J. Microbiol. Biotechnol. (2017), 27(11), 1907–1915 https://doi.org/10.4014/jmb.1708.08004 Research Article Review jmb

Bacterial Hormone-Sensitive (bHSLs): Emerging for Biotechnological Applications T. Doohun Kim*

Department of Chemistry, College of Natural Science, Sookmyung Women’s University, Seoul 04310, Republic of Korea

Received: August 3, 2017 Revised: September 11, 2017 Lipases are important enzymes with biotechnological applications in dairy, detergent, food, Accepted: September 16, 2017 fine chemicals, and pharmaceutical industries. Specifically, hormone-sensitive (HSL) is First published online an intracellular lipase that can be stimulated by several hormones, such as catecholamine, October 14, 2017 glucagon, and adrenocorticotropic hormone. Bacterial hormone-sensitive lipases (bHSLs),

*Corresponding author which are homologous to the C-terminal domain of HSL, have α/β- fold with a Phone: +82-2-2077-7806; catalytic triad composed of His, Asp, and Ser. These bHSLs could be used for a wide variety of Fax: +82-2-2077-7321; E-mail: doohunkim@sookmyung. industrial applications because of their high activity, broad substrate specificity, and ac.kr remarkable stability. In this review, the relationships among HSLs, the microbiological origins, the crystal structures, and the biotechnological properties of bHSLs are summarized. pISSN 1017-7825, eISSN 1738-8872 Keywords: Hormone-sensitive lipase, cap domain, promiscuity, substrate specificities, Copyright© 2017 by The Korean Society for Microbiology industrial applications and Biotechnology

Introduction hormone (ACTH), and glucagon [10, 11]. The activity of HSL is regulated by reversible cAMP-dependent phosphorylation Lipases (triacylglycerol , E.C. 3.1.1.3) catalyze [12]. In adipose tissue, HSL catalyzes the breakdown steps the hydrolysis of or -related compounds, which of triacylglycerol and diacylglycerol, which promotes the could release free fatty acids (FFAs), diacylglycerols, release of FFAs for transport to other organs as a major monoacylglycerols, and glycerols in the mobilization source of energy. In addition, HSL has been shown to process of fats or oils [1, 2]. In addition, these enzymes, hydrolyze or synthesize a broad range of substrates which are widespread in all three domains of life, are also containing ester linkages, which include cholesteryl esters, involved in synthetic reactions such as esterification, esters of steroid hormones, retinyl esters, and other lipid- transesterification, alcoholysis, or acidolysis [3, 4]. Most related substrates [13, 14]. This broad substrate specificity lipases belong to the α/β-hydrolase superfamily, which is makes them highly attractive for industrial applications. supposed to have a reaction mechanism similar to that of HSL has been comprehensively studied in mammalian serine proteases [5, 6]. In addition to their physiological species, and three isoforms are known, ranging in size from roles, lipases are highly useful for industrial applications in 84 to 130 kDa [15]. The human HSL is composed of a dairy, food, pharmaceutical, cosmetic, biofuel, and fine 786-amino-acid polypeptide without any membrane-spanning chemical industries owing to their high stability, broad region. This enzyme could form functional aggregates at substrate specificity, high regio-/stereo-selectivity, and high concentrations as well as bind to phospholipid vesicles remarkable stability [7-9]. Moreover, there are still great for its function [11, 13]. Using biochemical and biophysical demands for new lipases with great stability, high methods, including sedimentation analyses, denaturation selectivity, and strong activity, which could be applied to a studies, and limited proteolysis, HSL has been shown to be wide range of industrial processes. composed of two domains; an N-terminal binding domain Hormone-sensitive lipase (E.C. 3.1.1.79, HSL) is an and an identical C-terminal catalytic domain [15-17]. The intracellular lipase that can be stimulated by several N-terminal domain has been implicated in mediating hormones, such as catecholamine, adrenocorticotropic protein-protein interactions, whereas the C-terminal domain

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harbors a catalytic triad composed of Ser-Asp-His with proteins related to the HSL family have been found, several phosphorylation sites. Specifically, the N-terminal including an from Acinetobacter calcoaceticus, and domain could interact with adipocyte lipid binding protein N-acetyl hydrolases from Streptomyces hygroscopicus [24- [18, 19]. The C-terminal domain has an α/β-hydrolase fold, 26]. Over the last few years, there has been a substantial which has a highly conserved Ser residue in the Gly-x-Ser- increase in the number of publications dealing with the x-Gly sequence [20]. In addition, a regulatory module of isolation and characterization of bHSLs. ~150 amino acids, which is a main target of protein kinase Since the discovery of the bHSL family, many bHSLs A, is located in the C-terminal domain [21]. During have been identified in a wide range of bacteria, such as hydrolysis, this catalytic Ser could work as a nucleophile, Pseudomonas sp. B11-1 [27], Bacillus acidocaldarius [28], oil- which is assisted by the other two residues in close spatial degrading bacterium strain HD-1 [29], Escherichia coli [30], proximity. Archaeoglobus fulgidus [31], Psychrobacter sp. Ant300 [32], Mycobacterium tuberculosis [33-35, 42-44], Streptomyces Bacterial Hormone-Sensitive Lipases coelicolor [36], Oenococcus oeni [37], Rhodococcus sp. CR-53 [38], Rheinheimera sp. [39], Lactobacillus plantarum WCFS1 Besides the highly conserved catalytic motif (Gly-Xaa- [40, 41], and Bacillus halodurans [45]. Furthermore, many Ser-Xaa-Gly) that is observed in most lipases/, the bHSL genes were identified through metagenomic approaches HSL family shows no with other directly from environmental DNA samples [46-64]. These previously known mammalian lipases such as pancreatic metagenomic bHSLs were isolated from soil [46, 53, 55, 56], lipase or hepatic lipase [22]. Specifically, the N-terminal mud and sediment-rich water [47], environmental soils domain lacks any similarity with any other known proteins, [48], forest soil [49, 60, 64], marine sediments [50, 51, 58], which is a distinctive feature of HSL. However, several activated sludge [52, 54], mountain soil [57], tidal flat bacterial proteins, which are collectively called bacterial sediment [59], surface sediment [61], and permafrost [62, hormone-sensitive lipases (bHSLs), are homologous to the 63]. Interestingly, bHSL family members were shown to be C-terminal domain of HSL [22]. Initially, lipase 2 of selectively inhibited by 5-methoxy-3-(4-phenoxyphenyl)- Moraxella TA144 was noticed to be homologous to the C- 3H-[1,3,4]oxadiazol-2-one [65]. terminal domain of HSL [22, 23]. Then, other bacterial Although the number of bHSLs being discovered is

Fig. 1. Structural organization of human hormone-sensitive lipase (hHSL) and several bacterial HSLs (bHSLs) such as BFAE, Est25, EST2, and AFEST. The hHSL is composed of an N-terminal domain and a C-terminal catalytic domain. The C-terminal domain of hHSL, as well as bHSLs, harbors the catalytic triad (Ser-Asp-His) and conserved motifs (GXSXG, DPXXD, and HGX).

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largely increasing, there is still limited information about from the N-terminal residue and 30-50 residues between this protein family, and only a small number of them have β6 and β7 in the central β-sheet. Specifically, in Est25, the been characterized. The consensus amino acid sequences of first region of the cap domain is composed of 85 amino the representative members of the bHSL family members acids, which form two α-helices that cover the active site are shown in Fig. 1. For biochemical characterizations of [77]. In EST2, this N-terminal region of the cap domain has bHSLs, heterologous expression of the bHSL family been shown to play a significant role in maintaining members were achieved by using E. coli as a host [36-39]. enzyme activity, stability, and specificity [83]. Structural In addition, gene products from uncultured microorganisms comparisons revealed that the most striking differences were successfully expressed and purified [58-61, 63]. The among the bHSL family are in the cap region (Fig. 2). The products of Rv1399c (LipH), Rv3097c (LIPY), (Rv2970 (LipN), main variability resides in the different lengths and relative or Rv1076 (LipU) from M. tuberculosis were overexpressed orientations of helices and connecting loops. In crystal as inclusion bodies, and proteins were refolded with structures of bHSLs, this region has high B factors several modifications [33, 34, 42, 43]. Interestingly, PsyHSL compared with other regions [77-79]. The second region of from Psychrobacter sp. TA144 has been expressed in a the cap domain between β6 and β7 is clearly separated soluble form in the presence of trehalose or benzyl alcohol from the core β-sheets. This region (~150 residues) in the [66]. Moreover, an N-terminally his-tagged Cest-2923 was mammalian HSL family, which is notably longer than that marginally stable in solution, and a C-terminally his-tagged of bHSL proteins, contains several post-translational protein showed an oligomeric conformation suitable for phosphorylation sites for the regulation of these enzymes crystallization [40, 67]. After purification, molecular features [84, 85]. The regulatory mechanism that mediates the of the bHSL family members were studied in terms of movement of the cap domain toward efficient substrate crystal structures, thermostability, substrate specificities, binding is still not clearly understood. To date, in several enantioselectivity, and kinetic parameters. attempts to understand the conformational changes that occur in bHSLs, X-ray crystallography as well as molecular Structures of bHSLs

To date, several members of the bHSL family have been structurally described, including brefeldin A esterase (BFAE) from Bacillus subtilis [68], EST2 from Alicyclobacillus acidocaldarius [69], AFEST from A. fulgidus [70], EstE1 from a metagenomic library [71], EstE5 from a metagenomic library [72], Sto-Est from Sulfolobus tokodaii [73], EstE7 from a metagenomic library [74], PestE from Pyrobaculum calidifontis VA1 [75], Rv0045c from M. tuberculosis [76], Est25 from a metagenomic library [77], Cest-2923 from L. plantarum WCFS1 [40, 41], E40 from a marine sedimental metagenomic library [78], and Est22 from a deep-sea metagenomic library [79]. Although these bHSL family members showed relatively low primary sequence identity, all these crystal structures are composed of an α/β- hydrolase fold that is found in many hydrolases, including proteases, esterases, peroxidases, and dehalogenases [80, 81]. The α/β-hydrolase fold has a left-handed β-sheet containing parallel β-strands, although one strand (β2) strand is antiparallel to the other strands. These strands are Fig. 2. Electrostatic potential representations of bacterial connected by a bundle of helices in both concave and hormone-sensitive lipases. convex sides [82]. The electrostatic potentials of the negatively charged (red) and These bHSLs have a cap domain, which is composed of positively charged (blue) regions are shown. The cap domains above two or three α-helices in the upward region of the main β- the substrate-binding pocket are shown as ribbon diagrams (cyan). sheet. The cap domain is composed of 50-100 residues (A) BFAE, (B) AFEST, (C) Est25, (D) EST2.

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dynamics simulations were performed [75, 77, 86]. mesophilic temperatures, although a systematic approach Using site-directed mutagenesis, catalytic residues as was carried out to identify HSL genes from glacier soil [95]. well as the key residues in the substrate-binding region However, Blip from Bacillus halodurans C-125 showed high were identified [87-89]. The active site of the bHSL family thermal stability with a t1/2 of 35 min at 100°C, and complete members consists of a canonical catalytic triad, with Ser as refolding to its native conformation after 30 min at 90°C the nucleophile, His as the proton carrier, and Asp as the was observed [45]. In addition, EstE1 and EST2 were active acidic residue [80]. A tetrahedral intermediate, which could at high temperatures of up to 95°C [96, 97]. A structural be stabilized by residues in the oxyanion hole, is formed comparison of AFEST, BFAE, and EST2 has demonstrated when an alkoxide ion from the catalytic Ser attacks the that an increase in the number of electrostatic interactions electrophilic carbon of the ester linkage. The active site of and hydrogen bonds as well as a decrease in loop sizes and these enzymes is negatively charged in their optimal pH hydrophobic areas seemed to be a main reason for their range, which facilitates the separation of the products after thermostability [98]. Important residues in AFEST, such as the cleavage [90]. The crystal structure of the EstE5-PMSF Arg98 and Arg148, for thermostability were identified mostly complex showed a detailed atomic view around the catalytic in loops regions or α-helices [97]. In contrast, PsyHSL, Lipo1, triad of bHSLs [72]. Interestingly, a short sequences, motif and EstIM1 are cold-active enzymes with high catalytic of His-Gly-Gly-Gly, which is located 70-100 amino acids activity at low temperatures [52, 57, 99]. Interestingly, in upstream of the catalytic Ser, is largely conserved within PsyHSL, osmolytes were shown to increase its thermal the members of the bHSL family. This motif has been stability and its specific activity. In the presence of 3 M reported to stabilize the oxyanion hole during the hydrolysis trimethylamine N-oxide (TMAO), the specific activity of of tertiary alcohols [91, 92]. PsyHSL increased by ~2.5 fold at 50°C, whereas the Most bHSL family members, including BFAE, Est25, addition of 1 M TMAO increased its thermostability by 5- EstE1, and PestE, exist as dimers or larger oligomers, which fold at 45°C [66]. In addition, the activity of PMGL2 from a are stabilized mainly by hydrogen bonds and hydrophobic permafrost metagenomic library was reported to be interactions between two antiparallel β8 strands from the stimulated in the presence of 0.25-1.5 M NaCl [62]. α/β-hydrolase domain. These oligomers are further classified Interestingly, immobilization increased the thermal stability as subtype 1 and subtype 2 based on the differences in the and half-life of LipN [42]. The increased stability and dimeric interfaces [41]. In Aes, the two α-helices in the recyclability of cross-linked Est25 aggregates (CLEA-Est25) catalytic domain appear to be major factors in facilitating were also reported [77]. dimerization [93]. In EstE1, two residues of Val274 and It is widely known that bHSLs are promiscuous by Phe276 were identified to be important for its dimerization nature, and this is related to their physiology, evolution, [71]. In addition, Tyr182 in the wall of the active site was and metabolism [81]. Most of the bHSL family members largely responsible for the thermal stability of EstE1 [94]. In preferentially hydrolyze short-chain p-nitrophenyl esters Sto-Est, Arg267 was shown to be critical for maintaining dimer and tributyrin. Specifically, AFEST and EST2 hydrolyzed p- conformation [73]. In E25, dimerization is essential for the nitrophenyl hexanoate with KM and kcat values of 11 μM and catalytic function of Asp282 [61]. Interestingly, Cest-2923 220 μM, and 1,014 s-1 and 3,420 s-1, respectively [100, 101]. In exhibits a pH-dependent pleomorphic behavior, although a addition, PsyHSL was highly active against pNP-pentanoate, -1 -1 dimer is favored at neutral pH [40]. In E40, introduction of with kcat/KM of 215 mM s [99]. The KM and Vmax of LipU largely hydrophobic residues such as Trp or Phe in α7 were calculated to be 1.73 μM and 62.24 μM/min, respectively significantly improved the half-life of this protein to several [102]. LipN was able to synthesize methyl butyrate as well hundred folds, suggesting that hydrophobic interactions as hydrolyze 4-hydroxyphenylacetate to hydroquinone [42]. between the α/β-hydrolase fold and cap domain is a key EstDL26 and EstDL136 reactivated chloramphenicol from element for thermostability [78]. As dimerization is closely its acetyl derivatives by counteracting the chloramphenicol related to enzyme stability, bHSL proteins are qualified acetyltransferase activity in E. coli [56]. Substantial for biotechnological and industrial applications at high enantioselectivity of Est25 and EST2 was observed with the temperatures [36, 62, 71]. preference for (R)-product [103, 104]. Furthermore, Est4 and Est25 successfully hydrolyzed the tertiary alcohol Biotechnological Perspectives esters such as linalyl acetate and α-terpinyl acetate [38, 77]. In addition, PestE showed high enantioselectivity in the Most of the bHSL family members function optimally at kinetic resolution of racemic esters, which could be explain

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by its structural features [75]. In a recent report, EST2 was triacylglycerol lipases in cellular metabolism. Biochem J. used as a biosensor for the detection of organophosphate 414: 313-325. pesticides such as paraoxon [105] or foodborne bacteria 3. Stergiou PY, Foukis A, Filippou M, Koukouritaki M, [106]. Furthermore, EST2 was immobilized on a nitro- Parapouli M, Theodorou LG, et al. 2013. Advances in cellulose membrane for longer stability, good reproducibility, lipase-catalyzed esterification reactions. Biotechnol. Adv. 31: 1846-1859. and high sensitivity [105]. In this respect, HerE was shown 4. Jaeger KE, Eggert T. 2002. Lipases for biotechnology. Curr. to hydrolyze the acetyl groups from heroin to yield Opin. Biotechnol. 13: 390-397. morphine, which could be exploited to develop a heroin 5. Hui DY, Howles PN. 2002. Carboxyl ester lipase: structure- biosensor [107]. function relationship and physiological role in In conclusion, bHSLs from different bacteria and metabolism and . J. Lipid Res. 43: 2017-2030. metagenomic libraries exhibit a wide range of thermal and 6. Holmquist M. 2000. Alpha/beta-hydrolase fold enzymes: chemical stabilities, substrate specificities, and kinetic structures, functions and mechanisms. Curr. Protein Pept. properties. These varying properties allow bHSLs to be Sci. 1: 209-235. employed in a large spectrum of biotechnological applications, 7. Gupta R, Gupta N, Rathi P. 2004. Bacterial lipases: an such as in detergents, food additives, bioremediation, overview of production, purification and biochemical biofuels, and biotransformation. This review aimed to properties. Appl. Microbiol. Biotechnol. 64: 763-781. compile recent advances in the study of bHSLs, which 8. Kumar A, Khan A, Malhotra S, Mosurkal R, Dhawan A, Pandey MK, et al. 2016. Synthesis of macromolecular systems could pave a way for the preparation of more active and via lipase catalyzed biocatalytic reactions: applications and selective biocatalysts. Further studies on the binding future perspectives. Chem. Soc. Rev. 45: 6855-6887. mechanisms, interaction modes, cap domain movements, 9. Anobom CD, Pinheiro AS, De-Andrade RA, Aguieiras EC, and in vivo substrates of the bHSL family remain to be Andrade GC, Moura MV, et al. 2014. From structure to investigated. In addition, crystallographic studies of the catalysis: recent developments in the biotechnological bHSL family members are also required to comparatively applications of lipases. Biomed. Res. Int. 2014: 684506. elucidate the differences in structures, substrate-binding 10. Lampidonis AD, Rogdakis E, Voutsinas GE, Stravopodis DJ. mechanisms, and catalysis between them and other related 2011. The resurgence of hormone-sensitive lipase (HSL) in lipases. Moreover, protein engineering strategies could mammalian lipolysis. Gene 477: 1-11. also be employed in order to design enzymes that are more 11. Lass A, Zimmermann R, Oberer M, Zechner R. 2011. 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