|Professor Chris Abell|
Our initial focus was on the enzymes from E. coli. We cloned, sequenced and over-expressed the genes for the four enzymes, and solved the X-ray crystal structures of the enzymes.
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 [Structure 2003]. The protomer is a closed β-barrel, a member of the (βα)8 phosphoenolpyruvate/pyruvate superfamily [J. Bacteriol. 2003]. The decamer is toroidal comprising two closed superposed circles, each of five protomers.
We carried out extensive structural and calorimetric [J. Med. Chem. 2006] studies on KPR. We have solved the structure of the apo-enzyme [Biochemistry 2001], the KPR:NADP+ binary complex [Biochemistry 2005], and the KPR:NADP+:pantoate ternary complex [Chem. Commun. 2007], and used these to gain a structural understanding of the catalytic pathway.
Ternary complex: KPR: pantoate and NADP+[Chem. Commun. 2007]
Aspartate decarboxylase belongs to small group of decarboxylases that uses an integral pyruvoyl group attached to the N-terminus of one of its protein subunits. The protein is initially translated as a proenzyme, which is processed at the Gly24-Ser25 bond to produce a β-subunit with a carboxyl group at its C-terminus and a α-subunit with a pyruvoyl group at its N-terminus [Biochem. J. 1997]. We solved the structure of the tetrameric protein [Nature Structural Biology 1998]. 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 then kinetically and crystallographically [EMBO J. 2003]. We have determined the stereochemistry of the decarboxylation [Chem. Commun. 2001], developed a rapid screen to look at the substrate specificity using MALDI TOF MS [Chem. Commun. 2003] and developed a novel assay using isothermal titration calorimetry.
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 [Structure 2001] and synthesised a sulfonamide analogue of the pantoyl adenylate intermediate and showed it to be a potent inhibitor [Nature Nanotechnology 2008].
Pantothenate pathway in plants We used radiochemical feeding experiments to detect pathway intermediates in plants [Can. J. Chem. 1994]. We cloned two KPHMT genes from Arabidopsis, and demonstrated that they are functional [Plant J. 2004]. 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 [Biochem. J. 1999], wheat and Arabidopsis[Plant J. 2004], providing definitive evidence for the pathway in plants [Physiologia Plantarum 2006].