Research

Industrial Biocatalysis

Enzymes are Nature’s remarkable natural catalysts found in every living organism. They are being used with increasing frequency in synthetic chemistry as an attractive, environmentally friendly alternative to conventional chemical catalysts. Their main drawback is that they tend to be highly specific for a small number of substrates and/or are not stable in the conditions used in industrial processes. However, enzyme performance can be improved by a process known as “directed evolution”, a combination of protein engineering (by semi-rational or random mutagenesis) and selective screening of the best variants. We have successfully applied this technology to the evolution of an N-acetyl-amino acid racemase (NAAAR) involved in the synthesis of chiral α-amino acids, molecules of interest for the pharma industry. We have generated libraries of mutants by selecting important residues of the NAAAR active site and developed a colorimetric high-throughput assay to screen for variants with increased activity towards a range of novel substrates.

Research in the Campopiano group is divided into three main areas:

- Industrial Biotechnology.

- Natural Product Biosynthesis.

- Development of Novel Antimicrobials.

We have successfully applied this technology to the evolution of N-acetyl-amino acid racemase (NAAAR), involved in the synthesis of chiral α-amino acids, molecules of interest for the pharma industry (Baxter et al., 2012). We generated libraries of mutants by selecting important residues of the NAAAR active site and developed a colorimetric high-throughput assay to screen for variants with increased activity towards a range of novel substrates (Sánchez-Carrón et al., 2015).

We are also interested in a number of other enzymes which can be exploited for industrial biocatalysis such as nitrile and amide synthases and transaminases

Natural Product Biosynthesis

Sphingolipid Biosynthesis

We have been studying how bacteria, mammals and yeast produce sphingolipids – these are essential components of the cell membrane and play important roles in regulation and metabolism.

In collaboration with Prof. Jim Naismith (University of St. Andrews) and Prof. Teresa Dunn (USU), we have determined the structure of Serine palmitoyltransferase (SPT), the first enzyme in the sphingolipid biosynthetic pathway which catalyses a decarboxylative, Claisen-like condensation. This work has allowed us to explore the molecular basis of human disease and the mechanism of this complex enzyme. We have also studied how SPT interacts with a range of natural and synthetic inhibitors such as cycloserine. Most recently the novel, dual-mode, inhibitory mechanism of the SPT-specific natural product myriocin was elucidated. Myriocin not only forms a stable adduct with PLP in the active site but it also generates a long chain aldehyde suicide inhibitor via a “reverse-aldol” mechanism that reacts with an essential active site lysine residue (Wadsworth et al., 2013). Currently we are studying the structure and regulation of the membrane-bound, heterodimeric human SPT complex using mass spectrometry and x-ray crystallography.

We also study other enzymes on the sphingolipid biosynthesis pathway, such as sphingosine phosphate lyase, in collaboration with Dr Allan Brown (Exeter) and Dr Jon Marles-Wright (Newcastle).

Natural Products

Natural products (NPs) are low molecular weight organic molecules made by bacteria, fungi, lichens, marine invertebrates and plants. Their revolutionary applications in the Pharmaceutical, agricultural, food and cosmetic industries cannot be underestimated. There is untapped potential in undiscovered compounds from unexplored biological sources that are now more accessible, through the advances in synthetic biology, genomics and proteomics which allow for manipulation of biosynthetic pathways.

In our group we have interest in identifying and characterizing metabolic pathways involved in the production of natural molecules such as Tambjamines (with Prof. Mike Burkart), with antimicrobial, anticancer and immunosuppressive properties. Tambjamines are natural products produced by the marine organism Pseudoalteromonas tunicata. Tambajmines have a bi-pyrole strucutre which gives rise to their characteristic yellow colour. They are cytotoxic and have displayed ion transporter characteristics and so are being investigated for use in the treatment of cystic fibrosis.

Biotin is an essential vitamin in plants and mammals functioning as the carbon dioxide carrier within central lipid metabolism. The latter stages of biotin biosynthesis are conserved between different bacterial species. However, in the first step there are different pathways for the formation of the key pimeloyl-CoA thioester intermediate. In B. subtilis BioW is one of the last remaining unexplored enzymes in the biotin biosynthesis pathway. BioW catalyses the formation of pimeloyl-CoA from pimelic acid, MgATP and CoASH. We have determined the crystal structure of BioW with our collaborator Prof. Jim Naismith (Wang et al. 2016). Using this crystal structure we have been able to engineer PCAS to catalyse the formation of novel acyl-CoA derivatives that are useful chemical building blocks. We are now building on our work to further explore, exploit and evolve this enzyme.

Protein Drug Targets

We are interested in a range of protein targets which are involved in essential metabolic reactions such as drug detoxification, metal uptake and vitamin-dependent, post-translational modifications e.g. Glutathione S-Transferases (GSTs), Ferric binding proteins (FBPs), Biotin Protein Ligases (BPLs).

Dynamic covalent chemistry (DCC) uses reversible chemical reactions to set up an equilibrating network of molecules at thermodynamic equilibrium, which can adjust its composition in response to any agent capable of altering the free energy of the system. When the target is a biological macromolecule, such as a protein, the process results in the protein directing the synthesis/amplification of its highest affinity ligand.

Previously, we demonstrated that reversible acylhydrazone formation is an effective chemistry for biological dynamic combinatorial library (DCL) formation (Bhat et al., 2010). In the presence of aniline as a nucleophilic catalyst, DCLs equilibrate rapidly at pH 6.2, are fully reversible, and may be switched on or off by a change in pH.

Our current research focusses on developing this chemistry as a drug discovery platform by applying it to other biologically relevant targets and using a combination of HPLC, native mass spectrometry and NMR to deconvolute the DCLs. Most recently we have assembled and developed an acylhydrazone library to target FabH, a key condensation enzyme in bacterial fatty acid biosynthesis.