Ammonia-lyases (ALs) represent an interesting pool of targets for the ongoing research project aimed to engineer known enzymes to modify their substrate specificity and allow the biosynthesis of 6-aminohex-2-enoic acid (6-AHEA) from lysine (Lys). This achievement would pave the way to the bio-based production of adipic acid, the industrially most important di-carboxylic acid.
Indeed, ALs catalyze the deamination of amino acids by cleaving a C-N bond i.e. the reaction necessary to convert Lys to 6-AHEA. However, a lysine AL able to catalyze the desired reaction is not known. One of the most promising targets is the 3-methylaspartate ammonia-lyase (MAL) that we aim to engineer to be able to perform the biosynthesis of 6-AHEA. Despite the fact that a few structures of the active dimer-state of MAL are available, its structural biology, functional conformational changes and dynamics around the catalytic pocket are still unknown.
Here, in collaboration with the Computational Biology Laboratory led by Prof. Elena Papaleo, we aim to investigate the molecular details of the structural biology of MAL by using all-atom molecular dynamics (MD) simulations. Indeed, all-atom simulations have been proved useful on several enzymes to describe functional dynamics, from local motions in the binding pocket to conformational changes associated with opening and closing of lid loops. Our investigation will provide novel understanding into the unknown functional mechanisms of MAL that are crucial for our project. We will perform multi-replicate MD simulations in the microsecond timescale using accurate physical models, i.e. force fields (ffs) to obtain a statistically relevant and reproducible investigation of the conformational changes observed. As starting conformations, we will use the crystallographic structure of the dimeric state of MAL in its apo state (PDB 1KKO) and in its bound state (PDB 1KKR), after removal of the substrate. In this way, we will perform simulations of MAL dimer in apo state starting from both a bound and an unbound structure. We will compare the two structural ensemble collected with simulations to evaluate when they will reach a high similarity, giving us information about their convergence and the reproducibility of our results. In total we will perform at least ten different simulations (with 5 replicates each) of two different structures of MAL, which will be analyzed by monitoring different structural parameters. We will not only investigate local motions around the active site but also conformational changes occurring in other structural elements, such as possible opening/closing motions that can be relevant for the functional mechanisms.
We will shed light on dynamics of MAL, providing crucial knowledge not only to understand its activity at the molecular level but also to guide the design of mutations that could alter the enzymatic reaction, such as accommodating new substrates. Finally, the rational design of mutations will be validated by experiments, in a continuous crosstalk with the computational part of the project.