More than 50 halogenase sequences from species of macroalgae, fungi, and bacteria have been identified from the publicly available genome, transcriptome, and metagenome resources. These were searched using sequence similarity approaches and hidden Markov models. Promising candidates have been selected for further investigations by the other core groups. In particular, an Ascophylum nodosum sequence was extracted from an inherent genome assembly and forwarded to the other core groups. Furthermore, DNA from several species was sequenced. The most advanced genome is that of Ascophyllum nodosum; the genome was assembled and mined for vanadium-dependent bromoperoxidases. Several copies were identified where the most promising candidate was forwarded. For Fucus vesiculosus and Palmaria palmata, sequencing data was also obtained and draft assemblies obtained. In addition, data on Ulva (sea lettuce) was obtained as well.
Structural models of 20 different halogenase sequences identified above and/or already available in the toolbox of CombiCom were generated. The model quality is generally lower for algi models compared to fungi, and for bacteria models compared to algi. Still, the model quality ranges from high to average. The structural models of halogenases were classified based on their molecular recognition properties using DrugsScore2018 encoding and Zernike descriptors. Evolutionary/structurally different halogenases but with similar binding sites, therefore, able to bind similar substrates, were highlighted. This is particularly important when focusing on enzymes with previously not described reactivity and selectivity. Finally, the selectivity of representative halogenases was investigated with molecular docking of selected possible substrates. Syntheses of halo-phenyl-pyrazolidine-diones previously suggested as PEPC inhibitors were established and first syntheses finalized. Furthermore, new halohydrazin-based target compounds have been suggested based on retrosynthetic analysis, computationally evaluated, and a short synthesis was established. As to halogenases, synthetic genes for the proposed sequences have been ordered, but the supply was delayed due to the pandemic. Finally, successfully expressed halogenases are currently biochemically characterized, with the most promising currently being VPO-RR from Rhodoplanes roseus.
Overall, the project progressed as planned, although strict Corona lockdown policies at Forschungszentrum Jülich led to pronounced delays. We, therefore, applied for and got granted a cost-neutral extension of the project until the end of 2021.
Prof. Dr. Holger Gohlke
Computational Pharmaceutical Chemistry and Molecular Bioinformatics
Heinrich Heine University Düsseldorf
phone: +49 211 8113662
Prof. Dr. Jörg Pietruszka & Dr. Thomas Classen, Bioorganic Chemistry, Heinrich Heine University Düsseldorf
Prof. Dr. Björn Usadel, Botany and Molecular Genetics, RWTH Aachen University
01.09.2019 - 31.08.2021
The total budget of HaloEnz is 336,340 €. HaloEnz is is part of the NRW-Strategieprojekt BioSC and thus funded by the Ministry of Culture and Science of the German State of North Rhine-Westphalia.