Di Nardo, Giovanna

Gilardi, Gianfranco

King, Pw

Morra, Simone

Valetti, Francesca

English

DI NARDO, Giovanna

Institutional Research Information System University of Turin

Studies of Cysteine 298 Role in [FeFe]-hydrogenasesSimone Morra1, Francesca Valetti1*, Giovanna Di Nardo1, Paul W. King2 and Gianfranco Gilardi11Department of Life Sciences and Systems Biology, University of Torino, Torino, Italy.      2Biosciences Center, National Renewable Energy Laboratory, Golden, Co, USA*francesca.valetti@unito.itREFERENCESCornish AJ et al., 2011, J. Biol. Chem., 286, 38341-38347. King PW et al., 2006, J. Bacteriol., 188, 2163-2172.Knörzer P et al., 2012, J. Biol. Chem., 287, 1489-1499. Lautier T et al., 2011, Faraday Discuss., 148, 385-407.Morra S et al., 2012, PLoS ONE, 7, e48400. Patrick WM et al., 2003, Protein Eng., 16, 451-457.Peters JW et al., 1998, Science, 282, 1853-1858. Reetz MT et al., 2007, Nat. Protoc., 2, 891-903.Vignais PM et al., 2007, Chem. Rev., 107, 4206-4272.CONCLUSIONS AND FUTURE PERSPECTIVESThe exclusive functional replacement of cysteine with aspartic acid, an ionisable residue, and the shift in the pH activity profile demonstrate experimentally the central role of C298 in the proton transfer pathway to the active site during [FeFe]-hydrogenase catalysis. Since C298 is strongly conserved among all the known functional [FeFe]-hydrogenases, it is reasonable that the results obtained here for CaHydA have broader implications regarding its functional significance in the entire class.Future work will aim at a more detailed characterisation of the most relevant variants, application of site saturation mutagenesis to other residues within the enzyme core and study of other [FeFe]-hydrogenases.INTRODUCTIONCysteine 298 in Clostridium acetobutylicum HydA hydrogenase (CaHydA) is a highly conserved residue in all [FeFe]-hydrogenases and it is one of the residues that are closer to the Fedatom of the H-cluster. It has previously been proposed to be involved in the catalytic cycle during proton transfer and to be an important structural element of the cavity that hosts the active site H-cluster.In order to obtain a comprehensive study of its role, a semi-random approach was used. C298 was studied by site saturation mutagenesis, to simultaneously evaluate the functional effect of cysteine replacement with all the other 19 aminoacids. A specific activity screening protocol was developed in order to test the effect of the mutations directly in libraries composed of a large number of E. coli clones and the most relevant variants have been characterised.ACKNOWLEDGEMENTSThe study was partially funded by projects BIOH2 and HyStrEM (EU structural funds N.1083/2006 P.O.R. “Competitività regionale e occupazione” F.E.S.R. 2007/2013 Asse 1 «Innovazione e transizione produttiva »Misura I.1.3 Innovazione e PMI)PURIFICATION AND CHARACTERISATION OF SELECTED VARIANTSFive variants were selected on the basis of the screening results and structural similarity and compared to the wild type enzyme. Recombinant expression in E. coli and affinity purification were accomplished under strict anaerobic conditions taking special care to decrease variability.Figure 7. Summary of the 5 variants selected and the wild type enzyme. Models of the structures were obtained using PyMol.Figure 8. Coomassie stained SDS-PAGE of CaHydA WT and the 5 selected variants (65.5 kDa).Figure 9. Far UV circular dichroism spectra, showing no major structural effect due to the mutation in the selected variants.Figure 10. Relative hydrogenase activity with methyl viologen as artificial electron mediator. H2 evolution was assayed by gas chromatography; H2 uptake was assayed spectrophotometrically. Relative activity was calculated as the ratio with the WT activity.Figure 11. Hydrogen evolution activity dependence of the pH.The secondary structure was not importantly affected, as determined by circular dichroism spectroscopy in the far UV region.The enzymatic activity assays confirmed the results obtained during the library screening: only C298D had a remarkable activity (50% in comparison to the WT). C298E and C298S showed very little residual activity, while C298N and C298A did not show any detectable activity. Also, C298D caused a shift in the pH activity profile towards acidic conditions.RANDOM LIBRARY GENERATION AND SCREENINGThe site saturation library was generated using an NNK degenerated codon, thus allowing the generation of 32 possible DNA variants, encoding for all the 20 aminoacids and requiring a reasonably low screening effort. Screening of 315 clones allowed to cover all the possible variants with a probability of completeness of 99.79%.Figure 5. Representative set of the screened clones including CaHydA WT as a control (L54).Figure 4. DNA sequencing of the plasmid library showing the expected randomisation. Figure 6. DNA sequencing of all the active clones and of a representative subset of negative clones, demonstrating the diversity of screened clones.The screening revealed that most CaHydA variants did not show any detectable hydrogenase activity, while some showed wild type-like signals. No clones with an intermediate signal could be observed. DNA sequencing revealed that the active clones were variants encoding for cysteine (codon TGT) or aspartic acid (codon GAT) only.CYSTEINE 298 IN [FeFe]-HYDROGENASESCysteine 298 is part of the L1 motif (TSCCPxW) and it is highly conserved in all [FeFe]-hydrogenases. It is located very close to the Fed atom of the H-cluster. For these reasons it might have several roles in structure/function, catalytic mechanism and oxygen sensitivity. Figure 1. Sequence alignment of various [FeFe]-hydrogenases. Figure 2. Structure model of CaHydA with theH-cluster and cysteine 298.ACTIVITY SCREENING PROTOCOLA specific screening protocol was set up to directly assay [FeFe]-hydrogenase activity in E. coli colonies. The screening takes advantage of methyl viologen (MV) reduction coupled to H2 uptake by the enzyme. The specificity and the lowest detection threshold were also tested.WT100%activityMUTANT14%activityC-0%activityFigure 3. Scheme of the activity screening mechanism and representative tests demonstrating the specificity and the lowest detection threshold.

Studies of Cysteine 298 Role in [FeFe]-hydrogenases

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Studies of Cysteine 298 Role in [FeFe]-hydrogenases

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