In silico approach for amine dehydrogenase discovery – MODAMDH

MODAMDH is an innovative project combining bio-informatics, chemo-informatics and biocatalysis to identify diverse AmDHs among biodiversity. It aims to combine methodologies only scarcely used for biocatalytic purposes. Native AmDHs are searched both by sequence-driven analysis approaches using distant homology and by 3D structure-guided approaches. To enlarge the catalogue of AmDHs, the biodiversity is screened using not only the generalist UniProtKB database, but also publicly available genomic and metagenomics data resources, in addition to the Genoscope ones. The different steps are:
- definition of the reference AmDH family, clusterization, generation of hidden Markov models (HMM) profiles and catalophores (minimal active site topology) for each subgroup
- definition of the NAD(P)-dependent enzyme pool to be screened, clusterization, generation of HMM profiles and 3D models for each family
- selection of distant homologues based on HMM/HMM search between NAD(P)-dependent enzyme families and reference AmDHs ones
- identification of new structurally different AmDHs families from the screening of the reference catalophores within the 3D-enzyme models of NAD(P)-dependent enzyme families
- production and in vitro tests of selected enzymes
- iterative approach using newly experimentally validated AmDHs and their resolved structure

Biodiversity screening, i.e. 2.6 billion sequences retrieved from 8 publicly available (meta)genomic resources, yielded 20.3 million NAD(P)-dependent protein sequences, which were then grouped into 104,734 subfamilies and for each of them an HMM profile was generated. The former reference AmDH family (2,011 sequences) has been extended to 27,282 sequences thanks to the addition of homologous sequences present in the metagenomic resources. Members of this updated family were then classified using phylogeny (4 groups, 4 HMM profiles) and an active site classification (ASMC pipeline: 4 groups, 4 HMM profiles). Comparison of these reference HMM profiles with the HMM profiles of NAD(P)-dependent proteins identified 27 distant homologs which, when included in previous classifications, revealed no new phylogenetic or structural groups. The AmDH activity of the sequence presenting the lowest homology with known proteins was confirmed by in vitro activity tests, as well as for 42 enzymes representative of the diversity of the extended family. Several AmDHs showing drastically extended substrate spectra compared to the AmDHs identified at the beginning of the project are being characterized. The search for structural analogues could not be completed due to the complex structural dynamics of AmDHs during catalysis which, given the computational resources available, made the search for catalophores inappropriate.
These results have given rise to 2 publications in peer-reviewed journals and a main publication currently in progress, as well as 7 communications in French and European scientific congresses.

The MODAMDH project has allowed a considerable advance in the knowledge of the AmDH activity within biodiversity, which greatly extends the diversity of the portfolio of biocatalysts for the synthesis of amines. The new substrate spectrum of some AmDHs identified during the project and their very low homology with the previous reported AmDHs open the door to numerous applications in synthesis and structural studies. MODAMDH is also a solid base for the implementation of a generic workflow for the constitution and the subsequent analysis of a resource of enzyme families (ESR/EquipEx+ ALADIN project); MODAMDH highlights the need of combining bioinformatics and biocatalysis in order to expand enzymatic resources for the future of chemistry.

- Publication on main results : currently in progress
- Fossey-Jouenne et al; Front. Catal. ; 2023 ; 3:1105948 ; doi.org/10.3389/fctls.2023.1105948
- Ducrot et al; ChemCatChem ; 2022 ; 14, 22 ; doi.org/10.1002/cctc.202200880

7 communications (oral, poster) in scientific congresses

In a current context of waste reduction, catalysis and more particularly biocatalysis, meets the needs of a more sustainable chemistry. The presence of chiral amines in many synthetic intermediates of key pharmacological, agronomic or other industrial compounds, leads in the search for biocatalytic methods of access to these molecules in enantiomerically pure forms. Amine dehydrogenases (AmDHs), catalyzing the asymmetric reductive amination of ketones by only the use of an inexpensive amine source, ammonia, and a regenerable cofactor, are one of the most promising alternative to conventional synthesis. The discovery of native AmDHs, by the scientific coordinator of MODAMDH project and her collaborators, allowed assigning the first genes to this function, thus widening this panel of enzymes previously restricted to engineered enzymes. To meet the criteria for the development of the biocatalysts in the industry, particularly their stability, activity, selectivity, specificity or substrate spectra, new enzymes have to be found. Mining genomes and metagenomes constitutes a powerful and complementary way to access novel sequences offering high diversity and original features. Strategies dealing with the sole pairwise alignment of primary sequences make it possible to discover numerous enzymes that meet the needs but generally lead to enzymes belonging to already known families. The aim of the MODAMDH project is to screen biodiversity by two more innovative approaches using distant homology and three-dimensional topology of active sites within filtered genomic and metagenomic data. This will help to widen the enzymatic frameworks catalyzing reductive amination and to access to homologues with various structures, having extended characteristics, in particular in terms of substrate spectrum or complementary stereoselectivities. The structural analysis of all experimentally validated AmDHs will make it possible to propose targets for protein engineering required to improve their use in synthesis. The expected results will also allow these research methods to flourish within the biocatalysis community, by further promoting the progress of associated bio-informatics tools.