Structural enzymology

Enzyme mechanisms

For many years, Ribonucleases, N-ribohydrolases and thiamine-dependent decarboxylases have been our model systems of choice to study enzyme catalysis. Over the years, we coupled older physicochemical techniques (LFR, isotope exchange effects, steady state and fast kinetics) to modern protein engineering approaches to study the chemistry of the enzyme catalysed reaction in all its details. Current research topics include DHFR and tRNA editing enzymes.

Among cellular RNA's, transfer RNA's are by far the nucleic acids that are the most frequently and diversely posttranscriptionally modified by specific modifying enzymes. It is estimated that about 1% genome in E. coli encodes for proteins involved in RNA modification reactions. The complexity of the minimal substrates and the involved reactions make these very challenging proteins to work with, however, and have caused these enzymes to be overlooked somewhat in the past. As first systems we are focussing on enzymes involved in wobble modification and on enzymes belonging to the SPOUT methyltransferases (MTases), a large class of S-adenosyl-L-methionine-dependent enzymes that exhibit an unusual alpha/beta fold with a very deep topological knot.

We are also investigating the mechanisms by which antibodies can modulate the functions of proteins. Antibodies can elicit a number of responses in proteins of therapeutic interest, ranging from partial inhibition of enzyme activity, to allosteric activation of receptors and enzymes. However, the molecular mechanisms underlying these effects are often poorly understood. We wish to gain a better understanding of the inhibition and activation mechanisms of nanobodies by studying the effects of nanobodies on the model enzyme dihydrofolate reductase (DHFR).

In a more applied project, we are validating gated nanocontainers filled with enzymes as nanoreactors to activate particular compounds ‘in situ’. To deliver these nanocontainers to particular cells, specific camel single-domain antibodies (nanobodies) will be fused to these particles by chemical or biochemical means.

Redox Biology

Many cellular oxidation and reduction pathways rely on the relay of electrons between pairs of cysteines. Our research focusses on enzymes that rely on such pairs of cysteines for their function. Last years, we unraveled the reaction mechanism of pI258 arsenate reductase (ArsC) from S. aureus, a redox enzyme with four cysteines. Biochemistry, quantum chemistry, kinetic studies, NMR and X-ray crystallography have provided insight into the reduction mechanism of ArsC. This redox-enzyme combines a phosphatase-like nucleophilic displacement reaction with a unique intramolecular disulfide bond cascade. Currently, we focus on arsenate reductase from Corynebacterium glutamicum. This enzyme uses a completely novel redox cascade in which the sequential involvement of thiol nucleophiles are found on several partner molecules.

We also address questions regarding the specificity and promiscuity of the thioredoxin-fold. We study thioredoxin, nature’s ubiquitous reductant, and protein disulfide oxidases and isomerases. These enzymes ensure that the correct pairs of cysteine residues are joined during the oxidative folding process of cysteine containing proteins. We use wild type and disulfide mutants of RNase I as model substrates to study the Dsb oxidoreductases in E. coli. The kinetics of the oxidative folding process and the structures of redox proteins caught in action as a mixed disulfide complex present new challenges for the future. Further, in order to reach for the high hanging fruits of structural biology, we are developing novel enzyme based in vitro folding technology, Smooth Funnel technology. With this SF-technology we will efficiently fold recombinant proteins.

A close collaboration of the research group of Joris Messens with the group of Jean-Francois Collet at the de Duve Institute (UCL), resulted in the Brussels Center For Redox Biology.