Engineering the Mechanisms of CC Bond Forming ...

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Gavin Williams, Adam Nelson and Alan Berry. University of Leeds, teeds, LS2 9JT. Engidng theMecha&ms of C-CBond Forming Eryvms. Aldolases are a largeĀ ...
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Biochemical Society Transactions (2000) Volume 28, part 3 E n g i d n g theMecha&ms of C-CBond Forming Eryvms

Gavin Williams, Adam Nelson and Alan Berry University of Leeds,teeds,LS2 9JT Aldolases are a large p u p of enzymes that catalyze aldol condensation or cleavage. As such, these enzymes are becoming increasingly popular as biwtalysts for the synthesis of ran sugars with relevance to the phamtaceutical industry.

Fnrctose-l,6-b1sphosphate ddolase (FBP-aldolase) catalyzes the reversible aldol condensation betwem glyceraldehyde 3-phosphate and dihydroxyacetone phosphate to yield fructose 1,6-bisphosphate. The e n z y m m y s e d aldol reaction involves stabilisation of a nucleophilic enolate intermediate by either Schiff-base formation by an active site lysinc residue (Class I FBP-aldolascs), or by a divalent metal cation (Class I1 FBP-aldolases). The role of a number of residues involved in substrate recognition and catalysis on the Class I1 FBP-aldolase from Escherichia coli has been delineated by stnrctural studies and sitedirected mutagenesis. Forced evolution involving in vrtm DNA shuffling is being used to generate large libraries of mutant aldolases with novel activities. A number of high-throughput screens for novel aldolase activity are being evaluated for use in selecting desired Catalysts.

An Invwtigation into the Propertie of a Chaperone Domain and Analysis of the Non-covalently Bound Complex- with Peptide Subshates using M w s Spectrometry.

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Alison E. Ashcroft*'", Sheena E. Radford"', Joseph E. Coyle"', Mawen Pitkeathty"', Ulrich F. Hartl'", "'School of Biochemistry & Molecular Bioloev. The University of Leeds. Leeds. LS2 9JT. U.K; '*)Oxford Centre for Molecular Scknces, South Parks Road, Oxford, OX1 3QT, U.K; (')L)epartment of Cellular Biochemistry, Max Planck Institute for Biochemistry, Am Klopferspitz 18% D 8 2 152, Germany. The ability of chaperones to bind with a variety of proteins to aid folding mechanisms is an intriguing recognition event in protein chemistry. The chaperone GroEL functions alongside its cochaperonin GroES, and the interaction between the two depends on a series of residues in the "mobile loop" region of the GroES chain. The apical domain of GroEL, which has been reported to assist protein folding (l,2), was isolated to investigate the binding of GroEL with substrates. Electrospray mass spectrometry data are presented showing changes in the charge state distribution of the chaperone when acquired under difkrent pH, cone voltage and source temperature conditions, and inferences regarding the conformationalbehaviour of the apical domain are discussed. Protein-peptide binding is illustrated for both the mobile loop segment of GroES and another peptide substrate, despite these complexes being weakly bound, mainly with hydrophobic interactions. The variance of the percentage of h e and complexed apical domain protein within the charge state distribution is reported.

(I). J. Chatellier, F. Hill, P. A. Lund, A. R. Fersht, Proc. Natl. Acad. Sci. USA, 95, (17), 9861, 1998; (2). A. M. Buckle, R. Zahn, A. R. Fersht, ibid., 94, (8), 3571, 1997.

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Multi-dimensional Nuclear Magnetic Resonance Spectnwcopy to Study the Structure and Dynamics of the Class n Fructoee -1,Gbisphospbate Aldolase. Christine Hilcenko,Alan Berry, Amout P. Kalverda, Steve W. Homans. University of Leeds, Leeds LS2 9JT.

Fn~ctose-1,6-bisphosphate (FBP) aldolase catalyses the reversible aldol condensation of glyceraldehyde-3-phosphate (G3P) and dehydroxyacetone phosphate (DHAP)to form FBP. Thus FBP-aldolase plays a key role in the glycolytic and gluconeogenic metabolic pathways, and due to its ability to create carbon-carbon bonds it may prove very valuable as a catalyst in organic chemistry. However the use of this enzyme in organic chemistry is limited by the fact that the enzyme is highly specific t o w & the DHAP as the aldol donor. Thus if novel substrates were to be used the FBP-aldolase would need to be engineered to modie this specificity. This work therefore investigates the structure-function relationships of this enzyme using multidimensional nuclear magnetic resonance (NMR) spectroscopy, to aid understanding of substrate recognition and enzyme catalysis. This technique has shown to provide a malth of information about local structure, conformation and molecular dynamics, including the degree of flexibility in different regions of a molecule and the movement of different loops in the molecule upon substrate binding. Coupled with complete 'H IabeIling methods, this powerful technique gives improved resolution and sensitivity of theNMRSpeCtra.

Results from overexpression studies are presented which show that expression of the FBP-aldolase is maintained the labelling media (M9minimal media with 13C-glucoseand %&Cl) and the results from the initial NMR experiments.

0 2000 Biochemical Society

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Exploring the folding landscape of alpha helical proteins.

Andrew Capaldi', Neil Ferguson', Claire Friell, Stan Gorskil, Colin Kleanthous2and Sheena Radford'. I School of Biochemistry and Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK. School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ, UK.

The folding kinetics of a family of four small four-helical bundle proteins (the E colicin binding immunity proteins) and a number of mutants and chimers have been examined. While many of these proteins fold in simple two-state transitions, others fold either through multiple transition states or populate partially folded intermediates. This diverse behaviour is being examined in order to understand the effect of sequence, rather than topology, on the folding energy landscape. An overview of our current work will be presented.