University of Groningen Novel Halohydrin Dehalogenases by Protein

University of Groningen Novel halohydrin dehalogenases by protein engineering and database mining Schallmey, Marcus IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2015 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Schallmey, M. (2015). Novel halohydrin dehalogenases by protein engineering and database mining. University of Groningen. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 27-09-2021 ! "# ! $ ! % $ & ' ! 1 Junior Professorship for Biocatalysis, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany. 2 Enzymicals AG, Walther-Rathenau-Straße 49a, 17489 Greifswald, Germany. Schallmey M, Koopmeiners J, Wells E, Wardenga R, Schallmey A: Expanding the halohydrin dehalogenase enzyme family: Identification of novel enzymes by database mining. Applied and Environmental Microbiology 2014, 80 :7303–7315. © 2014 American Society for Microbiology ( ' Halohydrin dehalogenases are very rare enzymes which are naturally involved in the mineralization of halogenated xenobiotics. Due to their catalytic potential and promiscuity, many biocatalytic reactions have been described which have led to several interesting and also industrially important applications. Nevertheless, only a handful of these enzymes have been made available through recombinant techniques and hence it is of general interest to expand the repertoire of these enzymes to enable novel biocatalytic applications. After identification of specific sequence motifs, 37 novel enzyme sequences were readily identified in public sequence databases. All enzymes, which could be heterologously expressed, also catalyzed typical halohydrin dehalogenase reactions. Phylogenetic inference for enzymes of the halohydrin dehalogenase enzyme family confirmed that all enzymes form a distinct monophyletic clade within the short chain dehydrogenase/reductase superfamily. In addition, the majority of novel enzymes are substantially different to previously known phylogenetic subtypes. Consequently, four additional phylogenetic subtypes were defined which largely expand the halohydrin dehalogenase enzyme family. We show that the enormous wealth of environmental and genome sequences present in public sequence databases can be tapped for the in silico identification of very rare but nonetheless biotechnologically important biocatalysts. Our findings help to readily identify halohydrin dehalogenases in ever growing sequence databases and, in consequence, make even more members of this interesting enzyme family available to the scientific and industrial community. ' MS and AS designed the experiments. MS performed the homology searches and extracted the sequence motifs. MS designed the synthetic genes which were then subcloned, expressed and the enzymes were characterized together with JK and EW. MS, JK, EW, RW, and AS wrote the manuscript. ) * Halohydrin dehalogenases (also called haloalcohol dehalogenases, haloalcohol/ halohydrin epoxidases, or hydrogen-halide lyases; EC 4.5.1.-) (HHDHs) are biotechnologically relevant enzymes that catalyze the reversible dehalogenation of β-haloalcohols under epoxide formation [1, 2, 188]. Besides being useful for the production of enantiopure haloalcohols [24, 29, 63] and epoxides [29, 107, 174, 175], these enzymes can also be applied in the formation of novel carbon-carbon, carbon-nitrogen, or carbon-oxygen bonds. Due to their promiscuous epoxide ring-opening activity, cyanide, azide or nitrite are for example accepted as nucleophiles in the ring-opening reaction, thus leading to a diverse range of products [45]. Examples of relevant HHDH applications are the production of optically pure C3 or C4 fine chemical precursors [29, 31, 76, 174], including the multi-ton scale production of enantiopure ( R)-4-cyano-3-hydroxybutyrate esters for statin drugs [47], or the production of chiral tertiary alcohols [79, 80, 100, 101] for which conventional organic synthesis is rather challenging ( Figure 1 ) [104, 106]. " & " % !" )! ( !"! !"! % ! ' " ( ! "! !"! % # $ % Figure 1. Examples of HHDH-catalyzed reactions include A) the preparation of (optically pure) haloalcohols and epoxides [29] as well as B) synthetic routes towards statin side chain precursors [47] and C) tertiary alcohols [80, 100]. Despite their designated potential as biocatalysts, few HHDHs have thus far been made available to the scientific and industrial community since the initial discovery of bacterial enzymes with HHDH activities more than 45 years ago. Since then, a couple of bacterial species have been reported to possess HHDH activity but only very few HHDH enzymes have been purified and characterized biochemically, which has been recently reviewed elsewhere in detail [2, 188]. Of these, only six HHDH genes have been cloned and expressed recombinantly, namely hheA from Corynebacterium sp. strain N-1074 [32], hheA2 from + ( Arthrobacter sp. strain AD2 [9], hheB from Corynebacterium sp. strain N-1074 [32], hheB2 from Mycobacterium sp. strain GP1 [9] and two identical hheC sequences from Agrobacterium radiobacter AD1 [9] and Rhizobium sp. strain NHG3 [33]. All of the cloned HHDHs belong to the short-chain dehydrogenase/reductase (SDR) superfamily and exhibit several major features of this diverse enzyme class [9, 56, 58]. For example, all known HHDHs make use of a catalytic triad, share the commonly found homomultimeric quarternary assembly, and both crystallized HHDHs, namely HheA2 [39] and HheC [38], possess a tertiary structure similar to other Rossmann-fold proteins. Besides these overall similarities to SDR enzymes, HHDHs can be distinguished from SDR enzymes by a combination of mechanistic and sequence/structure characteristics. The concerted activity of Ser-Tyr-Lys in classical SDR enzymes abstracts a proton from the substrate’s hydroxyl group and an enzyme-bound NAD(P) + cofactor is responsible for hydride abstraction. In contrast, HHDHs possess a catalytic triad that is composed of Ser-Tyr-Arg and, instead of a cofactor-binding site, a spacious anion-binding pocket is present in the structures of HheA2 and HheC [38, 39]. In consequence, all known HHDH sequences form only a minute but well- defined fraction within the SDR superfamily with more than 163,000 SDR enzymes which can be retrieved from UniProt [10]. Based on activity profiles and sequence identities, the available HHDH enzymes have been classified into three different phylogenetic subgroups, namely type A, B, or C [9]. HHDHs within each type share more than 97% sequence identity while the identity between enzymes of different types is below 33%. Due to these high sequence identities within each of the three subtypes, only three different HHDH enzymes are currently available for biotechnological exploitation. Although these few known HHDHs have already given rise to many interesting applications, it is of great interest to increase the number of functionally diverse HHDH enzymes [2, 188]. A viable approach to generate novel and functionally diverse enzymes employs rational and random protein engineering strategies which can yield drastically improved and functionally diverse enzyme variants [190]. Such strategies have been successfully applied to HheA2 [41, 176, 203] and HheC [34, 37, 47, 110] addressing specific drawbacks of the respective parental enzymes and yielding HHDH mutants with sometimes substantial improvements towards target reactions. Nevertheless, parental sequences will always govern the overall accessible sequence space in every protein engineering study. For example, HheC has been shown to be extraordinarily tolerant to mutations at 153 of its total 254 residues with a maximum of 42 simultaneous substitutions per variant described [47]. However, these heavily engineered enzyme variants are still rather similar to parental HheC, e.g. HheC-2360 [177] with more than 85% sequence identity. Clearly, novel sequences would be a valuable addition to the functional diversity of the HHDH enzyme toolbox. Further, novel HHDH enzymes might already exhibit activities or characteristics which are difficult to engineer or even unlikely to be accessible by laboratory evolution. Herein, we report the identification of novel HHDHs in publicly available sequence databases by making use of specific sequence motifs which allow for the unambiguous discrimination of true HHDH sequences from the vast number of other SDR sequences. * % Sequence characteristics of HHDHs Firstly, in order to identify novel HHDH enzymes, all known HHDH sequences were inspected for distinctive residues which distinguish

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