Characterization of a Solvent-Tolerant Amidohydrolase Involved in Natural Product Heterocycle Formation
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catalysts Article Characterization of a Solvent-Tolerant Amidohydrolase Involved in Natural Product Heterocycle Formation Lea Winand, Dustin Joshua Vollmann, Jacqueline Hentschel and Markus Nett * Department of Biochemical and Chemical Engineering, TU Dortmund University, Emil-Figge-Str. 66, 44227 Dortmund, Germany; [email protected] (L.W.); [email protected] (D.J.V.); [email protected] (J.H.) * Correspondence: [email protected]; Tel.: +49-231-7554742 Abstract: Heterocycles are important building blocks in pharmaceutical drugs and their enzymatic synthesis is attracting increasing interest. In recent years, various enzymes of the amidohydrolase superfamily were reported to catalyze heterocycle-forming condensation reactions. One of these enzymes, MxcM, is biochemically and kinetically characterized in this study. MxcM generates an imidazoline moiety in the biosynthesis of the natural product pseudochelin A, which features potent anti-inflammatory properties. The enzyme shows maximal activity at 50 ◦C and pH 10 as −1 −1 well as a kcat/Km value of 22,932 s M at its temperature optimum. Experimental data suggest that the activity of MxcM does not depend on a catalytic metal ion, which is uncommon among amidohydrolases. MxcM is highly active in diverse organic solvents and concentrated salt solutions. Furthermore, we show that MxcM is also capable to introduce imidazoline rings into derivatives of its natural substrate myxochelin B. Overall, MxcM is a solvent-stable, halotolerant enzyme with promising biochemical and kinetic properties and, in future, might become a valuable biocatalyst for the manufacturing of pharmaceutical drugs. Citation: Winand, L.; Vollmann, D.J.; Hentschel, J.; Nett, M. Keywords: amidohydrolase; biocatalysis; heterocycle; imidazoline; MxcM; natural product; sol- Characterization of a Solvent-Tolerant vent tolerance Amidohydrolase Involved in Natural Product Heterocycle Formation. Catalysts 2021, 11, 892. https:// doi.org/10.3390/catal11080892 1. Introduction The amidohydrolases constitute a large enzyme family with more than 36,000 mem- Academic Editor: Frank Schulz bers [1]. They feature a common (β/α)8-barrel structural fold and typically possess a metal Received: 3 July 2021 center, which is required for the activation of a water molecule [2–4]. While most members Accepted: 22 July 2021 of the amidohydrolase family catalyze hydrolytic cleavage reactions, there are noteworthy Published: 24 July 2021 exceptions, as exemplified by the uronate isomerase, which interconverts aldoses and ketoses and does not depend on divalent cations for enzymatic activity [5]. In recent years, Publisher’s Note: MDPI stays neutral several amidohydrolases have been reported that are involved in the biosynthesis of phar- with regard to jurisdictional claims in macologically active natural products [6–10]. The corresponding enzymes are engaged in published maps and institutional affil- heterocycle-forming condensation reactions (Figure1A), which is consistent with a shifted iations. reaction equilibrium. Examples are the amidohydrolases BomN, CbxE, and NatAM, which generate benzoxazole moieties in the biosyntheses of antibiotic A-33853, caboxamycin and nataxazole (Figure1B) [ 6–8]. Additionally, the amidohydrolases CyrG and CyrH were proposed to assemble an uracil moiety in cylindrospermopsin biosynthesis [9]. Another Copyright: © 2021 by the authors. more distantly related homolog, MxcM, was discovered in the pseudochelin biosynthetic Licensee MDPI, Basel, Switzerland. gene cluster of the marine bacterium Pseudoalteromonas piscicida S2040 [10]. Heterologous This article is an open access article expression of mxcM in Myxococcus xanthus and in vitro analysis of the purified enzyme distributed under the terms and confirmed the assumed function in heterocycle formation [10]. MxcM was shown to cat- conditions of the Creative Commons alyze an intramolecular condensation of the β-aminoethyl amide moiety in myxochelin B, Attribution (CC BY) license (https:// thereby generating the characteristic imidazoline moiety of pseudochelin A (Figure1C). creativecommons.org/licenses/by/ 4.0/). Catalysts 2021, 11, 892. https://doi.org/10.3390/catal11080892 https://www.mdpi.com/journal/catalysts Catalysts 2021, 11, x FOR PEER REVIEW 1 of 11 Catalysts 2021, 11, 892 2 of 11 FigureFigure 1. 1.(A ()A General) General reaction reaction mechanism mechanism of heterocycleof heterocycle formation formation catalyzed catalyzed by amidohydrolases. by amidohydrolases. (B) Examples (B) Examples of natural of productsnatural products containing containing amidohydrolase-produced amidohydrolase-produced heterocycles. heterocycles. (C) MxcM-catalyzed (C) MxcM-catalyzed formation formation of the imidazolineof the imidazoline ring in pseudochelinring in pseudochelin A biosynthesis. A biosynthesis. (D) Examples (D) Examples of approved of approved drugs featuring drugs featuring an imidazoline an imidazoline scaffold. scaffold. SubsequentSubsequent studiesstudies revealed revealed that that the introductionthe introduction of the imidazolineof the imidazoline ring contributes ring tocontributes the potent to anti-inflammatory the potent anti-inflammatory activities of this activities natural of product this natural [11–13 product]. Furthermore, [11–13]. it becameFurthermore, evident it thatbecame MxcM evident possesses that someMxcM substrate possesses flexibility, some substrate which can flexibility, be exploited which for thecan precursor-directedbe exploited for biosynthesisthe precursor-directed of diverse lipoxygenase biosynthesis inhibitors of diverse [11, 12lipoxygenase]. inhibitorsIn fact, [11,12]. imidazolines and imidazoles are found in many pharmaceutical drugs [14,15]. SomeIn examples fact, imidazolines are clonidine, and moxonidine, imidazoles tizanidine,are found naphazoline,in many pharmaceutical oxymetazoline, drugs and tetryzoline[14,15]. Some (Figure examples1D) [ 16]. Althoughare clonidine, thesecompounds moxonidine, are currentlytizanidine, made naphazoline, by chemical synthesisoxymetazoline, [17], there and is tetryzoline an increasing (Figure interest 1D) in the [16]. development Although ofthese biocatalytic compounds production are processes,currently inmade general, by andchemical for enzymatic synthesis heterocycle [17], there formation, is an increasing in particular interest [18,19 in]. the developmentHere, we reportof biocatalytic the biochemical production characterization processes, ofin the general, imidazoline-forming and for enzymatic amido- hydrolaseheterocycle MxcM. formation, For this in particular purpose, [18,19]. optimal reaction parameters were determined under in vitroHere,conditions we report and the reactionbiochemical kinetics charac wereterization recorded. of Additionally,the imidazoline-forming we describe theamidohydrolase tolerance of MxcM MxcM. against For this organic purpose, solvents optimal and reaction the impact parameters of non-aqueous were determined solvents, asunder well in as salt-containingvitro conditions systems and the on reaction the enzymatic kinetics activity.were recorded. The substrate Additionally, specificity we of MxcM,describe which the tolerance was previously of MxcM explored against in organicin vivo solventsstudies [11and], wasthe impact analyzed of andnon-aqueous quantified insolvents, vitro. The as datawell presentedas salt-containing can be considered systems on to laythe the enzymatic foundation activity. for possible The substrate future en- gineeringspecificity studies of MxcM, and which the integration was previously of heterocycle-forming explored in in vivo amidohydrolases studies [11], was in analyzed chemical processand quantified development. in vitro. The data presented can be considered to lay the foundation for possible future engineering studies and the integration of heterocycle-forming 2.amidohydrolases Results in chemical process development. 2.1. Characterization of MxcM 2. ResultsMost amidohydrolases possess a catalytic zinc ion in their active center [2,3]. The amidohydrolase2.1. Characterization NatAM of MxcM represents the closest homolog of MxcM for which a structure was determinedMost amidohydrolases [8]. A sequence possess alignment a catalytic shows zinc that ion the in threetheir histidineactive center residues [2,3]. which The areamidohydrolase responsible for NatAM the coordination represents ofthe the closest zinc ionhomolog in NatAM of MxcM (His75, for His77,which anda structure His253), arewas similar determined in MxcM [8]. A and sequence in the alignment other heterocycle-forming shows that the three amidohydrolases histidine residues (Figure which2). Onlyare responsible CyrH lacks for a the histidine coordination residue of at the position zinc ion 77 in and NatAM features (His75, an asparticHis77, and acid His253), residue instead.are similar Since in supplementationMxcM and in the ofother 5 mM heterocycle-forming EDTA was reported amidohydrolases to eliminate the (Figure enzymatic 2). activityOnly CyrH of NatAM lacks a [ 8histidine], we also residue evaluated at position the effect 77 of and this features complexing an aspartic agent onacid the residue activity ofinstead. MxcM. Since Unexpectedly, supplementation the addition of 5 mM of EDTA didwas notreported significantly to eliminate affect the the enzymatic enzymatic conversionactivity of NatAM of the tested [8], we MxcM also substrateevaluated myxochelin the effect of B this (residual complexing activity agent 93.6 ±on2.6%). the After