Jan Metske van der Laan

Structures of an Isopenicillin N Converting Ntn-Hydrolase Reveal Different Catalytic Roles for the Active Site Residues of Precursor and Mature Enzyme

Penicillium chrysogenum Acyl coenzyme A:isopenicillin N acyltransferase (AT) performs the last step in the biosynthesis of hydrophobic penicillins, exchanging the hydrophilic side chain of a precursor for various hydrophobic side chains. Like other N-terminal nucleophile hydrolases AT is produced as an inactive precursor that matures upon posttranslational cleavage. The structure of a Cys103Ala precursor mutant shows that maturation is autoproteolytic, initiated by Cys103 cleaving its preceding peptide bond. The crystal structure of the mature enzyme shows that after autoproteolysis residues 92-102 fold outwards, exposing a buried pocket. This pocket is structurally and chemically flexible and can accommodate substrates of different size and polarity. Modeling of a substrate-bound state indicates the residues important for catalysis. Comparison of the proposed autoproteolytic and substrate hydrolysis mechanisms shows that in both events the same catalytic residues are used, but that they perform different roles in catalysis.

Peroxicretion: a novel secretion pathway in the eukaryotic cell

BackgroundEnzyme production in microbial cells has been limited to secreted enzymes or intracellular enzymes followed by expensive down stream processing. Extracellular enzymes consists mainly of hydrolases while intracellular enzymes exhibit a much broader diversity. If these intracellular enzymes could be secreted by the cell the potential of industrial applications of enzymes would be enlarged. Therefore a novel secretion pathway for intracellular proteins was developed, using peroxisomes as secretion vesicles.ResultsPeroxisomes were decorated with a Golgi derived v-SNARE using a peroxisomal membrane protein as an anchor. This allowed the peroxisomes to fuse with the plasma membrane. Intracellular proteins were transported into the peroxisomes by adding a peroxisomal import signal (SKL tag). The proteins which were imported in the peroxisomes, were released into the extra-cellular space through this artificial secretion pathway which was designated peroxicretion. This concept was supported by electron microscopy studies.ConclusionOur results demonstrate that it is possible to reroute the intracellular trafficking of vesicles by changing the localisation of SNARE molecules, this approach can be used in in vivo biological studies to clarify the different control mechanisms regulating intracellular membrane trafficking. In addition we demonstrate peroxicretion of a diverse set of intracellular proteins. Therefore, we anticipate that the concept of peroxicretion may revolutionize the production of intracellular proteins from fungi and other microbial cells, as well as from mammalian cells.

Protein redesign by learning from data

Protein redesign methods aim to improve a desired property by carefully selecting mutations in relevant regions guided by protein structure. However, often protein structural requirements underlying biological characteristics are not well understood. Here, we introduce a methodology that learns relevant mutations from a set of proteins that have the desired property and demonstrate it by successfully improving production levels of two enzymes by Aspergillus niger, a relevant host organism for industrial enzyme production. We validated our method on two enzymes, an esterase and an inulinase, creating four redesigns with 5-45 mutations. Up to 10-fold increase in production was obtained with preserved enzyme activity for small numbers of mutations, whereas production levels and activities dropped for too aggressive redesigns. Our results demonstrate the feasibility of protein redesign by learning. Such an approach has great potential for improving production levels of many industrial enzymes and could potentially be employed for other design goals.