Taking Advantage of Promiscuity of Cold-Active Enzymes

: Cold-active enzymes increase their catalytic e ﬃ ciency at low-temperature, introducing structural ﬂexibility at or near the active sites. Inevitably, this feat seems to be accompanied by lower thermal stability. These characteristics have made cold-active enzymes into attractive targets for the industrial applications, since they could reduce the energy cost in the reaction, attenuate side-reactions, and simply be inactivated. In addition, the increased structural ﬂexibility could result in broad substrate speciﬁcity for various non-native substrates, which is called substrate promiscuity. In this perspective, we deal with a less addressed aspect of cold-active enzymes, substrate promiscuity, which has enormous potential for semi-synthesis or enzymatic modiﬁcation of ﬁne chemicals and drugs. Further structural and directed-evolutional studies on substrate promiscuity of cold-active enzymes will provide a new workhorse in white biotechnology. carbohydrate esters, and p-nitrophenyl esters. Ha SGNH1 speciﬁcally hydrolyzed butyl esters such as p -nitrophenyl butyrate, 1-naphthyl acetate, tert -butyl acetate, and glyceryl tributyrate. To improve the catalytic activity of Ha SGNH1 six mutants, Asn83Leu (1), Met86Glu (2), Met86Arg (3), Arg87Leu (4), Phe131Ala (5), and Ile173Phe (6) were generated using site-directed mutagenesis. Only four showed 120% enhanced hydrolysis of p -nitrophenyl butyrate than wild-type Ha SGNH1. Two showed higher activity towards carbohydrate acetates, while three showed for larger substrates like p-nitrophenyl hexanoate. The bulky phenyl side chain of six preferred bindings to short-chain fatty esters like p-nitrophenyl acetate. The immobilized Ha SGNH1-CELA was applied for the synthesis of petroleum, food, and cosmetic products (butyl and oleic esters) in a nonpolar solvent. The successful synthesis of butyl acetate, butyl butyrate, and oleic acid butyl ester was conﬁrmed by gas chromatography analysis.

In the context of industrial applications of cold-active enzymes, the features mentioned above have been well exploited. However, less investigated has been the broadening of the substrate specificity of cold-active enzymes due to their intrinsic characteristics, which may lead to increased enzyme promiscuity, compared to mesophilic orthologs [2,3,[11][12][13][14][15]. Notably, enzymes with broad substrate specificity frequently have large active site to embrace substrates of varying sizes and shapes [16][17][18]. Especially, it is likely that the hydrophobic substrate binding sites could promote binding of the non-native substrates by hydrophobic effects. The increased exposure of hydrophobic residues in cold-active enzymes may stimulate substrate promiscuity [19][20][21]. The broader substrate specificity of cold-active enzymes, another intriguing property, could expand their application to the molecular design or semi-synthesis for fine chemicals and drugs. In this article, we focus on promiscuity of cold-active enzymes with a few recent reports for the possible application in organic synthesis. Note that we mainly refer to the cold-active enzymes as a cold-adapted enzymes from psychrophilic microorganisms.
Mesophilic and/or thermophilic enzymes showing broad substrate specificity have been well reviewed [18,[22][23][24]. A partial list of mesophilic/thermophilic promiscuous enzymes along with psychrophilic orthologs are listed in Table 2. The critical features of enzymes to show promiscuity are the presence of an highly mobile active site loop [25], highly reactive active sites [23], active site embedded with cofactors [23], and highly hydrophobic substrate [26]. Some of these are similar to natural characteristics of cold-active enzymes. Hence, cold-actives enzymes could be promiscuous furthermore or fortuitously, compared to their mesophilic orthologs. In addition, it is probable that the substrate promiscuity of cold-active enzymes could be escalated or arisen by thermal fluctuation compared to mesophilic orthologs, without losing their advantages mentioned above. This may occur by chance due to the experimental conditions, which are different from natural environment. In the condition of in vivo and low temperature, cold-active enzymes should have strict substrate specificity. However, cold-active enzymes examined at room temperature may undergo an increased thermal fluctuation at the flexible loop regions near the active site and it may result in an unregulated substrate binding of xenobiotic compounds. This unexpected property of cold-active enzymes could be useful for enzyme-mediated semi-synthesis of fine chemicals and drugs. However, we should consider that the structural flexibility of the active sites is very similar between cold-active and mesophilic enzymes, meaning that substrate promiscuity applies to a very limited number of cold-active enzymes, since the active site residues are highly conserved [27][28][29]. Through comparative computational studies of cold-and warm-active enzymes, Åqvist and his coworkers supposed that structural flexibility of cold-active enzymes generally located at surface loops of the protein may confer higher reaction rates at low temperature [7,[27][28][29]. Except for Candida antarctica Lipase B [24,30], to date, only a handful of reports on enzyme promiscuity and possible application of cold-active enzymes have been investigated [14,15,31,32].
Similar to other acylaminoacyl peptidases (AAPs), the cold-active AAP (SpAAP) from psychrophilic bacterium Sporosacrina psychrophila possesses promiscuous ester hydrolysis activity [85,95]. The SpAAP exhibited hydrolytic activity on native N-acylated amino acids p-nitroanilide substrates, and on several promiscuous p-nitrophenyl esters of fatty acids of different chain length (C4-C18). Similar characteristics in the primary sequence were found in the primary sequence SpAAP with a lack of disulfide bonds, which probably confers structural flexibility for broader substrate specificity [95]. Contrary to hyperthermophilic AAP from Aeropyrum pernix (ApAAP) [84,96,97], SpAAP had a lower K m and k cat for N-acyl amino acids, but, interestingly, higher K m and k cat for short-chain fatty acids esters. This discrepancy was supported by finding that the diameter of a tunnel in the β-propeller domain to the active site is wider in SpAAP than in mesophilic and thermophilic orthologs [98].
The cold-active acetyl xylan esterase (PbAcE) from psychrophilic soil microbe Paenibacillus sp. had a broad substrate specificity (Figure 2A) [32]. Structural analysis of PbAcE suggested that flexible subunit movements and different active site loop conformations might allow PbAcE to have these characteristics different from other mesophilic and thermophilic homologs. PbAcE forms hexamer in solution and each monomer has α/β hydrolase fold with conserved catalytic triad residues of Ser185, Asp274, and His303. Activity assay data showed that PbAcE has typical properties of cold-active enzymes with strong low-temperature activity and broad substrate specificity. PbAcE has moderate and strong deacetylation activity for glucose penta-acetate and xylan acetate as well as reversible acetylation activity for xylan. In addition, PbAcE exhibited strong deacetylation activity for lipids and tertiary alcohol esters. These results proposed that PbAcE can accommodate various substrates and it could be useful for deacetylation biocatalyst of other xenobiotic ligands. For example, acetyl group of beta-lactam antibiotics such as cefotaxime, 7-Aminocephalosporic acid (7-ACA), and cephalosporin C were successfully removed by PbAcE ( Figure 2B). The enzymatic modification of antibiotics is of significant importance to overcome the antibiotic resistance issue by increasing the chemical diversity. Furthermore, the possibility of commercial application of PbAcE was assessed by ensuring that the activity was maintained more than 80% even after immobilization followed by repetitive use of 18 times.
Le et al. reported that the recombinant HaSGNH1-type lipase from Halocynthiibacter arcticus catalyzed the synthesis of biodiesel and flavor compounds [31]. HaSGNH1 had a sandwich structure containing five central parallel β-sheets between two layers of four α-helices per layer. The catalytic triad of S18, D171, and H174 was embedded near the surface within a catalytic pocket. The solvent-exposed substrate-binding pocket offered the substrate flexibility to HaSGNH1. The substrate-binding pocket encircled with five amino acids, Asn83, Met86, Arg87, Phe131, and Ile173, regulated the entry of substrates via noncovalent interactions. HaSGNH1 was applied for the hydrolysis of tert-butyl esters, carbohydrate esters, and p-nitrophenyl esters. HaSGNH1 specifically hydrolyzed butyl esters such as p-nitrophenyl butyrate, 1-naphthyl acetate, tert-butyl acetate, and glyceryl tributyrate. To improve the catalytic activity of HaSGNH1 six mutants, Asn83Leu (1), Met86Glu (2), Met86Arg (3), Arg87Leu (4), Phe131Ala (5), and Ile173Phe (6) were generated using site-directed mutagenesis. Only four showed 120% enhanced hydrolysis of p-nitrophenyl butyrate than wild-type HaSGNH1. Two showed higher activity towards carbohydrate acetates, while three showed for larger substrates like p-nitrophenyl hexanoate. The bulky phenyl side chain of six preferred bindings to short-chain fatty esters like p-nitrophenyl acetate. The immobilized HaSGNH1-CELA was applied for the synthesis of petroleum, food, and cosmetic products (butyl and oleic esters) in a nonpolar solvent. The successful synthesis of butyl acetate, butyl butyrate, and oleic acid butyl ester was confirmed by gas chromatography analysis.
The cold-active lipase B (CALB) from psychrophilic Candida Antarctica is one of the most studied cold-active enzymes with broad substrate specificity [4]. CALB has a structure similar to α/β hydrolases containing both α-helices and β-strand. The catalytic triad of Ser, His, and Asp/Glu is embedded near the surface within a catalytic pocket [88]. CALB has open, large, and medium substrate-binding pockets, which imparts structural flexibility and broad substrate specificity [89]. The flexible structure of CALB allows the efficient catalysis of various industrially important reactions. CALB catalyzes the epoxidation of α,β-unsaturated aldehydes with hydrogen peroxide to give α,β-epoxy aldehydes ( Figure 1A) [90]. Aldol addition of ethanethiol to α,β-unsaturated hexanal gives diastereomeric addition products 2-methyl-3-(ethylthio)pentanal by CALB mutant (Ser105Ala) ( Figure 1B) [91]. CALB also efficiently catalyzes aldol addition reaction between tricyclic diketone to give bis-product within 4 h ( Figure 1C) [92]. Under polar and nonpolar solvents, CALB catalyzes different reactions [93]. For example, CALB catalyzes the amidation reaction between α,βunsaturated ester and amine to give α,β-unsaturated amide under a polar solvent, whereas Michael addition is favored under a non-polar solvent to give β-amino ester ( Figure 1D) [93]. The Baeyer-Villiger oxidation reaction of α-alkyl cyclic ketone to give ethyl 5-acetoxypentanoate can also be catalyzed by CALB ( Figure 1E) [94]. These results imply that CALB could accommodate various substrates, which could be useful for the catalysis of industrially important reactions. of beta-lactam antibiotics such as cefotaxime, 7-Aminocephalosporic acid (7-ACA), and cephalosporin C were successfully removed by PbAcE ( Figure 2B). The enzymatic modification of antibiotics is of significant importance to overcome the antibiotic resistance issue by increasing the chemical diversity. Furthermore, the possibility of commercial application of PbAcE was assessed by ensuring that the activity was maintained more than 80% even after immobilization followed by repetitive use of 18 times.  Another example is Cytochrome P450 monooxygenases (CYPs). CYPs are heme-containing enzymes that catalyze the hydroxylation of a wide variety of substrate molecules [99]. Bacterial steroid hydroxylase CYPs are particularly useful for effectively introducing hydroxyl groups to a wide range of steroids in the pharmaceutical industry. Thus, these CYPs have received increasing attention in recent years for industrial applications. Kim et al. (2017) reported a newly identified CYP steroid hydroxylase (BaCYP106A2) from the bacterium Bacillus sp. PAMC 23377 isolated from Kara Sea of the Arctic Ocean, and its crystal structure was determined at a resolution of 2.7 Å. Furthermore, structural comparisons with other CYPs showed that the α8-α9 loop region of BaCYP106A2 was intrinsically mobile and might be important for broad substrate specificity. The hydroxylase activity of BaCYP106A2 was examined with two different kinds of steroid substrates, 4-androstenedione and nandrolone, and the reaction was analyzed with HPLC and LC-MS [99]. CYPs oxidized 4-androstenedione and nandrolone to 15β-hydroxyandrostenedione and 15β-hydroxynandrolone, respectively. Unlike the mesophilic CYP orthologs from Bacillus meagterium [100], BaCYP106A2 seemed to mainly monohydroxylate steroid substrates with high conversion yields ( Figure 3). The specific modification of pharmaceutically important compounds by the cold-active CYP sheds light on drug development and production.  [31]. HaSGNH1 had a sandwich structure containing five central parallel β-sheets between two layers of four α-helices per layer. The catalytic triad of S18, D171, and H174 was embedded near the surface within a catalytic pocket. The solventexposed substrate-binding pocket offered the substrate flexibility to HaSGNH1. The substratebinding pocket encircled with five amino acids, Asn83, Met86, Arg87, Phe131, and Ile173, regulated the entry of substrates via noncovalent interactions. HaSGNH1 was applied for the hydrolysis of tertbutyl esters, carbohydrate esters, and p-nitrophenyl esters. HaSGNH1 specifically hydrolyzed butyl esters such as p-nitrophenyl butyrate, 1-naphthyl acetate, tert-butyl acetate, and glyceryl tributyrate. To improve the catalytic activity of HaSGNH1 six mutants, Asn83Leu (1), Met86Glu (2), Met86Arg (3), Arg87Leu (4), Phe131Ala (5), and Ile173Phe (6) were generated using site-directed mutagenesis. Only four showed 120% enhanced hydrolysis of p-nitrophenyl butyrate than wild-type HaSGNH1. Two showed higher activity towards carbohydrate acetates, while three showed for larger substrates like p-nitrophenyl hexanoate. The bulky phenyl side chain of six preferred bindings to short-chain fatty esters like p-nitrophenyl acetate. The immobilized HaSGNH1-CELA was applied for the synthesis of petroleum, food, and cosmetic products (butyl and oleic esters) in a nonpolar solvent. The successful synthesis of butyl acetate, butyl butyrate, and oleic acid butyl ester was confirmed by gas chromatography analysis.
Another example is Cytochrome P450 monooxygenases (CYPs). CYPs are heme-containing enzymes that catalyze the hydroxylation of a wide variety of substrate molecules [99]. Bacterial steroid hydroxylase CYPs are particularly useful for effectively introducing hydroxyl groups to a wide range of steroids in the pharmaceutical industry. Thus, these CYPs have received increasing attention in recent years for industrial applications. Kim et al. (2017) reported a newly identified CYP steroid hydroxylase (BaCYP106A2) from the bacterium Bacillus sp. PAMC 23377 isolated from Kara Sea of the Arctic Ocean, and its crystal structure was determined at a resolution of 2.7 Å. Furthermore, structural comparisons with other CYPs showed that the α8-α9 loop region of BaCYP106A2 was intrinsically mobile and might be important for broad substrate specificity. The hydroxylase activity of BaCYP106A2 was examined with two different kinds of steroid substrates, 4-androstenedione and nandrolone, and the reaction was analyzed with HPLC and LC-MS [99]. CYPs oxidized 4androstenedione and nandrolone to 15β-hydroxyandrostenedione and 15β-hydroxynandrolone, respectively. Unlike the mesophilic CYP orthologs from Bacillus meagterium [100], BaCYP106A2 seemed to mainly monohydroxylate steroid substrates with high conversion yields ( Figure 3). The specific modification of pharmaceutically important compounds by the cold-active CYP sheds light on drug development and production. In conclusion, structural flexibility of cold-active enzymes could enhance or give rise to enzyme promiscuity. Consequently, cold-active enzymes have merits for the enzymatic modification and semi-synthesis of fine chemicals and medicines by virtue of their broad substrate specificity on top of their existing merits as industrial catalysts. To take advantage of the under-utilized facet of coldactive enzymes, structure-based protein engineering and directed evolution should be employed in the near future to develop enzymes with improved substrate promiscuity for various chemical reactions. In conclusion, structural flexibility of cold-active enzymes could enhance or give rise to enzyme promiscuity. Consequently, cold-active enzymes have merits for the enzymatic modification and semi-synthesis of fine chemicals and medicines by virtue of their broad substrate specificity on top of their existing merits as industrial catalysts. To take advantage of the under-utilized facet of cold-active enzymes, structure-based protein engineering and directed evolution should be employed in the near future to develop enzymes with improved substrate promiscuity for various chemical reactions.