Aldoxime dehydratases (Oxd) are enzymes, which catalyze the dehydration of aldoximes to nitriles in an aqueous environment under mild reaction conditions. These enzymes enable a biocatalytic, cyanide and co-factor-free access towards a variety of different nitriles. The first enzymes were discovered by the Asano group about two decades ago and naturally these enzymes play a role in the aldoxime-nitrile pathway in bacteria (Asano and Kato, 1998, Asano, 2002, Oinuma et al., 2003, Rädisch et al., 2022). Up to now thirteen aldoxime dehydratases are known (Betke et al., 2018, Chen et al., 2021, Rädisch et al., 2018, Křístková et al., 2023). The X-ray structure of three of these aldoxime dehydratases are solved, namely from OxdRE, an aldoxime dehydratase from Rhodococcus sp. N-771 (Kato et al., 2004) (PDB 3a15, 3a16, 3a17) (Sawai et al., 2009), from OxdA an aldoxime dehydratase from Pseudomonas chlororaphis B23 (Oinuma et al., 2003) (PDB 3W08) and OxdB an aldoxime dehydratase from Bacillus sp. OxB-1 (Matsui et al., 2022) (PDB 7F30). Aldoxime dehydratases carry a heme b as a prosthetic group in their active site, which is surrounded by a catalytic triade. Usually, the catalytic triade is formed by an arginine, histidine and serine. In the catalytic cycle the nitrogen of the aldoxime substrate is coordinated by the iron(II) in the heme b group, while the hydroxyl group of the aldoxime function is fixed by the serine, which is part of the catalytic cycle. First, the histidine is protonated by the arginine located in close proximity. This increases the electrophilicity of the histidine and it can protonate the hydroxyl function of the aldoxime. The protonation allows removal of a water molecule and the α-carbon of the aldoxime orients towards the histidine. The dehydrated substrate with a resulting linear structure of Fe=N=C is interacting with serine, which forms a hydrogen bond with the hydrogen of the α-carbon. In a last step, the acidic α-carbon hydrogen is transferred to the histidine, and a nitrile is formed and released (Nomura et al., 2013). This mechanism enables a dehydration of an aldoxime to a nitrile in an aqueous environment under mild reaction conditions. This mechanism is enabled by a catalytic triad, which is conserved among the known aldoxime dehydratases, but very recently a novel aldoxime dehydratase was found, namely OxdFv from Fusarium vanettii. This novel enzyme has a different catalytic triad consisting of two glutamine and one arginine (Křístková et al., 2023). Aldoxime dehydratases show a broad substrate scope starting from aliphatic aldoximes over aryl-aliphatic aldoximes, cyclic aliphatic aldoximes, to aromatic aldoximes (Chen et al., 2021, Betke et al., 2017, Domínguez de María, 2021). They show remarkable activities towards aliphatic aldoximes with a certain chain length (C6-C10) and process stability when used as whole-cell catalyst. This could be shown for the aldoxime dehydratase OxdB from Bacillus sp. OxB-1, which could convert n-octanaloxime (8) with a substrate loading of 1.4 kg/L in a feeding approach with whole-cells as catalyst (Hinzmann et al., 2019). This high process stability and product tolerance of OxdB was used to established a chemo- and bio-catalytic cascade reaction for the production of aliphatic nitriles from alcohols in the product nitrile as solvent (Hinzmann et al., 2020a). Another approach towards nitriles starting from carboxylic acids was established very recently. In this approach the carboxylic acid is reduced to the aldehyde by carboxylate reductase enzymes (CARs). The oxime is then formed in situ chemically by condensation with hydroxylamine and in a last step the oxime is dehydrated via aldoxime dehydratases (Horvat et al., 2022, Winkler et al., 2023). Besides a broad substrate scope and high productivities, aldoxime dehydratases show an interesting behavior in chiral nitrile syntheses. Depending on the E/Z ratio of the aldoxime substrate the enantioselectivity changes. With one enzyme both enantiomers can be produced from a racemic substrate mixture, and this behavior could recently be rationalized via molecular docking (Yavuzer et al., 2021). These examples revealed aldoxime dehydratases as useful enzymes with industrial relevance, but they still have their limitations. The substrate scope of aldoxime dehydratase is broad, but they show activity only for very few aromatic aldoximes. Only two enzymes are known to convert a limited number of aromatic aldoximes, namely an aldoxime dehydratase from Rhodococcus sp. YH3-3 (OxdYH3) with limited activity for para-methyl and para-chloro-substituted benzaldoxime as well as for 2-furfuryl aldoxime. This limited activity could be increased up to 6 fold by a mutagenesis study by Choi et al. (1998); Choi et al., (2020) The second enzyme with activity for aromatic aldoximes is an aldoxime dehydratase from Pseudomonas putida F1 (OxdF1). This enzyme can efficiently catalyze the dehydration from 2-bromobenzaldoxime and 2-chloro-6-fluorobenzaldoxime (Chen et al., 2021). Besides the challenging aromatic aldoximes, sterically hindered aldoximes and heteroaromatic aldoximes turned out to be difficult substrates for aldoxime dehydratases. Furthermore, aldoxime dehydratases can convert chiral nitriles enantioselectively, but there is no enzyme known, which can selectively produce only one enantiomer out of an E/Z substrate mixture. These examples show that there is a need to find new aldoxime dehydratases with enhanced activity, enantioselectivity, stability and/or different substrate scope.

There are various ways to find new enzymes, one of them is genome mining based on the primary sequence of the proteins. This was recently successfully applied to identify and characterize a new aldoxime dehydratase from Pseudomonas putida F1 (OxdF1) (Chen et al., 2021). However, this method has the limitation that only primary sequence information is included, but no structural information. An alternative is provided by a 3DM-database, which is based on the three-dimensional structure of the proteins. 3DM is a web-based software developed by the company Bio-Prodict (Kuipers et al., 2010). This company generates a customized library, which is accessed by the software 3DM. The library is based on structural alignment enabled by a matrix based numbering scheme. The protein structures are either available in the protein database or generated as models. Based on this numbering, large metagenome databases are screened and protein structures and models are aligned and compared with the target protein. This results in a large database based on three-dimensional similarities of the included proteins. Because those multiple structure alignments contain evolutional information, such as conserved regions or correlated mutations, they can also be placed in phylogenetic context with the generation of subfamilies. The 3DM system was already used for identification of novel enzymes like the discovery of a thermostable amine transaminase from Burkholderia multivorans (Kollipara et al., 2022). In this work, we used the 3DM system to identify novel Oxds from a 3DM database based on aldoxime dehydratase OxdB, which has been provided by Bio-Prodict.