Nitrile Metabolism in Fungi: a Review of Its Key Enzymes Nitrilases with Focus on Their Biotechnological Impact

fungal biology reviews 33 (2019) 149e157

journal homepage: www.elsevier.com/locate/fbr

Review Nitrile metabolism in fungi: A review of its key enzymes nitrilases with focus on their biotechnological impact

Ludmila MARTıNKOVA

Institute of Microbiology, Academy of Sciences of the Czech Republic, 142 20 Prague 4, Czech Republic article info abstract

Article history: Nitriles are abundant in the plant kingdom. The ability to detoxify them is beneficial for mi- Received 25 April 2018 crobes living in the plant environment. Nitrilases (NLases; EC 3.5.5.-), which hydrolyze ni- Received in revised form triles to carboxylic acids, have been well characterized in bacteria, and classified into 8 November 2018 various substrate-specificity subtypes (aromatic NLases, aliphatic NLases, arylacetoN- Accepted 13 November 2018 Lases). NLases also occur in filamentous fungi, mainly in Ascomycota (subdivision Pezizomy- cotina), as documented by genome mining. However, the investigation of NLases in fungi Keywords: has been delayed compared to bacteria. Only a few NLases (aromatic NLases) were purified Biotransformation from native fungal strains (mainly Fusarium), which were grown under suitable induction Fungi conditions. Over a few past years, the spectrum of known fungal NLases was broadened Heterologous production by expressing fungal NLase genes in Escherichia coli. Thus functional NLases were reported Nitrilase for the first time in fungi of genera Auricularia, Macrophomina, Nectria, Neurospora, Pichia, Ta- Nitrile metabolism laromyces, Trichoderma and Trichophyton. Two major substrate-specificity subtypes were Phylogenetic distribution identified in them, i.e. aromatic NLases and arylacetoNLases, apart from a few NLases with broad substrate specificities. The biotechnological impact of fungal arylacetoNLases Abbreviations: was explored with a focus on the enantioselective hydrolysis of (R,S )-mandelonitrile, the aa selective hydrolysis of one cyano group in dinitriles and the hydrolysis of nitrile precursors amino acid of the taxol sidechain. Despite recent advances, the wealth of fungal NLases whose se- NLase quences have been deposited in databases has not yet been fully exploited. Overproduction nitrilase in E. coli has the potential to bring these NLases to life. This will enable to estimate the natural roles of NLases in fungi and will also provide new catalysts for biotechnological uses. ª 2018 Published by Elsevier Ltd on behalf of British Mycological Society.

1. Introduction and consist of nitrile-forming and nitrile-converting enzymes (Fig. 1). The former are aldoxime dehydratases (Oxds; EC Nitriles are abundant in nature, mainly occurring as cyano- 4.99.1.5e7) in bacteria and fungi, and cytochrome P450 (CYP) genic glycosides (defense compounds) or b-cyano alanine enzymes in plants. Both of them catalyze the dehydration of (HCN assimilation intermediate) in plants. The enzymes of nitrile precursors, aldoximes, which, in turn, are derived from nitrile metabolism are widespread but not ubiquitous in nature amino acids. The knowledge of Oxds in fungi is limited, as

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O In the hypothetical reaction mechanism of NLases, the cat- aldehyde alytic cystein interacts with the carbon atom of the C^N moi- R1 H PLANTS ety, and a tetrahedral intermediate is thus formed, which is NH2 R=R1CH(OH)- HNL OH ROH then cleaved into carboxylic acid or amide (Fig. 2). The former R CYP OH CYP NLase RN RN is largely the major product of the reaction, while the latter is O O amino acid aldoxime nitrile carboxylic acid formed from specific substrates such as those bearing electro- philic substituents at a-position. However, the tendency to OxD NLase FUNGI form amide also depends on the origin of the enzyme NLase ROH RN (Sosedov and Stolz 2014). O Microbial NLases have been usually classified into a few subtypes (aromatic NLases, arylacetoNLases and aliphatic Fig. 1 e Biosynthesis and bioconversion of nitriles in plants NLases) designated according to their preferential substrates and fungi. CYP [ cytochrome P450 enzymes; (aromatic nitriles, arylacetonitriles, aliphatic nitriles; OxD [ aldoxime dehydratase; NLase [ nitrilase; Kobayashi and Shimizu 1994; O’Reilly and Turner, 2003; HNL [ hydroxynitrile lyase. Thuku et al., 2009). The latter study also defined the bromoxynil-specific NLases (Thuku et al., 2009). Plant NLases are comprised of subtypes NIT1-3 with preference for some only two fungal Oxds were characterized in some detail. One of arylaliphatic and aliphatic nitriles and NIT4 with preference them was isolated from the mycelium of Sclerotinia sclerotiorum for b-cyanoalanine (Vorwerk et al., 2001; Piotrowski et al., and the other, from Fusarium graminearum, was overproduced 2001). in E. coli. The former transformed a few aldoximes such as The interest in NLases has been prompted by their ability indolyl-3-acetaldoxime, 4-hydroxyphenylacetaldoxime and to hydrolyze nitriles under ambient conditions and at mild analogues (Pedras et al.,2010), while the latter exhibited a broad pH values, to discriminate between enantio- and substrate specificity for aliphatic and arylaliphatic aldoximes regioisomers, and to selectively hydrolyze cyano groups in (Kato and Asano 2005). presence of other functional groups. These biotransforma- Some specific nitriles (cyanohydrins in plants, toyocamy- tions were mainly examined with the bacterial NLases (for re- cin in Streptomyces) are synthesized by other nitrile-forming views see Martınkova and Kren, 2010; Martınkova et al., 2017) enzymes. The synthesis of cyanohydrins from aldehydes and, partly, the plant NLases (Osswald et al., 2002), while and HCN is catalyzed by hydroxynitrile lyases (HNLs; 4.1.2.-), fungal NLases have been largely neglected for years. which have also found a number of applications in the synthe- The first fungal NLase (aromatic NLase) was purified from sis of chiral nitriles (for reviews see Bracco et al. 2016 and Fusarium solani by Harper (1977) but its amino acid (aa) Bhalla et al. 2018). The nucleosidic antibiotic toyocamycin is sequence was never determined. Later, using 2- produced via its nitrile precursor PreQ(0) (7-cyano-7- cyanopyridine and valeronitrile as powerful inducers enabled deazaguanin; Tao et al., 2015), while 7-cyano-7-deazaguanine to isolate two aromatic NLases from this species and to synthetase (EC 6.3.4.20) participates in the ATP dependent partially determine their sequences (for a review see synthesis of the nitrile precursor (McCarty et al., 2009; Liu Martınkova et al., 2009). In addition, a NLase with a broad sub- et al., 2018). strate specificity was induced in Fusarium proliferatum by The key enzymes of the nitrile catabolism in fungi and ε-caprolactam (Yusuf et al., 2013a, b). However, isolating plants are NLases (EC 3.5.5.-). The probable roles of NLases NLases from fungi has its limits, as the enzyme production in plants consist of e.g. cyanide detoxification, catabolism of may be low, inducer difficult to find or the cultivation of the natural nitriles, supply of nitrogen for growth, and, under spe- fungus time consuming. Therefore, heterologous expression cific conditions, production of the growth hormone indole-3- has been used in recent years to obtain new fungal NLases. acetic acid (for a review see Piotrowski, 2008). The natural These studies not only resulted in the characterization of functions of NLases in microbes are less understood but it is further aromatic NLases, but also enabled it to prepare a num- justified to hypothesize on their key roles in nitrile detoxifica- ber of arylacetoNLases, which until that time had only been tion and nitrogen recycling, which may facilitate the organ- found in bacteria. isms’ participation in dead plant decay or plant The properties and applications of NLases have been pathogenesis. In addition to NLases, bacteria produce an alter- reviewed from many viewpoints. For instance, some native enzymatic system enabling the hydrolysis of nitriles in two steps: 1. transformation of nitrile to amide by nitrile S-E hydratase, EC 4.2.1.84, and 2. transformation of amide to car- S-E RS-E R OH + E-SH +H2O - NH3 +H2O R N R NH2 boxylic acid by amidase, EC 3.5.1.4 (for reviews see Kobayashi -E-SH R NH OH O O and Shimizu 1999; Martınkova and Kren 2002; Prasad and nitrile carboxylic acid Bhalla 2010). -E-SH NLases are classified into branch 1 of the “NLase-super- R NH2 family” proteins, typified by their E-K-E-C catalytic tetrade O (Thuku et al. 2009). The NLase superfamily consists of 13 amide branches, whose members are largely known to act on non- Fig. 2 e Hypothetical reaction mechanism of nitrilase (ac- peptide CeN bonds, with different substrate specificities. cording to O’Reilly and Turner, 2003). E [ enzyme. The NLase activity was only found in branch 1 (Brenner 2002). Nitrile metabolism in fungi 151

comprehensive reviews summarized the knowledge of NLases Yusuf et al., 2015). The typical size of their subunits was (Gong et al., 2012b), the nitrile-converting enzymes (Ramteke similar to those in bacterial NLases (29.8e45.8 kDa; Thuku et al., 2013) or the aldoxime-nitrile pathway (Bhalla et al., et al., 2009), e.g. 36e37 kDa in NLases from Trichophyton benha- 2018). Other reviews focused on specific aspects such as the miae (formerly Arthroderma benhamiae) and Nectria haemato- metagenome and database mining for NLases (Gong et al., cocca (Vesela et al. 2013). The native molecular weights in 2013), integration of nitrile-hydrolyzing enzymes in cascade these NLases were determined to be 336e360 kDa (Vesela reactions (Martınkova et al., 2014), biological degradation of et al. 2013), suggesting a multimeric structure similar to bacte- cyanide (Luque-Almagro et al., 2016), enantioselective bio- rial NLases with 130e650 kDa (Thuku et al., 2009). All the en- transformations of nitriles and amides (Wang, 2015) or NLase zymes examined were moderately thermostable and applications with industrial impact (Martınkova et al., 2017). exhibited pH profiles with maximum activities and stabilities The focus of these studies was on bacterial enzymes, while in the neutral or slightly alkaline pH range similar to the ma- the properties or applications of fungal NLases were only jority of bacterial NLases. However, the NLase from G. monili- marginally discussed. The only review which focused exclu- formis was more stable at alkaline pH (up to 11; Petrıckov a sively on fungal NLases was published ca. 9 y ago et al., 2012b) than the majority of other NLases (Ramteke (Martınkova et al., 2009). The heterologous production of these et al., 2013), which was in accordance with the high activity enzymes was in its early stage at that time. Therefore, the aim of the NLase from Gibberella intermedia at pH 10 (Gong et al., of this work is to summarize the advances in this research 2012a). Similar pH profiles were obtained for the NLase from þ area, i.e. the production methods, the biochemical character- T. benhamiae. The sensitivity of the fungal NLases to Ag ization and the biotechnological uses of the enzymes. The (Gong et al., 2012a; Yusuf et al., 2015) was similar to their bac- phylogenetic distribution of their subtypes will also be dis- terial counterparts, reflecting the conserved reaction centre, cussed. Cyanide hydratases, which form an evolutionarily specifically the key role of the active cystein residue in their distant type of enzymes, and cyanide dihydratases (nitrilase reaction mechanism. homologues) have been recently reviewed elsewhere (Martınkova et al., 2015, Park et al., 2017) and will not be included in this work. 3. Substrate specificity subtypes

The spectrum of aromatic NLases, which were previously ob- 2. Heterologous expression of fungal nitrilase tained directly from fungi Aspergillus niger, F. solani, Fusarium genes and biochemical characterization of the oxysporum, Penicillium multicolor (for a review see Martınkova enzymes et al., 2009) and Exophiala oligosperma (Rustler et al., 2008), was extended with heterologously produced NLases from Fungal NLases are probably not glycosylated or at least do not Aspergillus kawachii (Vesela et al., 2016), Penicillium rubens require glycosylation for their activities. Therefore, E. coli can (formerly Penicillium chrysogenum)(Kaplan et al., 2013), Talaro- be used as a suitable host for the expression of fungal NLase myces marneffei (formerly Penicillium marneffei), Gibberella moni- genes. Thus the expression systems used for bacterial and liformis (Petrıckov a et al., 2012b; Kaplan et al., 2013) and fungal NLases were similar. Low concentrations of the inducer Gibberella intermedia (Gibberella fujikuroi var. intermedia)(Gong IPTG (0.5 mM) and relatively low temperatures (e.g., 25 C) had et al., 2012a). The designation of this subtype as aromatic to be maintained to avoid excessive translation rates, and, NLases is based on the finding that aromatic or heteroaro- hence, to minimize protein misfolding (Petrıckov a et al., matic nitriles (often 4-cyanopyridine) are their preferential 2012b). An optimized protocol even used IPTG concentrations substrates but their ability to hydrolyze straight-chain as low as 0.02 mM. Both E. coli BL21 and E. coli Origami B (DE23) aliphatic nitriles or dinitriles was also demonstrated were acceptable as the host strains but the latter provided (Goldlust and Bohak, 1989; Gong et al., 2012a; Kaplan et al., higher activity yields. The genes were cloned into expression 2013; Vesela et al., 2016). The turnover number (TON) of the ar- vectors of the pET type. To achieve high cell densities, 2 YT omatic NLase from G. moniliformis for its best substrate benzo- and particularly EnPresso media were suitable (Vesela et al., nitrile was ca. 5.8 s 1, as calculated from kinetic data 2016). determined by Petrıckov a et al. (2012b). Thus this value was The effect of the co-expression of bacterial chaperones on significantly lower than in NLases from Rhodococcus rhodochr- the production of fungal NLases was examined, and the ous (ca. 129 s 1; Yeom et al., 2010) or even Aeribacillus pallidus GroEL/ES chaperone was found to have positive effects on (formerly Bacillus pallidus; 14 444 s 1; Almatawah et al., 1999). the production of two aromatic NLases. The total activities The first two fungal arylacetoNLases were obtained by of these NLases increased almost three to seven times and expressing the genes from A. niger and Neurospora crassa in the specific activities of the whole cells more than four times. E. coli (Kaplan et al., 2011; Petrıckov a et al., 2012b). Aromatic At the same time, the percentage of activity in the soluble and heteroaromatic nitriles were poor substrates of these en- fractions increased ca. 1.2 to 1.7 times. The effect of this chap- zymes, while the preferential ones were phenylacetonitrile erone was enzyme-specific (only efficient in aromatic NLases). and (R,S )-mandelonitrile. Similar enzymes were later found Other chaperones such as Hsp70 or trigger factor failed to to be encoded in T. benhamiae, A. kawachii, Aspergillus oryzae, exhibit similar effects (Petrıckov a et al., 2012b). Auricularia subglabra (formerly Auricularia delicata), N. haemato- Over 10 of the recombinantly produced fungal NLases were cocca and Macrophomina phaseolina (Kaplan et al., 2013; Vesela largely purified by metal affinity chromatography (Petrıckov a et al., 2013, 2016). The TONs of fungal arylacetoNLases, in et al., 2012b; Gong et al., 2012a; Vesela et al., 2013, 2016; which kinetic data were determined (NLases from A. niger, 152 L. Martınkova

N. crassa, N. haematococca and T. benhamiae; Petrıckov a et al., Close homologues of the NLase from F. proliferatum occur in 2012b; Vesela et al., 2013), were calculated to be 6.5e68.5 s 1 other species of Fusarium but also in other genera of subdivi- for phenylacetonitrile and 6.1e19.3 s 1 for (R,S )-mandeloni- sion Pezizomycotina (Trichoderma, Metarhizium, Colletotrichum, trile. Those of bacterial NLases covered a broad range of Penicillium etc.) and are largely designated carbon-nitrogen hy- 0.072e175 s 1 for mandelonitrile and 1.1e18.85 s 1 for phenyl- drolases in databases. This group of NLase family proteins acetonitrile (brenda-enzymes.org). Thus the TONs of fungal exhibit significant levels of identity to the carbon-nitrogen hy- NLases were largely in this range except for the TON of N. hae- drolase from Saccharomyces cerevisiae (ca. 50% in NLase from F. matococca NLase for phenylacetonitrile, which was the highest proliferatum; Yusuf et al., 2015). The putative hydrolase from S. (68.5 s 1). cerevisiae was crystallized (Kumaran et al., 2003) which enabled A different NLase with a broad substrate specificity (for ar- to use it as the template for modeling and substrate docking omatic and aliphatic nitriles but also mandelonitrile) was ob- studies of the NLase from F. proliferatum (Yusuf et al., 2015). tained by expressing a gene from Fusarium proliferatum. This Basidiomycota did not contain any NLases similar to the protein was evolutionarily distant from the aforementioned aforementioned subtypes except for the aforementioned fungal NLases. Its molecular mass (theoretically 33 kDa) was NLase from Auricularia. It is possible that the gene was ac- slightly lower than in the majority of other NLases with typical quired by this fungus through horizontal transfer. The puta- masses of 35e45 kDa (Yusuf et al., 2015). tive NLase superfamily proteins, which are found in Different sequence patterns could be recognized in the Basidiomycota (unpublished results), are distant in evolution proximity of the catalytic cystein in different substrate- from the NLases discussed here and their functions or activ- specificity subtypes (Fig. 3). For instance, all proteins with a ities are still unclear. short insert of six aa residues upstream of the catalytic cystein exhibited arylacetoNLase activities. However, there were also other arylacetoNLases lacking this pattern. The broad sub- 5. Biotechnology potential strate specificity NLases from Meyerozyma contained an insert comprised of five aa residues. Notably, the F. proliferatum In organic synthesis, NLases have been often found to be lacked the typical NLase motif CWEH, containing a CYDV suitable alternatives of chemical catalysts, operating at mild motif instead. temperatures and near neutral pH, and, in many cases, in a stereo- or a regioselective mode. However, the industrial applications of NLases are still few, comprising e.g. of the pro- 4. Phylogenetic distribution duction of pyrazine-2-carboxylic acid, picolinic acid (Liese et al., 2000), nicotinic acid (Gong et al., 2012b) or mandelic The aforementioned characterized NLases largely originated acid and its derivatives (Brady et al., 2004; Gong et al., from division Ascomycota (subdivision Pezizomycotina). Similar 2012b). Although the first-generation catalysts have been putative NLases were also predicted in many other members already applied for the industrial synthesis of (R)-mandelic of Pezizomycotina. These fungi belonged to the classes Dothideo- acid (synthetic intermediate, chiral resolving agent) from mycetes, Eurotiomycetes, Leotiomycetes and Sordariomycetes. The (R,S )-mandelonitrile, new and improved catalysts of this re- searches also indicated a rare occurrence of similar NLases action are being constantly sought. Recently, whole cells of in subdivision Saccharomycotina. Various types of NLases E. coli producing fungal arylacetoNLases were found to be were found in almost each of the classes, and very often suitable candidates for such catalysts. The cells hydrolyzed also in the same genus (Table 1). up to 500 mM mandelonitrile (Vesela et al., 2015), while the

ArylacetoNLase Auricularia delicata gb|EJD42068.1 FGGEIGTVKVGALACWEHTQPLLK Macrophomina phaseolina gb|EKG14506.1 FGGDVGVVKVGALACWEHTQPLLK Trichophyton benhamiae gb|AFF60190.1 FGGDIGVVKVGTLACWEHALPLLK Neurospora crassa emb|CAD70472.1 FGSELGSIKVGTLNCWEHAQPLLK Nectria haematococca gb|AFF60191.1 FGAEHGKIKVGCFACWEHTQPLLK Aspergillus niger gb|AEH52057.1 IG------KVGALACWEHIQPLLK emb|CAK47246.1 AG------RVGALSCWEHIQPLLK Aspergillus kawachii dbj|GAA83217.1 AG------RVGALSCWEHIQPLLK Aspergillus oryzae dbj|BAE63579.1 VG------RVGALSCWEHIQPLLK Aromatic NLase Aspergillus kawachii dbj|GAA90167.1 FG------NIGGLNCWEHTQTLLR Gibberella moniliformis gb|ABF83489.1 FG------RVAGLNCWEHTQTLLR Penicillium rubens gb|KZN90726.1 FG------RIGGLNCWEHTQPLLR Talaromyces marneffei gb|AEH52060.1 FG------KVGGLNCWEHLQPLLR Broad-substrate Meyerozyma guilliermondi gb|AFF60192.1 FK-EAGPVEVGCLSCWEHMQPLLY -specificity Trichoderma virens gb|EHK18468.1 FG------RVGSLSCWEHIQPLLK NLases Fusarium proliferatum gb|AGW81831.1| YG------KIAVAICYDVRFPELA

Fig. 3 e Multiple alignment of sequences surrounding catalytic region in fungal nitrilases; NLase [ nitrilase. Identical residues are highlighted in red. Strongly similar residues and weakly similar residues are in blue and green, respectively. The catalytic cystein is underlined. irl eaoimi fungi in metabolism Nitrile

Table 1 e Distribution of aromatic nitrilase, arylacetonitrilase and broad-substrate-speciﬁcity nitrilase homologues in ﬁlamentous fungi. Division Subdivision Class Generaa containing

Aromatic NLases ArylacetoNLases Broad-substrate-speciﬁcity NLases

Ascomycota Pezizomycotina Dothideomycetes Cercospora, Clohesyomyces, Neofusicoccum, Baudoinia, Cenococcum, Cercospora, Clohesyomyces, Pyrenochaeta, Verruconis Diplodia, Glonium, Macrophomina, Neofusicoccum, Paraphaeosphaeria, Pyrenochaeta Eurotiomycetes Aspergillus, Capronia, Cladophialophora, Exophiala, Aspergillus, Capronia, Cladophialophora, Emmonsia, Penicilliopsis, Penicillium, Phialophora, Talaromyces Exophiala, Microsporum, Nannizzia, Paracoccidioides, Penicilliopsis, Penicillium, Phialophora, Talaromyces, Trichophyton Leotiomycetes Coleophoma, Oidiodendron, Pezoloma, Phialocephala Botrytis, Coleophoma, Hyaloscypha, Oidiodendron, Pezoloma, Phialocephala, Pseudogymnoascus, Rutstroemia, Sclerotinia Sordariomycetes Colletotrichum, Daldinia, Diaporthe, Fusarium, Colletotrichum, Daldinia, Diaporthe, Fusarium, Trichoderma Gibberella,Hypoxylon, Nectria, Neonectria, Hypoxylon, Nectria, Neonectria, Neurospora, Pestalotiopsis, Phaeoacremonium, Pestalotiopsis Rosellinia, Tolypocladium, Verticillium Saccharomycotina Saccharomycetes Meyerozyma, Wickerhamomyces Basidiomycota Agaricomycetes Auricularia a The genera in which NLases were experimentally conﬁrmed (Rustler et al., 2008; Kaplan et al., 2013; Vesela et al., 2016) are in bold. Other genera shown contain homologues with at least 50% identity to experimentally conﬁrmed NLases. 153 154 L. Martınkova

bacterial NLase was already inhibited by 300 mM of this sub- lower activities for these substrates. In contrast, its activity strate and toluene (10%, v/v) had to be added to alleviate this for fumaronitrile was higher than in arylacetoNLases. inhibition (Zhang et al., 2011). The tolerance of fungal NLase Different from arylacetoNLases, the aromatic NLase trans- towards high product concentrations was also significant, formed this substrate not into cyano acid, but cyano amide the reaction rate being almost constant at up to ca. 500 mM (Fig. 5). of (R)-mandelic acid. E. coli whole cells producing fungal arylacetoNLases were All fungal NLases examined were selective for (R)-mande- also used in the chemo-enzymatic synthesis of taxol deriva- lonitrile, but their selectivities were of different degrees. The tives with modified side chains (Wilding et al., 2015). Taxol NLases from e.g. A. niger, N. crassa or N. haematococca exhibited (Paclitaxel) is a well-known anticancer drug, and its deriva- high selectivity for (R)-mandelonitrile. The hydrolysis of (R,S )- tives may be useful for studies of its structureeactivity rela- mandelonitrile into (R)-mandelic acid must be performed un- tionships. All six NLases examined were active for the trans- der conditions of rapid racemization of the substrate (pH 8). isomer of the cyanodihydrooxazole precursor (trans-1) which This ensures that the “correct” enantiomer (R-nitrile) is they transformed into a mixture of acid and amide. The ratio permanently available for the catalyst and a high enantiopur- of both products (7:3 through 9:1) depended on the enzyme. ity of the product is maintained (Fig. 4). The ee values (95.6%) None of these enzymes transformed the corresponding cis- obtained with fungal NLases acting on high substrate concen- isomer (cis-1). Thus regioselectivity of the fungal NLases for trations were slightly lower than with bacterial NLases (up to this type of nitriles was demonstrated (Fig. 6). ca. 99%) but ee can be increased by optimizing the substrate Protein engineering was used in several bacterial NLases feed or by recrystallization (Zhang et al., 2010). In contrast, to improve their catalytic properties (for a review see the catalyst productivity of the fed-batch process (ca. Martınkova et al. 2017). The replacements of amino acids 40 g g 1) was largely higher than with non-recycled bacterial in the catalytic region, in particular, were demonstrated to NLases. This allowed low catalyst loads of ca. 2 g of dry cell have significant effects on the enantioselectivities, specific weight L 1 to be used (Vesela et al., 2015). activities and amide formation in these enzymes (Kiziak In contrast to the aforementioned NLases, the NLase from and Stolz, 2009; Sosedov et al.,2010). Although fungal NLases T. benhamiae was only moderately selective (Vesela et al., 2015; only share moderate similarities with bacterial NLases (with Fig. 4). Its ability to also hydrolyze (S )-mandelonitrile can be identities largely not exceeding 40%), the region in the prox- used for the production of (S )-mandelic acid from this sub- imity of the catalytic centre is more conserved (Kaplan et al., strate under stereoretentive conditions, i.e. a low pH hinder- 2013). Therefore, it may be predicted that similar amino acid ing substrate racemization. (S )-Mandelonitrile can be replacements will change the catalytic properties in bacte- obtained in the S-oxynitrilase-catalyzed reaction of benzalde- rial and fungal NLases in a similar way. This was experi- hyde and HCN (Chmura et al., 2013). mentally confirmed by modifying the sequence in the There is also a great demand for NLases catalyzing the hy- proximity of the catalytic cystein in the arylacetoNLases drolysis of a single cyano group in dinitriles, as the resulting from A. niger and N. crassa. In fact, the W168A mutant of cyano acids or cyano amides are useful synthons (Bayer the NLase from N. crassa gave a much higher amide:acid ra- et al., 2011; Vergne-Vaxelaire et al., 2013). Whole cells produc- tio (85:15) than the wild-type enzyme (40:60) (Petrıckov a ing fungal arylacetoNLases transformed cyanophenylacetoni- et al., 2012a), which was in accordance with the amide for- triles and phenylenediacetonitriles into the corresponding mation by analogous variants of the Psedomonas fluorescens cyano acids (Fig. 5) at a preparative scale. A member of the ar- NLase (Kiziak and Stolz, 2009). The amide-forming mutants omatic NLase subtype (NLase from A. kawachii) exhibited may be useful as alternatives for nitrile hydratases, which

OH OH OH NLase mutant NLase NH2 OH (Neurospora crassa) (Aspergillus niger) N O pH 8 O

(S)-mandelamide (R,S)-mandelonitrile (R)-mandelic acid e.e. 32% e.e.> 90% pH 8 pH 5 NLase (Trichophyton OH benhamiae) OH OH OH

O O

(R)-mandelic acid (R,S)-mandelic acid e.e. 63%

Fig. 4 e Transformations of (R,S )-mandelonitrile into different products by various fungal nitrilases (examples); NLase [ nitrilase. Nitrile metabolism in fungi 155

OH n ArylacetoNLase n N N N O

OH ArylacetoNLase N N N O n n

n=0 Cyanophenyl acetonitriles; n=1 Phenylenediacetonitriles

Aromatic N N N ArylacetoNLase NLase OH NH2 N O Fumaronitrile O

Fig. 5 e Transformations of dinitriles into cyano acids or cyano amide by fungal nitrilases; NLase [ nitrilase.

Hypothetical members of both subtypes were found to be abundant in specific taxa of division Ascomycota and ca. 15 of O O them were overproduced and characterized. In contrast, the CN COOH N N arylacetoNLase from A. subglabra was the only characterized ArylacetoNLase NLase from Basidiomycota. Databases contain a wealth of sequences coding for putative NLases with unknown properties. It seems worth unravel- ling the substrate specifities and other biochemical properties (±)-trans-1 O of these proteins, as this may provide new catalysts but also help to elucidate the roles of these enzymes in the metabolism CONH2 N of nitriles in nature. Knowledge of the structureeactivity rela- O tionships enables us to predict the properties of some of the CN N putative enzymes, close homologues of which were character- ArylacetoNLase no ized. However, other of these putative enzymes exhibit low product similarities to characterized NLases. This is the case for the majority of NLases in Basidiomycota. These proteins must be overproduced to determine their functions. The methods for (±)-cis-1 the heterologous production of fungal NLases were estab- lished in the aforementioned works and will be useful in Fig. 6 e Transformation of taxol precursor, (±)-trans-2,4- further studies of the related enzymes, although optimum diphenyl-4,5-dihydrooxazole-5-carbonitrile ((±)-trans-1), by conditions will have to be fine-tuned for each new member fungal nitrilases; (±)-cis-1 was not accepted as a substrate. of this enzyme group. Fungal NLases enlarged the portfolio NLase [ nitrilase. Only one of the enantiomers is depicted of NLases with possible applications for synthetic uses but for each compound. their industrial potential for e.g. the cyanohydrin or dinitrile hydrolysis will require further evaluation, including the prep- form amides from nitriles, but are largely less stable and aration of immobilized cells or enzymes and optimization of less enantioselective than NLases. the process conditions on lab and pilot scale.

6. Conclusions Conﬂict of interest

Fungal NLases can be classified into two main subtypes e ar- None. omatic NLases and arylacetoNLases. Although aromatic NLases generally hydrolyze not only aromatic but also aliphatic nitriles, the designation is justified, as their signifi- Acknowledgements cant activities for aromatic nitriles such as benzonitrile make them different from arylacetoNLases. Another criterion This study was supported by the Czech Science Foundation to distinguish between both types is the arylacetoNLase activ- (project 18-00184S) and the Ministry of Education, Youth and ity for a-substituted arylacetonitriles such as mandelonitrile. Sports of the Czech Republic (project COST LD15107). 156 L. Martınkova

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