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The Abell Group

 

Our initial focus was on the enzymes from E. coli. We cloned, sequenced and over-expressed the genes for the four enzymes. X-ray crystal structures of the enzymes were solved in a collaboration in the laboratory of Professor Tom Blundell at the Department of Biochemistry, University of Cambridge.

KPHMT

The structure of E. coli KPHMT with product bound was solved to 1.8 Å resolution using a combination of native and selenomethionine proteins. This was a significant technical achievement as there were 160 selenium atoms in the unit cell. 

Ketopantoate reductase

We carried out extensive structural and calorimetric studies on KPR. We have solved the structure of the apo-enzyme, the KPR:NADP+ binary complex and the KPR:NADP+:pantoate ternary complex, and used these to gain a structural understanding of the catalytic pathway.

Ternary complex: KPR: pantoate and NADP+:

Aspartate decarboxylase

We solved the structure of the tetrameric protein and remarkably, the structure included a backbone ester, a key intermediate in protein processing. In order to study both processing and catalysis, we made a large number of mutants and characterised them kinetically and crystallographically. We have determined the stereochemistry of the decarboxylation and developed a rapid screen to look at the substrate specificity using MALDI TOF MS and developed a novel assay using isothermal titration calorimetry.

Pantothenate synthetase

Pantothenate synthetase catalyses the ATP-dependent condensation of pantoate and β-alanine. The reaction proceeds via a pantoyl adenylate intermediate. We solved the unliganded structure of E. coli pantothenate synthetase to 1.7 Å and synthesised a sulfonamide analogue of the pantoyl adenylate intermediate and showed it to be a potent inhibitor.

Pantothenate pathway in plants: we used radiochemical feeding experiments to detect pathway intermediates in plants. We cloned two KPHMT genes from Arabidopsis and demonstrated that they are functional. We have verified the identity of a putative yeast panC gene by functional complementation, and isolated a cyanobacterial panC gene. In addition we have isolated cDNAs for pantothenate synthetase from several higher plants including Lotus japonicus and rice, wheat and Arabidopsis, providing definitive evidence for the pathway in plants.

Related Publications 

Structure of E-coli ketopantoate hydroxymethyl transferase complexed with ketopantoate and Mg2+, solved by locating 160 selenomethionine sites
F von Delft, T Inoue, SA Saldanha, HH Ottenhof, F Schmitzberger, LM Birch, V Dhanaraj, M Witty, AG Smith, TL Blundell, C Abell – Structure (London, England : 1993) (2003) 11, 985
Comparative Analysis of the Escherichia coli Ketopantoate Hydroxymethyltransferase Crystal Structure Confirms that It Is a Member of the (  )8 Phosphoenolpyruvate/Pyruvate Superfamily
F Schmitzberger, AG Smith, C Abell, TL Blundell – Journal of bacteriology (2003) 185, 4163
Probing hot spots at protein-ligand binding sites: A fragment-based approach using biophysical methods
A Ciulli, G Williams, AG Smith, TL Blundell, C Abell – Journal of Medicinal Chemistry (2006) 49, 4992
Crystal Structure of Escherichia coli Ketopantoate Reductase at 1.7 Å Resolution and Insight into the Enzyme Mechanism †
D Matak-Vinković, M Vinković, SA Saldanha, JL Ashurst, F von Delft, T Inoue, RN Miguel, AG Smith, TL Blundell, C Abell – Biochemistry (2001) 40, 14493
The crystal structure of Escherichia coli ketopantoate reductase with NADP+ bound
CMC Lobley, A Ciulli, HM Whitney, G Williams, AG Smith, C Abell, TL Blundell – Biochemistry (2005) 44, 8930
Crystal structure of Escherichia coli ketopantoate reductase in a ternary complex with NADP+and pantoate bound: Substrate recognition, conformational change, and cooperativity
A Ciulli, DY Chirgadze, AG Smith, TL Blundell, C Abell – Journal of Biological Chemistry (2007) 282, 8487
Escherichia coli l-aspartate-α-decarboxylase: preprotein processing and observation of reaction intermediates by electrospray mass spectrometry
MK Ramjee, U Genschel, C Abell, AG Smith – Biochem J (1997) 323 ( Pt 3), 661
Crystal structure of aspartate decarboxylase at 2.2 A resolution provides evidence for an ester in protein self-processing.
A Albert, V Dhanaraj, U Genschel, G Khan, MK Ramjee, R Pulido, BL Sibanda, F von Delft, M Witty, TL Blundell, AG Smith, C Abell – Nat Struct Biol (1998) 5, 289
Structural constraints on protein self-processing in L-aspartate-α -decarboxylase
F Schmitzberger, ML Kilkenny, CMC Lobley, ME Webb, M Vinkovic, D Matak-Vinkovic, M Witty, DY Chirgadze, AG Smith, C Abell, TL Blundell – EMBO Journal (2003) 22, 6193
Identification of Tyr58 as the proton donor in the aspartate-α-decarboxylase reaction
SA Saldanha, LM Birch, ME Webb, BK Nabbs, F von Delft, AG Smith, C Abell – Chemical Communications (2001) 1, 1760
Rapid screening by MALDI-TOF mass spectrometry to probe binding specificity at enzyme active sites.
ME Webb, E Stephens, AG Smith, C Abell – Chemical communications (Cambridge, England) (2003) 9, 2416
The crystal structure of E. coli pantothenate synthetase confirms it as a member of the cytidylyltransferase superfamily.
F von Delft, A Lewendon, V Dhanaraj, TL Blundell, C Abell, AG Smith – Structure (London, England : 1993) (2001) 9, 439
Nanomechanical detection of antibiotic-mucopeptide binding in a model for superbug drug resistance.
JW Ndieyira, M Watari, AD Barrera, D Zhou, M Vögtli, M Batchelor, MA Cooper, T Strunz, MA Horton, C Abell, T Rayment, G Aeppli, RA McKendry – Nat Nanotechnol (2008) 3, 691
Evidence for the pathway to pantothenate in plants
CE JONES, JE DANCER, AG SMITH, C ABELL – Canadian Journal of Chemistry (1994) 72, 261
Organisation of the pantothenate (vitamin B 5 ) biosynthesis pathway in higher plants
HH Ottenhof, JL Ashurst, HM Whitney, SA Saldanha, F Schmitzberger, HS Gweon, TL Blundell, C Abell, AG Smith – Plant J (2004) 37, 61
The final step of pantothenate biosynthesis in higher plants: Cloning and characterization of pantothenate synthetase from Lotus japonicus and Oryza sativum (rice)
U Genschel, CA Powell, C Abell, AG Smith – Biochem J (1999) 341 ( Pt 3), 669
Pantothenate biosynthesis in higher plants: Advances and challenges
E Chakauya, KM Coxon, HM Whitney, JL Ashurst, C Abell, AG Smith – Physiologia Plantarum (2006) 126, 319