycle via the key enzyme ribulose-1,5-bisphosphate carboxylase/ oxygenase (RubisCO). The efficiency of this enzyme is severely limited because it is unable to discriminate between carbon dioxide and molecular oxygen. The incorporation of oxygen instead of carbon dioxide results in photorespiration and leads to the formation of 2‑phosphoglycolate. This toxic compound has to be removed or, ideally, recycled. The first step of detoxification is the dephosphorylation of 2-PG to glycolate. To salvage glycolate cyanobacteria can use of several strategies (Zarzycki et. al., 2013). In any case, photorespiration always results in a significant loss of previously fixed carbon and nitrogen. Underscoring the impact of photorespiration, disruptions of these pathways in cyanobacteria or plants usually result in growth retardation and/or high CO2 requiring phenotypes. We are engineering a glycolate/glyoxylate salvaging pathway that circumvents the loss of carbon and actually fixes CO2 instead. This is accomplished by making use of enzymes that are involved in the 3‑hydroxipropionate bi-cycle, an alternative autotrophic CO2 fixation pathway found in the thermophilic filamentous anoxygenic phototroph Chloroflexus aurantiacus (Zarzycki et al. 2009). One turn of this designed cyclic photorespiratory bypass consumes glyoxylate, fixes bicarbonate, and finally provides pyruvate which can be used directly for biosynthesis or to replenish the CB cycle (Shih et al., 2014).
As in plants, autotrophic CO2 fixation in cyanobacteria occurs primarily by the Calvin-Benson cycle via the key enzyme ribulose-1,5-bisphosphate carboxylase/ oxygenase (RubisCO). The efficiency of this enzyme is severely limited because it is unable to discriminate between carbon dioxide and molecular oxygen. The incorporation of oxygen instead of carbon dioxide results in photorespiration and leads to the formation of 2‑phosphoglycolate. This toxic compound has to be removed or, ideally, recycled. The first step of detoxification is the dephosphorylation of 2-PG to glycolate. To salvage glycolate cyanobacteria can use of several strategies (Zarzycki et. al., 2013). In any case, photorespiration always results in a significant loss of previously fixed carbon and nitrogen. Underscoring the impact of photorespiration, disruptions of these pathways in cyanobacteria or plants usually result in growth retardation and/or high CO2 requiring phenotypes. We are engineering a glycolate/glyoxylate salvaging pathway that circumvents the loss of carbon and actually fixes CO2 instead. This is accomplished by making use of enzymes that are involved in the 3‑hydroxipropionate bi-cycle, an alternative autotrophic CO2 fixation pathway found in the thermophilic filamentous anoxygenic phototroph Chloroflexus aurantiacus (Zarzycki et al. 2009). One turn of this designed cyclic photorespiratory bypass consumes glyoxylate, fixes bicarbonate, and finally provides pyruvate which can be used directly for biosynthesis or to replenish the CB cycle (Shih et al., 2014).
Multifunctional malyl-CoA/β-methylmalyl-CoA/citramalyl-CoA lyases
Malyl-CoA lyases (MCLs) are promiscuous enzymes that catalyzes the reversible carbon-carbon- bond cleavage of structurally related Coenzyme A thioesters (Zarzycki and Kerfeld, 2013). They play important, multifunctional roles for example in the 3‑hydroxypropionate bi-cycle for autotrophic CO2 fixation in Chloroflexus aurantiacus, as well as in the ethylmalonyl-CoA pathway for acetate assimilation as studied in Rhodobacter sphaeroides. MCLs belong to the large superfamily of CitE-like enzymes, which also includes the β-subunit of citrate lyase (CitE), malyl-CoA thioesterases, and other C-C bond lyases. The CitE-like enzyme superfamily also bears sequence and structural similarities to malate synthases. All of these different enzymes share highly conserved catalytic residues, although they catalyze distinctly different reactions: C-C bond formation and cleavage, thioester hydrolysis, or even both. We solved the crystal structures of C. aurantiacus and the R. sphaeroides MCLs in apo- and substrate-bound forms (Zarzycki and Kerfeld, 2013). Although both MCLs share only relatively low amino acid sequence identity (~37%), their tertiary and quaternary structures are very similar. Comparison to known malate synthase structures raised the question why the MCLs are not also able to hydrolyze CoA thioester bonds. Site directed mutagenesis studies as well as further structural studies of other CitE-like structures may help to solve this question.
Malyl-CoA lyases (MCLs) are promiscuous enzymes that catalyzes the reversible carbon-carbon- bond cleavage of structurally related Coenzyme A thioesters (Zarzycki and Kerfeld, 2013). They play important, multifunctional roles for example in the 3‑hydroxypropionate bi-cycle for autotrophic CO2 fixation in Chloroflexus aurantiacus, as well as in the ethylmalonyl-CoA pathway for acetate assimilation as studied in Rhodobacter sphaeroides. MCLs belong to the large superfamily of CitE-like enzymes, which also includes the β-subunit of citrate lyase (CitE), malyl-CoA thioesterases, and other C-C bond lyases. The CitE-like enzyme superfamily also bears sequence and structural similarities to malate synthases. All of these different enzymes share highly conserved catalytic residues, although they catalyze distinctly different reactions: C-C bond formation and cleavage, thioester hydrolysis, or even both. We solved the crystal structures of C. aurantiacus and the R. sphaeroides MCLs in apo- and substrate-bound forms (Zarzycki and Kerfeld, 2013). Although both MCLs share only relatively low amino acid sequence identity (~37%), their tertiary and quaternary structures are very similar. Comparison to known malate synthase structures raised the question why the MCLs are not also able to hydrolyze CoA thioester bonds. Site directed mutagenesis studies as well as further structural studies of other CitE-like structures may help to solve this question.
Publications (*corresponding author):
- Gaudana, S.B., Zarzycki, J., Moparthi, V.K., and Kerfeld, C.A.* 2014. Bioinformatic Analysis of the Distribution of Inorganic Carbon Transporters and Prospective Targets for Bioengineering to Increase Ci uptake by Cyanobacteria. Photosynth. res. submitted
- Shih, P.M., Zarzycki J., Niyogi, K.K.*, and Kerfeld, C.A.* 2014. Introduction of a Synthetic CO2 Fixing Photorespiratory Bypass into a Cyanobacterium. J. Biol. Chem. Feb 20. [Epub ahead of print] LINK
- Zarzycki J. and Kerfeld, C.A.* 2013. The Crystal Structures of the tri-functional Chloroflexus aurantiacus and bi-functional Rhodobacter sphaeroides Malyl-CoA Lyases and Comparison with CitE-like Superfamily Enzymes and Malate Synthases. BMC Struct. Biol. 13:28 LINK
- Zarzycki, J., Axen, S.D., Kinney, J.N., and Kerfeld, C.A.* 2013. Cyanobacterial-based approaches to improving photosynthesis in plants. J. Exp. Bot. 64:787-798 LINK
- Kleiner, M., Wentrup, C., Lott, C., Teeling,H., Wetzel, S., Young, J., Chang, Y.J., Shah, M., VerBerkmoes, N.C., Zarzycki, J., Fuchs, G., Markert, S., Hempel, K., Voigt, B., Becher, D., Liebeke, M., Lalk, M., Albrecht, D., Hecker, M., Schweder, T., and Dubilier, N.* 2012. Metaproteomics of a gutless marine worm and its symbiotic microbial community reveal unusual pathways for carbon and energy use. Proc. Natl. Acad. Sci. U.S.A. 109:E1173-E1182 LINK
- Zarzycki, J. and Fuchs, G.* 2011. Co-assimilation of organic substrates via the autotrophic 3-hydroxypropionate bi-cycle in Chloroflexus aurantiacus. Appl. Environ. Microbiol. 77:6181-6188 LINK
- Kroeger, J.K., Zarzycki, J., and Fuchs, G.* 2011. A spectrophotometric assay for measuring acetyl-coenzyme A carboxylase. Anal. Biochem. 411:100-105 LINK
- Berg, I.A., Kockelkorn, D., Ramos-Vera, W.H., Say, R.F., Zarzycki, J., Hügler, M., Alber, B. E., and Fuchs, G.* 2010. Autotrophic carbon fixation in archaea. Nat. Rev. Microbiol. 8:447-460. LINK
- Berg, I.A., Kockelkorn, D., Ramos-Vera, W.H., Say, R.F., Zarzycki, J., and Fuchs, G.*. 2010. Autotrophic Carbon Fixation in Biology: Pathways, Rules, and Speculations. In M. Aresta (ed.), Carbon Dioxide as Chemical Feedstock. Wiley-VCH, Weinheim, Germany. LINK
- Zarzycki, J., Brecht, V., Müller, M., and Fuchs, G.* 2009. Identifying the missing steps of the autotrophic 3-hydroxypropionate CO2 fixation cycle in Chloroflexus aurantiacus. Proc. Natl. Acad. Sci. U.S.A. 106:21317–21322 LINK
- Zarzycki, J., Schlichting, A. Strychalsky, N., Müller, M., Alber, B.E., and Fuchs, G.* 2008. Mesaconyl-coenzyme A hydratase, a new enzyme of two central carbon metabolic pathways in bacteria. J. Bacteriol. 190:1366-1374. LINK