Studies on enzymes mechanism and selectivity using synthetic substrate analogues

Organic chemistry is a valuable tool for studying enzyme mechanisms. Upon incubation with a specific enzyme, synthetic substrate analogues labeled with heavy atoms or carrying extra functional groups can provide mechanistic insights. In the present work, new compounds were synthesised in order to study the mechanism and substrate selectivity of two enzymes: human γ-butyrobetaine hydroxylase and bacterial carboxymethylproline synthase.
γ-Butyrobetaine hydroxylase (BBOX) is an Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenase that catalyses the stereospecific hydroxylation of γ-butyrobetaine, the final step of L-carnitine (L-Car) biosynthesis in mammals. Substrate analogues were synthesised to probe BBOX specificity in vitro. Some of those unnatural substrates were oxidised by BBOX and the products identified using a range of analytical techniques. 3-(2,2,2-Trimethylhydrazinium)propionate (THP) is a clinically used BBOX inhibitor. Under standard assay conditions, THP was oxidised by BBOX. NMR studies have identified the products of this reaction to be malonic acid semialdehyde, formaldehyde, dimethylamine and 3-amino-4-(methylamino)butanoic acid. The formation of 3-amino-4-(methylamino)butanoic acid suggests that BBOX can catalyse a Stevens type rearrangement involving N-N bond cleavage and C-C bond formation. The proposed structures and mechanisms were confirmed by mass spectrometric and NMR analyses using [13C]-labeled THP as well as synthetic standards of both enantiomers of 3-amino-4-(methylamino)butanoic acid. Although the structure of the rearrangement product was confirmed, the stereochemistry remains unknown. Altogether, these studies revealed the unprecedented nature of a BBOX-catalysed C–C bond formation reaction upon THP oxidation and may inspire the design of improved inhibitors for BBOX and other 2OG oxygenases.
Pectobacterium carotovorum CarB and Streptomyces cattleya ThnE are two carboxymethylproline synthases (CMPS) that catalyse an early step in carbapenem antibiotics biosynthesis. CMPS produces (2S,5S)-carboxymethylproline (t-CMP) from malonyl-CoA and L-glutamate semi-aldehyde. L-Glutamate semi-aldehyde exists in equilibrium with L-5-hydroxyproline and L-pyrroline-5-carboxylate in solution (collectively abbreviated L-GHP). Because of the high stereoselectivity of t-CMP formation and the growing interest in novel carbapenem antibiotics, CMPS is potentially an interesting biocatalyst. A series of L-GHP analogues were synthesised and tested as CMPS substrates in an attempt to produce unnatural t-CMP derivatives enzymatically. Methyl-substituted L-GHP analogues were accepted by CMPS and the t-CMP products could be further carried through to the corresponding bicyclic carbapenams using CarA, a β-lactam synthetase. These results demonstrate the versatility of the early carbapenem biosynthetic pathway and the possibility of introducing structural diversity using synthetic substrate analogues. A crystal structure of S. cattleya ThnE was obtained in complex with L-proline and coenzyme A, giving the first insight into substrate binding. This structural information will potentially allow further rational mutagenesis studies aiming to broaden the range of unnatural L-GHP analogues accepted by CMPS.

Keywords: Carbapenems
Carnitine
structural biology
biosynthesis
enzymology
Advisor: Schofield, C