Biochem. J.

(1999) 343, 177–183 (Printed in Great Britain)


Bacterial lipolytic enzymes : classification and properties
Jean Louis ARPIGNY and Karl-Erich JAEGER1
Lehrstuhl fu$ r Biologie der Mikroorganismen, Ruhr-Universita$ t Bochum, Universita$ tsstrasse 150, D-44780 Bochum, Germany

Knowledge of bacterial lipolytic enzymes is increasing at a rapid and exciting rate. To obtain an overview of this industrially very important class of enzymes and their characteristics, we have collected and classified the information available from protein and nucleotide databases. Here we propose an updated and extensive classification of bacterial esterases and lipases based mainly on a comparison of their amino acid sequences and some fundamental biological properties. These new insights result in the identification of eight different families with the largest being further divided into six subfamilies. Moreover, the classification

enables us to predict (1) important structural features such as residues forming the catalytic site or the presence of disulphide bonds, (2) types of secretion mechanism and requirement for lipase-specific foldases, and (3) the potential relationship to other enzyme families. This work will therefore contribute to a faster identification and to an easier characterization of novel bacterial lipolytic enzymes. Key words : alignment, carboxylesterase, classification, lipase, structure.

Bacteria produce different classes of lipolytic enzyme, including carboxylesterases (EC, which hydrolyse small estercontaining molecules at least partly soluble in water, true lipases (EC, which display maximal activity towards waterinsoluble long-chain triglycerides, and various types of phospholipase. This paper deals with the former two classes of enzyme. For a description of phospholipases we refer the reader to recent review articles [1,2]. Our knowledge of the structure of lipases and esterases has increased considerably in recent years through the elucidation of many gene sequences and the resolution of numerous crystal structures [3,4]. Efforts accomplished the classification of this large set of data and the identification families and subfamilies of lipolytic enzymes [5,6] (see also the ESTHER database at http :\\cholinesterase). Many attempts have been made to identify sequence motifs conserved in lipolytic enzymes originating from a broad variety of organisms, including higher and lower vertebrates, invertebrates, fungi and bacteria, and to relate them to three-dimensional (3D) structural elements involved in substrate recognition and catalysis, and therefore being essential for the enzyme’s function. The structural superfamily of α\β-hydrolases defined by Ollis et al. [7] comprises a wide variety of enzymes whose activities rely mainly on a catalytic triad usually formed by Ser, His and Asp residues. This triad is functionally (but not structurally) identical with that of trypsin and subtilisin. In the amino acid sequences of α\β-hydrolases the three residues follow the order Ser-AspHis. The serine residue usually appears in the conserved pentapeptide Gly-Xaa-Ser-Xaa-Gly. α\β-Hydrolases notably include lipolytic enzymes, among which true lipases demand special attention because their peculiar catalytic properties make them very attractive for industrial applications. Their marked preference for water-insoluble substrates and their adsorption on the oil\water interface before hydrolysis involve substantial conformational changes of the enzyme’s architecture during ca-

talysis ; these have been particularly well documented for lipases of eukaryotic origin [8]. Lipolytic enzymes are currently attracting enormous attention because of their biotechnological potential [9–11]. Most of the lipases used in industry are microbial enzymes, of both fungal and bacterial origin. The great versatility of fungal lipases (from genera such as Candida, Geotrichum, Rhizopus and Thermomyces) in biotechnology is illustrated extensively by Gandhi [12], Benjamin and Pandey [13] and Pandey et al. [14]. Among bacterial lipases, attention has usually been focused on particular classes of enzymes such as the lipases from the genus Pseudomonas, which are especially interesting for biotechnology [15,16], or esterases possibly involved in bacterial pathogenicity [17]. Unfortunately, information on the relatedness of the numerous bacterial lipases and esterases studied so far is incomplete and scattered in the literature. Many new bacterial lipolytic enzymes have been studied since the publication of a comprehensive review article in 1994 [18]. However, no attempt has been undertaken to organize this information. Some biochemical properties (such as the dependence of activity on Ca#+ ions, pH and temperature) of the best studied families of lipases (from the genera Bacillus, Pseudomonas and Staphylococcus) have been summarized previously [15,16, 18,19]. Usually, lipolytic enzymes are characterized by their ability to catalyse a broad range of reactions. Unfortunately, the wide diversity of methods used for lipase assays (such as the hydrolysis of p-nitrophenyl esters, the pH-stat method and the monolayer technique) prevents a direct comparison of results on substrate specificities. In an effort to standardize the measurements, comparative studies have been performed [20,21]. However, only a limited number of bacterial lipases [16] were investigated in these studies. In the present paper, 53 sequences of bacterial lipases and esterases are compared and classified according to conserved sequence motifs and the biological properties of these enzymes. Relevant information obtained from the 3D structures is also highlighted when available. This work presents an overview of

Abbreviations used : 3D, three-dimensional ; HSL, hormone-sensitive lipase ; PAF-AH, platelet-activating-factor acetylhydrolase. 1 To whom correspondence should be addressed (e-mail karl-erich.jaeger! # 1999 Biochemical Society

this similarity is not restricted to the vicinity of functionally important residues but is distributed over the entire sequence.178 J. The propeptide (207–267 residues) presumably acts as an intramolecular chaperone and facilitates the translocation of the lipase across the cell membrane [19]. until the publication of the crystal structures of the lipases from Chromobacterium iscosum [27] and from Burkholderia cepacia [28. Arpigny and K. aeruginosa lipase. ulgaris lipases contain only one Cys residue. archaea. subtilis and B. WI. notably in this region (Figure 1). Ps. marcescens. Because the residues involved in the formation of both the Ca#+-binding site and the disulphide # 1999 Biochemical Society The various Bacillus lipases known have in common that an alanine residue replaces the first glycine in the conserved pentapeptide : Ala-Xaa-Ser-Xaa-Gly. Jaeger and B. lipase. E. by using the keywords ‘ bacteria. they are believed to be important in the stabilization of the active centre of these enzymes [28]. The central region of these proteins (residues 50–150) is approx. Madison. fluorescens and Serratia marcescens. Lang. The final presentation of results was prepared with the MEGALIGN program from the Lasergene software package (DNASTAR.1 and I. Lipases from Vibrio cholerae. bacterial lipases and esterases currently known and permits the classification of newly isolated lipolytic enzymes. DATA SEARCH AND ANALYSIS Sequences were retrieved from protein and nucleotide databases by means of the Entrez server at NCBI (http :\www. The acidic side chain. which are shown in Figure 1. Convincingly. Because some Pseudomonas species that produce important lipases have recently been renamed Burkholderia [25] and because many lipases originate from various other genera. cinnamoneus lipase and other Streptomyces lipases known so far. Acinetobacter calcoaceticus. However. sorting and alignment were obtained with the help of the Match-Box server [23] and the CLUSTAL W program [24]. Ps. Ps. wisconsinensis and Proteus ulgaris have molecular masses in the range 30–32 kDa and display a higher sequence similarity to the Ps. Two cysteine residues forming a disulphide bridge are conserved in a majority of sequences. 50 % similarity). subtilis and from subfamily I. B. Ps. Staphylococcal lipases are larger enzymes (approx.ncbi.1 and I.28].S. 2 and 3 because Pseudomonas lipases were probably the first to be studied and have a preponderant role in industry. L.nlm.2 of true lipases.A. 400 residues. The expression in an active form of lipases belonging to subfamilies I. carboxylesterase ’. such specific helper proteins have yet not been described for Ps.16].29]. Their molecular mass is approx. fluorescens C9 lipase do not lie at equivalent positions and no information is available on the possible existence of a disulphide bridge in this molecule. In these proteins this important residue lies much closer to the N-terminus than in other lipolytic enzymes [17]. These lipases have in common a higher molecular mass than lipases from subfamilies I. the catalytic centre of Strep.31]. Dijkstra. Enzymes from subfamily I. thermocatenulatus and B. thereby giving a hint about their general characteristics as a starting point to their investigation. No similarity was found between the Strep.1 and I. stearothermophilus produce lipases with similar properties. Very recently the crystal structure of the Ps. The Burkholderia glumae lipase was for a long time the only bacterial lipase with a known 3D structure [26]. fluorescens. The two Cys residues of the Ps.2 depends on a chaperone protein named lipase-specific foldase (‘ Lif ’). fragi and Ps. Apart from the residues forming the catalytic triad. the sequences of lipases from several bacterial genera were reported that are obviously related to families I. yielding a mature protein of approx. 75 kDa) that are secreted as precursors and cleaved in the extracellular medium by a specific protease. U.33]. scabies because of its significant similarity to the Aeromonas hydrophila esterase (30 %). W. All these enzymes belong to family I.2 (Ps.). Sequence similarity searches were performed with the BLAST 2. 45 kDa and they display maximal activity at approx. we propose a revised classification of true lipases on the basis of six subfamilies (Table 1). S. the lipases from the two mesophilic strains B. aeruginosa lipase was solved (D. Jaeger bridge are located in the vicinity of the catalytic His and Asp residues.0 and 65 mC [32. ulgaris and Ps. As shown by its crystal structure [37]. unpublished work) providing the first structure in the lipase family I. esterase. nih. The secretion of these enzymes occurs in one step through a three-component ATP-binding-cassette transporter system [30. K. two aspartic residues involved in the Ca#+-binding site described in the crystal structures are found at homologous positions in all sequences. Since the publication of comparative studies on Pseudomonas lipases [15. Both subfamilies also share important structural features.1. fragi. 15 %) with the other Bacillus and Staphylococcus lipases. Other lipases The lipases from Propionibacterium acnes (339 residues) [35] and from Streptomyces cinnamoneus (275 residues) [36] show significant similarity to each other (39 % identity. fluorescens C9.3 contains enzymes from at least two distinct species : Ps. which usually stabilizes the positive charge . luteola. the lipase from Staphylococcus hyicus also displays a remarkable phospholipase activity [34]. 65 kDa) and the absence of an N-terminal signal peptide and of Cys residues. Subfamily I. luteola lipase possesses this insertion (residues 254–272 in the preprotein) and shows a high similarity to the Burkholderia enzymes. The GDSL family The enzymes grouped in family II do not exhibit the conventional pentapeptide Gly-Xaa-Ser-Xaa-Gly but rather display a GlyAsp-Ser-(Leu) [GDS(L)] motif containing the active-site serine residue (Figure 2). scabies esterase has a particular architecture in that it forms a catalytic dyad instead of a triad. pumilus stand apart because they are the smallest true lipases known (approx. Lipases from Gram-positive organisms RESULTS AND DISCUSSION True lipases Pseudomonas lipases Bacterial true lipases were formerly ordered in the so-called Pseudomonas groups 1. We included in this family the esterase from Strep.2 on the basis of amino acid sequence comparison (Table 1 and Figure 1). Sequence comparison.0 program [22]. Interestingly. which is unique among true\Entrez\). 50 kDa . 50 % similar to lipases from B. 20 kDa) and share very little sequence similarity (approx. However. pH 9.2 are characterized by a slightly larger size (33 kDa) owing to an insertion in the amino acid sequence forming an anti-parallel double β-strand at the surface of the molecule [26. The Ps.

Brumlik and Buckley # 1999 Biochemical Society . Both enzymes have an α\β tertiary fold substantially different from that of the α\β-hydrolase family and share conserved sequence blocks with at least four other bacterial esterases. as shown in Figure 2. Pseudomonas oleovorans Haemophilus influenzae Psychrobacter immobilis Moraxella sp. B11-1 Archaeoglobus fulgidus Alcaligenes eutrophus Escherichia coli Moraxella sp. Similarity (%) Family I Subfamily 1 Enzyme-producing strain Pseudomonas aeruginosa* Pseudomonas fluorescens C9 Vibrio cholerae Acinetobacter calcoaceticus Pseudomonas fragi Pseudomonas wisconsinensis Proteus vulgaris Burkholderia glumae* Chromobacterium viscosum* Burkholderia cepacia* Pseudomonas luteola Pseudomonas fluorescens SIK W1 Serratia marcescens Bacillus subtilis Bacillus pumilus Bacillus stearothermophilus Bacillus thermocatenulatus Staphylococcus hyicus Staphylococcus aureus Staphylococcus epidermidis Propionibacterium acnes Streptomyces cinnamoneus Aeromonas hydrophila Streptomyces scabies* Pseudomonas aeruginosa Salmonella typhimurium Photorhabdus luminescens Streptomyces exfoliatus* Streptomyces albus Moraxella sp. Sulfolobus acidocaldarius Acetobacter pasteurianus Synechocystis sp. For the Aeromonas hydrophila esterase. a second enzyme displaying the GDSL motif. namely Trp-315. Abbreviations : OM. D50587 AF031226 X16945 X80800 X14033 U88907 U33845 X70354 Q05489 M58494 AF050153 D11455 D13253 M74010 A34992 U78785 X95309 X02844 M12715 AF090142 X99255 U80063 P10480 M57297 AF005091 AF047014 X66379 M86351 U03114 X53053 X62835 AF034088 AE000985 L36817 AE000153 X53868 M58445 U32704 X67712 X53869 AF071233 AB013096 D90904 S70419 S79600 Y11778 AE001287 Q01470 P37967 CAA22794 AAA99492 CAA78842 AAC60471 Family 100 95 57 43 40 39 38 35 35 33 33 14 15 16 13 15 14 15 14 13 14 14 100 36 35 28 28 100 82 33 100 54 48 40 36 25 100 41 34 34 32 20 100 50 24 20 16 100 48 45 100 43 40 Subfamily Properties True lipases 2 3 4 5 6 II (GDSL) 100 100 78 77 100 51 100 80 100 94 29 28 26 100 50 Phospholipase III IV (HSL) V VI Secreted acyltransferase Secreted esterase OM-bound esterase OM-bound esterase Secreted esterase Extracellular lipase Extracellular lipase Extracellular esterase 1 Esterase Lipase Carboxylesterase Putative lipase Carboxylesterase Extracellular esterase 2 PHA-depolymerase Putative esterase Extracellular esterase Extracellular esterase 3 Esterase Esterase Carboxylesterases VII VIII Carbamate hydrolase p-Nitrobenzyl esterase Putative carboxylesterase Stereoselective esterase Cell-bound esterase Esterase III * Lipolytic enzyme with known 3D structure. is replaced by the backbone carbonyl of the residue located three positions upstream of the histidine itself. polyhydroxyalkanoate. Alicyclobacillus acidocaldarius Pseudomonas sp. outer membrane . the α1 subunit of the plateletactivating-factor acetylhydrolase (α1PAF-AH) from bovine brain.Bacterial lipolytic enzymes Table 1 Families of lipolytic enzymes 179 Amino acid sequence similarities were determined with the program MEGALIGN (DNASTAR). shows a catalytic triad in which an aspartic residue also lies three positions upstream of the active-site histidine [38]. Spirulina platensis Pseudomonas fluorescens* Rickettsia prowazekii Chlamydia trachomatis Arthrobacter oxydans Bacillus subtilis Streptomyces coelicolor Arthrobacter globiformis Streptomyces chrysomallus Pseudomonas fluorescens SIK W1 Accession no. with the first member of each family (subfamily) arbitrary set at 100 %. PHA. Interestingly. of the active-site histidine residue.

amino acid residues belonging to the catalytic triad . Ps. cysteine residues forming the disulphide bridge . Obviously. unpublished work). more structural information is needed to establish whether these enzymes share a common fold and a # 1999 Biochemical Society common architecture of their catalytic triad (or dyad) in addition to the similarity of their sequences. which was also reported. fragi and Proteus vulgaris lipases are underlined. aeruginosa. Arpigny and K. fluorescens C9. and S. Cysteine residues in Ps. Another interesting feature of the GDSL esterases from Ps. Jaeger Figure 1 Alignment of amino acid sequences of true lipases from subfamilies I. Jaeger. it was shown in the above-mentioned bovine α1PAFAH [38] that a second conserved aspartic residue located three positions upstream from the active histidine (Figure 2) can take part in the active site and that this third acidic residue is not essential for the enzyme’s function. Wilhelm. =.-E. might be due to the misfolding of the protein in the periplasm and to its subsequent proteolytic degradation without implying that Asp-116 belongs to the catalytic triad. location of the active-site Asp-116 (position 134 in the precursor) proposed by Brumlik and Buckley [39] for the Aeromonas hydrophila enzyme. In these proteins the C-terminal domain is presumably folded into approx. Figure 2 Blocks of sequence conserved in the GDSL esterase family (family II . .41].1 and I. 12 amphipathic β-sheets forming an aqueous pore in the outer membrane. both the absence of activity of the enzyme and its severely impaired secretion. arguing that the Asp-116 Asn mutant is completely inactive. J. see Table 1) Symbols : $. Tommassen and K.2 (see Table 1) Symbols : $. However. However. amino acid residues belonging to the catalytic triad . Salmonella typhimurium and Photorhabdus luminescens is an additional C-terminal domain that encompasses approximately one-third of their entire sequence and is similar to that of a newly identified family of autotransporting bacterial virulence factors ([40. L. scabies esterase [37]. as in the Strep.-E. #. The catalytic N-terminal domain transits through this pore and is in some instances released in the extracellular medium by a specific proteolytic process.180 J. aspartic residues involved in the Ca2+-binding site. [39] proposed that the active-site aspartic residue is Asp-116 located in block III (Figure 2). #.

Their tertiary fold was modelled on the basis of the Strep. Compare the motifs surrounding the active-site serine residue. # 1999 Biochemical Society . The proposed active-site residues. was shown to be located in the sequence at a position non-equivalent to that found in the Strep. 20 % amino acid sequence identity with the intracellular and plasma isoforms of the human PAFAH. 40 % sequence similarity to eukaryotic lysophospholipases (Ca#+-independent phospholipases A ). Family III This family of lipases was identified primarily by Cruz et al. enzymes grouped in family V originate from mesophilic bacteria (Pseudomonas oleo orans. namely epoxide hydrolases. Very little is known about the other enzymes in this family.Bacterial lipolytic enzymes 181 Figure 3 Sequence blocks conserved in families IV. Family V Like proteins in the HSL family. exfoliatus enzyme. The sequence patterns conserved around the active-site residues of family V enzymes are presented in Figure 3. Their active-site aspartic residue. again underlining the great functional versatility of the α\β-hydrolase scaffold. Alcaligenes eutrophus) and thermophilic (Alicyclobacillus acidocaldarius. Acetobacter pasteurianus) as well as from cold-adapted (Moraxella sp. The relatively high activity at low temperature (less than 15 mC) retained by HSL and the lipase from Moraxella sp. The 3D structure of the Ps. identified primarily by site-directed mutagenesis.. Archeoglobus fulgidus) origins indicates that temperature adaptation is not responsible for such an extensive sequence conservation. are also highlighted. A Family VI With a molecular mass in the range 23–26 kDa. Figure 3 shows sequence blocks that are highly conserved in HSL and six lipolytic enzymes from distantly related prokaryotes. which were inferred from the threedimensional model of the human HSL [46]. merged with an additional N-terminal domain and a regulatory module inserted in the central part of the sequence. [44]. fluorescens carboxylesterase was solved [52]. amino acid residues belonging to the catalytic triad. in contrast with the heterotrimeric PAF-AH from bovine brain. exfoliatus (M11) lipase. Psychrobacter immobilis). The mammalian HSL seems to derive from a catalytic domain. homologous with the bacterial enzymes.. the tertiary fold and the active site residues of the Psy. On the basis of the crystal structure of the Xanthobacter autotrophicus dehalogenase [49]. mesophilic (Escherichia coli. Their # major conserved sequence motifs are shown in Figure 3. This enzyme displays the canonical fold of α\β-hydrolases and contains a typical catalytic triad. The active form of this enzyme is a dimer. This carboxylesterase hydrolyses small substrates with a broad specificity and displays no activity towards long-chain triglycerides [53]. Psy. They share significant amino acid sequence similarity (20–25 %) to various bacterial non-lipolytic enzymes. The subunit has the α\β-hydrolase fold and a classical Ser-Asp-His catalytic triad. immobilis lipase were predicted by molecular modelling [51]. Their amino acid sequences were derived from whole-genome sequences except that for the Spirulina platensis esterase. which also possess the typical α\β-hydrolase fold and a catalytic triad [49. Haemophilus influenzae. The enzymes in family VI display approx. However. [43] and mentioned by Wei et al. exfoliatus lipase structure [44]. immobilis) or heatadapted (Sulfolobus acidocaldarius) organisms. [47] was once thought to derive from the conserved sequence motifs of these enzymes [48]. These PAF-AHs are monomer proteins. comparative study of these enzymes would therefore be very valuable for the determination of the distinctive properties of this family of hydrolases.50]. the enzymes presented here are among the smallest esterases known. which was cloned specifically [54]. V and VI (see Table 1) Symbol : $. dehalogenases and haloperoxidase. It also shows approx. who solved the 3D structure of the Strep. The hormone-sensitive lipase (HSL) family A number of bacterial enzymes (family IV) display a striking amino acid sequence similarity to the mammalian HSL [45]. the marked sequence similarity (Table 1 and Figure 3) between esterases from psychrophilic (Moraxella sp.

P. and Saier.-E. Mol. Environ. L. 25. M. Lipids 93.. Madden. Lipids 93. Spener.. Biochem. and Soccol. C. J.. Dijkstra. R.. O. and Buckley. Johnson. M. and Benedik. Atomi. Nigam. Petersen. Serre. Higgins. which involves a Ser-Xaa-Xaa-Lys motif conserved in the N-terminal part of both enzyme categories [58. S. Y. 253. 28–61 Ollis. (1997) Appl. Y. This implies that the order of the catalytic residues in the sequence (Ser-Asp-His) that is conserved throughout the entire superfamily of lipases and esterases would not be respected in this case. 113–123 Henderson. M. Inoue... notably.. Conclusions Despite a highly conserved tertiary fold and obvious sequence similarities. Cygler.. Briffeuil. 173–185 Schrag. F. F. The genome sequencing project of Strep. M. (1997) J. Schimossek.. W. Frolow. M.. and Holland... Aoki. Wei. Lee. T.. van Heuvel. L. F. (1995) Nat. P. Schomburg. Reginster. R. L. Y. 85–107 Anthonsen.. and Buckley. K. Hwang. Acta 1301. et al. Shin. P. 3–14 Gandhi. and Cygler. Patkar. 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