Catalytic Dyad in the SGNH Hydrolase Superfamily: In-depth Insight

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Catalytic Dyad in the SGNH Hydrolase Superfamily: In-depth Insight into Structural Parameters Tuning the Catalytic Process of Extracellular Lipase from Streptomyces rimosus Ivana Lešcǐ ć Ašler,† Zoran Štefanić,*,† Aleksandra Maršavelski,‡ Robert Vianello,*,‡ and Biserka Kojić-Prodić† †

Division of Physical Chemistry, Rudjer Bošković Institute, Bijenička cesta 54, 10002 Zagreb, Croatia Division of Organic Chemistry and Biochemistry, Rudjer Bošković Institute, Bijenička cesta 54, 10002 Zagreb, Croatia

ACS Chem. Biol. 2017.12:1928-1936. Downloaded from pubs.acs.org by UNIV OF SOUTH DAKOTA on 08/24/18. For personal use only.

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S Supporting Information *

ABSTRACT: SrLip is an extracellular enzyme from Streptomyces rimosus (Q93MW7) exhibiting lipase, phospholipase, esterase, thioesterase, and tweenase activities. The structure of SrLip is one of a very few lipases, among the 3D-structures of the SGNH superfamily of hydrolases, structurally characterized by synchrotron diffraction data at 1.75 Å resolution (PDB: 5MAL). Its crystal structure was determined by molecular replacement using a homology model based on the crystal structure of phospholipase A1 from Streptomyces albidof lavus (PDB: 4HYQ). The structure reveals the Rossmann-like 3-layer αβα sandwich fold typical of the SGNH superfamily stabilized by three disulfide bonds. The active site shows a catalytic dyad involving Ser10 and His216 with Ser10-OγH···NεHis216, His216-NδH···OC-Ser214, and Gly54NH···Oγ-Ser10 hydrogen bonds essential for the catalysis; the carbonyl oxygen of the Ser214 main chain acts as a hydrogen bond acceptor ensuring the orientation of the His216 imidazole ring suitable for a proton transfer. Molecular dynamics simulations of the apoenzyme and its complex with p-nitrophenyl caprylate were used to probe the positioning of the substrate ester group within the active site and its aliphatic chain within the binding site. Quantum-mechanical calculations at the DFT level revealed the precise molecular mechanism of the SrLip catalytic activity, demonstrating that the overall hydrolysis is a two-step process with acylation as the rate-limiting step associated with the activation free energy of ΔG⧧ENZ = 17.9 kcal mol−1, being in reasonable agreement with the experimental value of 14.5 kcal mol−1, thus providing strong support in favor of the proposed catalytic mechanism based on a dyad.

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It has been known2,11 that the lipolytic SGNH-superfamily is significantly different from α/β-hydrolases by the protein fold and by the location of the active-site serine. In the 3-layered αβα sandwich-like fold, the active-site serine residue is positioned in a conserved Gly-Asp-Ser-Leu (GDSL) motif, close to the N-terminal end of an enzyme in contrast to the canonical α/β-fold, where a pentapeptide bearing the catalytic serine residue, usually Gly-Xaa-Ser-Xaa-Gly (where Xaa denotes any amino acid), is located in the middle of the amino acid sequence.2,11 Since then, the SGNH superfamily has been continuously expanded at the level of their primary sequences with the SGNH hydrolase-type esterase domain (88.921 recorded entries InterPro 62.0, 16th March, 2017), whereas their three-dimensional structures have been known in modest number (the Protein Data Bank currently contains 27 SGNH-unique structures). From the conserved active site residues found to be localized in four blocks, the name SGNH-

INTRODUCTION The SGNH−hydrolase superfamily was recognized about 20 years ago and originally named the GDSL-family upon the conserved motif comprising the active site serine.1 A broad phylogenetic distribution of these enzymes in archaea, bacteria, mammals, plants, fungi, and even viruses is closely related to their structural evolution accomplishing efficient biological functions.2 To ascertain phylogenetic relationships and to explore the evolution of the large GDSL-family, comparative sequence and genomic data have been used, so far, for lipases from land plants.3 This research revealed that most GDSLhydrolase genes are not unique and belong to multigene families. Such research for lipases/esterases of this superfamily from bacteria is very limited due to the problems related to the use of bacterial GenBank Data (draft genomes, incomplete data, and lack of appropriate software to handle Big Data).4 The SGNH-hydrolases are involved in many biological processes such as bacterial virulence, plant development and morphogenesis, and in plant defense mechanisms. The SGNH enzymes have been detected in different compartments: extracellularly,5,6 in the periplasm,7−9 and in the cytoplasm.10 © 2017 American Chemical Society

Received: December 22, 2016 Accepted: May 30, 2017 Published: May 30, 2017 1928

DOI: 10.1021/acschembio.6b01140 ACS Chem. Biol. 2017, 12, 1928−1936

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ACS Chemical Biology

Figure 1. Overall structure of Streptomyces rimosus lipase (SrLip) with the secondary structure annotations. (a) The enzyme fold can be described as the compact αβα-fold comprising 5 parallel β-strands surrounded by 11 α-helices and two 310 helices. (b) Overlap of the SrLip structure with the homologous structures of Streptomyces albidof lavus phospholipase A1 (PDB: 4HYQ, magenta) with the RMS deviation of 0.63 Å, and (c) Streptomyces scabies esterase (PDB: 1ESC, blue) with the RMS deviations of 1.32 Å indicates the high protein-fold similarity; the observed differences in 1ESC are mainly in the loop regions related to its longer protein chain (306 amino acid residues compared to 234 in SrLip and 236 in 4HYQ, respectively).

hydrolases has been used: block I, catalytic serine (serving as nucleophile and a proton donor to the oxyanion hole); block II, oxyanion hole glycine; and block III, oxyanion hole asparagine, both stabilizing the negatively charged intermediate by the hydrogen bond formation; block V, catalytic histidine, whereas the catalytic acid (aspartate or glutamate) is not entirely conserved throughout the superfamily, and thus, it is not included in the family name.12 Indeed, the crystal structures of a few SGNH enzymes, exemplified by the phospholipase A1 from S. albidof lavus,5 the esterase from S. scabies,6 and the 9-Oacetyl N-acetylneuraminic acid esterase from E. coli O157:H7,13 revealed a nucleophile serine and histidine in their active sites, whereas an acid residue (Asp/Glu) in the proximity of a catalytic histidine has not been observed. In the SGNHsuperfamily, the catalytic mechanism based on a dyad has not been clarified yet. One more characteristic feature of the SGNH-superfamily, related to the catalytic triad Ser-His-Asp (or Glu), is the distance of catalytic residues histidine and aspartate, which are separated by two amino acids in a sequence, whereas by 50 and more in α/β−hydrolases. The enzymes of SGNH-superfamily display substrate promiscuity, wide regio-specificity, and catalyze chemically different reactions.7,8,12,14−22 Accordingly, adaptability and flexibility of the active site can be expected, which can partly explain substrate and catalytic promiscuities. However, the natural substrates for most of the SGNH enzymes have remained unknown so far. In addition to important biological roles of SGNH enzymes, there is also a high potential in their biotechnological applications. Often detected substrate and enzyme promiscuities, optimal thermal stability, and wide pH-range have attracted attention because researchers want to explore and modify them to reach the maximal effect in biocatalysis.10,23−25 SGNH esterases and lipases obtained from extremophiles are of importance due to their excellent performance under extreme physicochemical conditions being of potential use in laundry detergents, finishing fabrics, and pulp and paper industries.25 In spite of a wide distribution of SGNH-hydrolases in living organisms, neither their biological functions nor catalytic mechanisms have been revealed so far. The focus of our research is to shed some light on the functional role of a Ser-

His catalytic dyad in the promiscuous enzyme from Streptomyces rimosus (SrLip, EC: 3.1.1.3) belonging to the SGNH-superfamily. Therefore, X-ray structure analysis and computational methods including molecular dynamics simulations and quantum-mechanical calculations within a cluster model of the enzyme were used.

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RESULTS AND DISCUSSION On the basis of sequence homology and conserved catalytic amino acid motif,1,2,11 we previously predicted Ser10 and His216 as catalytic amino acids,26 but Asp or Glu, two residues commonly present upstream of the catalytic His in SGNHhydrolases as a third member of a catalytic triad, was not observed in the SrLip protein sequence (Figure S1). Earlier, the nucleophile Ser10 was confirmed by the binding of a 3,4dichloroisocoumarin inhibitor and a detection of the corresponding covalent adduct by mass spectrometry.27 According to the BLAST tool,28 the enzymes with known 3D-structures most similar to SrLip are Streptomyces albidof lavus phospholipase A15 (SaPLA1, 64.4% protein sequence identity) and Streptomyces scabies esterase6 (SsEst, 22.0% protein sequence identity). Variations in the consensus sequences around the catalytic amino acids and amino acid residues of the oxyanion hole in these three homologous enzymes (Figure S1), and, in particular, a lack of a conserved catalytic acid (Asp/Glu), suggest somewhat different catalytic mechanisms, possibly based on a catalytic dyad. Among the determined 3D structures of SGNH hydrolases, in most cases, their active sites revealed the catalytic triad of Ser, His, and Asp,7 but details of catalytic mechanisms have not been resolved yet. Overall Structure of SrLip. The 3D-structure of SrLip was determined at the resolution of 1.75 Å (Figure 1A and Table 1). The monomeric SrLip enzyme comprises two molecules in an asymmetric unit (A and B). The two monomers are connected by a noncrystallographic symmetry (translation in the [101] direction). This leads to a very significant Patterson peak at (0.5, 0.0, 0.5) that amounts to 57% of the origin peak. Such an arrangement of molecules leads to an exceptionally low solvent content of 28%, only.29 The two monomers do not reveal significant conformational differences: the secondary structural elements overlapped well (RMSD of 0.54 Å).30 1929


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ACS Chemical Biology Table 1. Data Collection and Refinement Data wavelength (Å) resolution range (Å) space group unit cell total reflections unique reflections multiplicity completeness (%) mean I/σ(I) Wilson B-factor Rmerge Rmeas CC1/2 reflections used for refinement Rwork Rfree number of non-hydrogen atoms macromolecules water RMS (bonds) RMS (angles) Ramachandran favored (%) Ramachandran allowed (%) Ramachandran outliers (%) clash score average B-factor

1.0 24.01−1.71 (1.80−1.71) P21 38.09 78.69 56.56 90 104.49 90 131164 (15512) 34875 (4915) 3.76 (3.16) 0.99 (0.96) 9.3 (3.1) 14.69 0.09 (0.33) 0.11 (0.39) 0.98 (0.87) 34853 (3285) 0.1647 (0.2556) 0.2142 (0.3179) 3922 3420 468 0.007 1.27 97 2.4 0.21 5.07 17.14

Figure 2. Active site of the SrLip enzyme is compared with the one of SsEst, exhibiting the first catalytic dyad observed in the SGNH-family (in both active sites, hydrogen bonded water molecule chains are shown). (a) The analogous interactions to ones in SsEst are observed in SrLip (Streptomyces rimosus lipase, green): Ser10-OγH···NεHis216, His216-NδH···OC-Ser214, and Gly54-NH···Oγ-Ser10. The striking fact is that the relative positions of the carbonyl O atoms (of the main chains) acting as the neutral hydrogen bond acceptors in both enzymes are maintained. In both enzymes with catalytic dyad, these hydrogen bonds ensure the orientation of imidazole rings of Hisresidues suitable for double proton transfer involving a neutral proton acceptor (carbonyl group) in contrast to the carboxyl O atom of the aspartate residue, a typical member of the catalytic triad. (b) In the structure of SsEst (Streptomyces scabies esterase, PDB: 1ESC, blue), the hydrogen bonds Ser14-OγH···NεHis283, His283NδH···OCTrp280, and Gly66-NH···Oγ-Ser14 are essential for the catalysis.

Therefore, molecule A is used for all figures and subsequent computations reported here. All residues are found within the allowed regions of the Ramachandran plot. The mature enzyme comprises 234 amino acid residues, and all are visible in the Fourier maps. The enzyme reveals the Rossmann-like 3-layered αβα-sandwich fold typical of the SGNH-superfamily31 (CATH 3.40.50.1110) stabilized by the three disulfide bonds: Cys27− Cys52, Cys93−Cys101, and Cys151−Cys198, which were previously mapped by mass spectrometry.32 A structure similarity search using the DALI server33 indicated 4HYQ (SaPLA1 from S. albidof lavus)5 and 1ESC (SsEst from S. scabies)6 to be the closest ones to SrLip (Figure 1B,C). Their Z-scores of 38.7 and 27.3 can be expected from the BLAST search showing 64.4% and 22.0% sequence identity with SrLip, respectively. As with SrLip, both of these enzymes contain three disulfide bridges, which contribute to the compactness of the structure. Active Site of SrLip Involves a Catalytic Dyad. The active site is located in front of the entrance to the hydrophobic binding pocket. It comprises the catalytic Ser10 and His216 residues connected by a Ser10-Oγ-H···NεHis216 hydrogen bond (Figure 2A). The nucleophile Ser10 is placed within a short α1 helix connecting the strand β1 and the loop 13−22, whereas a motif of the α/β-hydrolases,34 the nucleophilic elbow, is supported by a less flexible and long helix (Figure S2). An insertion of about 20 amino acids after the short α1 helix containing Ser10 makes this the most flexible part of the enzyme; this might be correlated with the multifunctionality common to the SGNH enzymes. The catalytic His216 is located under the loop formed by residues 192−211. It is striking that residues 211−213 form a turn that orients the Asn213 away from the catalytic site and prevents it from participating in catalysis (Figure 2A); in the hydrolases with the catalytic triad, this very site is occupied by the catalytic Asp.

The highly hydrophobic and voluminous binding pocket of irregular shape has two compartments connected by a very narrow passage (situated between helices α6, α7, and α9, Figures 1A and S3). This passage is formed by aromatic rings of Tyr11, Tyr141, and Phe144, which could be closed by slight movements of their side chains and thus enable reshaping of the binding pocket; the first compartment, the smaller one, can accommodate short aliphatic chains; this passage can be relaxed using the whole binding pocket for longer aliphatic chains (Figures 3 and S3). Such a shape of the hydrophobic binding site and its dynamics are consistent with the demonstrated substrate promiscuity.14 To probe the characteristics of the active and binding sites, molecular dynamics simulations of both the apoenzyme and enzyme−substrate complex with pnitrophenyl caprylate (C8) were performed. The choice of this particular substrate was prompted by our recent work,14 which identified it as one of the best substrates tested against SrLip. In the active site microenvironment, there are hydrophobic residues (Val15 and Leu204) influencing the catalytic properties of His216 and perturbing pKa values of its imidazole 1930


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Figure 3. Insight into the active site and the binding pocket (gray shaded) of the SrLip complex with the p-nitrophenyl caprylate (C8) substrate, prior to the catalytic reaction, obtained by MD simulations. The C8 tail of the substrate is anchored in the hydrophobic pocket lined by Tyr11, Gly81, Phe86, Tyr141, Pro142, Phe144, Ile163 (extending behind the helix), Ala167, and Ile170. The location of the octanoyl aliphatic chain coincides with pentaethylene glycol bound in the binding pocket of phospholipase A1 from Streptomyces albidof lavus (PDB: 4HYQ).

carbonyl oxygen of Trp280 functioning as a neutral hydrogen bond acceptor (Figure 2B). Close overall structural similarity of SrLip with both SaPLA1 and SsEst (Figure 1) also reflects their functional similarity exhibiting a catalytic mechanism based on a dyad;5,6,35 catalytic residues in their active sites are conserved, Ser10/11/14 and His216/218/283, respectively (Figures S1 and 2). Interestingly, mutations of Ser216Ala and Ser216Asp in SaPLA1 revealed a total loss of enzyme activity.35 In the case of SrLip investigated here, our MD simulations show that an in silico Ser214Asp mutation does not produce a catalytic triad. During the entire 150 ns of MD simulations of the Ser214Asp mutant enzyme, the deprotonated Asp214 carboxyl group forms a persistent hydrogen bond with Thr208 and is permanently oriented away from the catalytic His216 residue (Figure S5), thus making the formation of a canonical catalytic triad very unlikely. It is important to emphasize that the hydrogen bonding essential for the SrLip catalysis, involving the catalytic dyad Ser10-OγH···NεHis216, His216-NδH···OC-Ser214, and Gly54-NH···Oγ-Ser10, is also present in the homologous enzymes discussed. The carbonyl oxygen atom of a main chain of Ser214/216 (in SrLip and SaPLA1) acts as the neutral hydrogen bond acceptor ensuring the orientation of the imidazole ring of His216/218 suitable for a proton transfer. However, in SsEst, Trp280 takes over the role of Ser214/216 (Figure 2B). Analogous MD simulations for apoenzyme and its complex with the p-nitrophenyl caprylate substrate confirmed the characteristic hydrogen bonds. Interestingly, a hydrogen bond between the main chain carbonyl group of Ser214 and NδH donor in His216, in both the apoenzyme and substrate− enzyme complex was maintained over the entire MD simulations (Figure 3 and Table S1). The same applies to the backbone amino group of the oxyanion-hole residue Gly54, which interacts with the hydroxyl oxygen of Ser10. Importantly, the crucial hydrogen bond between the Ser10 hydroxyl and His216 imidazole Nε occurs ten times more frequently in the enzyme with the bound substrate than in the apoenzyme, which is a significant observation (Table S1). This indicates that substrate binding promotes essential hydrogen bond formation between Ser10 and His216.

moiety. The crystal structure and molecular dynamics simulations (MD) of both the apoenzyme and enzyme/ substrate complex have not shown any charged residues in the vicinity of His216. Thus, because of the lack of a third residue of a catalytic triad (commonly in serine hydrolases completed by the Asp residue), the orientation of the imidazole moiety of His216 appropriate for catalysis is ensured by a His216 NδH···OC-Ser214 hydrogen bond (Figure 2A, Table S1). In other words, the pattern of a HisNδH···OOC-Asp hydrogen bond, typical of the catalytic triad is replaced by a hydrogen bond to the backbone carbonyl oxygen atom of Ser214. This is the most striking difference between the SGNH-hydrolases with the catalytic dyad and those with the triad. In this environment, our MD simulations at constant pH estimated the pKa value of the His216 imidazole moiety to be 5.2 (Figure S4), being significantly reduced from the value of 6.6 in aqueous media. This fact has several important implications for the activity of SrLip. First, it indicates the hydrophobic nature of the active site, thus suggesting that the binding of the predominantly apolar ester substrate will likely be exergonic and that the release of the formed polar products, an alcohol and carboxylic acid in particular, which will likely be favorable. Second, it convincingly shows that before the enzymatic reaction, His216 is in its un-ionized neutral form, thus ruling out the possibility of a Ser−His proton transfer prior to the substrate binding, which was demonstrated to occur in several triads.34 Third, a lowered His216 basicity implies that the initial Ser10 deprotonation will be high in energy, defining the rate-limiting step of the overall transformation. All of these aspects are presented in the section SrLip Catalytic Mechanism. For the first time in the SGNH-superfamily, a novel combination of the catalytic residues representing a dyad was observed in the 3D-structure of an esterase (PDB: 1ESC) from Streptomyces scabies.6 The active site of SrLip is compared to the one of the homologous S. scabies esterase (SsEst) (Figures 2A and B). In the active site of SsEst, the nucleophile Ser14 is hydrogen bonded to His283, and this hydrogen bond is not affected by the binding of the inhibitor.6 A carboxylic acid (Asp/Glu) is not present in the active site; thus, a suitable orientation of the His283 imidazole ring is ensured by a hydrogen bond between its Nδ-H fragment and a main-chain 1931


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ACS Chemical Biology SrLip Catalytic Mechanism. In order to elucidate the most feasible mechanism of the SrLip catalytic activity, quantummechanical calculations of several chemically possible reaction pathways were performed. From MD simulations, the snapshot among the lowest energy structures, having the substrate properly oriented for the reaction, was selected; geometries of the p-nitrophenyl caprylate substrate together with the following residues: Ser10 and His216 (the catalytic dyad), Gly54 and Asn82 (the oxyanion hole), and Asp9 and Tyr11 forming a tripeptide with the Ser10 residue were extracted (Figure 4). According to the estimated pKa values of Asp9 and

In order to minimize the errors associated with the initial selection of starting geometries from MD trajectories, we tried several conformations of the p-nitrophenyl caprylate substrate within the so-formed cluster and proceeded with the subsequent mechanistic calculations using the most stable complexes. After geometry optimization, the substrate remained in the active site predominantly stabilized through hydrogen bonds involving its carbonyl oxygen atom and the amino nitrogens of both Asn82 (2.995 Å) and Gly54 (3.53 Å); Gly54 completes its double proton donor function in the hydrogen bond to the Oγ oxygen of Ser10 (3.01 Å). While we inspected several different conformations, the most favorable position of the reactive water molecule reveals hydrogen bonding with Asn82 and Gly54 carbonyl atoms of the oxyanion-hole residues. Interestingly, the binding of the substrate and water molecule calculated in this way is favorable and exergonic at ΔGbind = −2.7 kcal mol−1, which is consistent with placing the p-nitrophenyl caprylic ester into the predominantly hydrophobic active site of the SrLip enzyme. Distances from the Ser10 reactive hydroxyl Oγ oxygen to the His216 imino Nε2 atom and substrate carbonyl carbon atoms are 2.795 and 3.959 Å, respectively. From this stationary point, we first investigated the feasibility of a direct nucleophilic attack of a water molecule toward the substrate carbonyl center. This produces a tetrahedral intermediate with the water−OH group bonded to the substrate, while the Asn82 carbonyl oxygen abstracts the remaining proton (Scheme S1). The reaction has the barrier of 27.3 kcal mol−1, which turned out to be 9.4 kcal mol−1 higher than that for a more feasible mechanism presented in Figure 5 later. In addition, the formed intermediate is very stable, and all attempts to model various subsequent steps of its degradation gave no indications for either transition states or stable intermediates, or were associated with barriers too high for feasible processes, which, taken all together, led us to conclude that direct hydrolysis by a water molecule is improbable. Second, a much more feasible mechanism is initiated through the deprotonation of the Ser10 hydroxyl group to the His216 imino Nε2 atom followed by the bond formation with the substrate carbonyl carbon atom (Figure 5). The mentioned proton transfer and nucleophilic attack proceed in a concerted way, being in agreement with earlier reports.41,42 Moreover, a very recent study by Uritsky and co-workers underlined that a

Figure 4. Schematic representation of the computational cluster model of the SrLip enzyme employed here (R1 = C8 aliphatic chain of the caprylic ester). Dashed lines indicate hydrogen bonds in the optimized reactant complex.

His216 (3.0 and 5.2, respectively), these residues were considered as deprotonated anion and un-ionized imidazole, respectively. This allowed us to build a cluster model of the SrLip enzyme on which all calculations were performed at the (CPCM)/M06-2X/6-311++G(2df,2pd)//(CPCM)/M06-2X/ 6-31G(d) level of theory. The choice of such a computational setup was prompted by its success in reproducing thermodynamic and kinetic parameters of various organic36,37 and enzymatic reactions.38−40 Further computational details are included in the Supporting Information.

Figure 5. Free energy profile for the most feasible pathway of the SrLip catalytic hydrolysis of p-nitrophenyl caprylate calculated at the (CPCM)/ M06-2X/6-311++G(2df,2pd)//(CPCM)/M06-2X/6-31G(d) level of theory. Only stationary point structures are presented due to clarity. 1932


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Figure 6. Complete pathway of SrLip reveals the overall enzyme reaction in the two steps: the formation of a covalent enzyme−alkoxyester intermediate (acylation) and its hydrolysis (deacylation) to restore the enzyme cycle. Each step consists of two processes, giving in total four distinct steps used by the SrLip enzyme to hydrolyze an ester to the corresponding alcohol and carboxylic acid. These include (i) deprotonation of Ser10− OH to His216 and the concerted addition of the nucleophilic hydroxide Ser10−O− toward the substrate carbonyl carbon atom, (ii) proton transfer from the protonated His216−H+ residue to the alkoxy group to generate and liberate an alcohol, (iii) nucleophilic attack of the water molecule to the substrate carbonyl carbon atom promoted by deprotonation to His216, and (iv) regeneration of the enzyme through the His216−Ser10 proton transfer to produce and liberate a carboxylic acid.

stepwise process will be operative only if pKa(His) ≥ pKa(Ser) in the Michaelis complex,34 otherwise the classical concerted mechanism will dominate, as our results indicate is the case for the SrLip enzyme. The free energy barrier for this process is ΔG⧧ = 15.2 kcal mol−1 (imaginary vibrational frequency, νIMAG = 298i cm−1), which increases the charge on the substrate carbonyl oxygen from −0.63 to −0.85 |e| that becomes an alkoxide in the tetrahedral intermediate. The transition state is predominantly stabilized with the oxyanion hole residues as evidenced in the shortening of the corresponding O(substrate)−N(Asn82) and O(substrate)−N(Gly54) bonds (3.066 and 2.870 Å, respectively). The reduction in the mentioned bond lengths is not excessive, particularly for the former bond, which is in line with earlier observations and rationalized by the enzyme design which allows only suboptimal stabilization of the oxyanions in order to avoid overstabilization of the initial reactants.43 This step is enderogonic, ΔGR = 12.2 kcal mol−1, and produces an anionic tetrahedral intermediate (Figure 6), which weakens and significantly elongates the C(carbonyl)−O(phenol) bond from 1.364 Å in the reactants to 1.535 Å in the intermediate. In the next step, ionized His216 acts as an acid and protonates the substrate phenol oxygen atom enabling the leaving group to be an alcohol instead of an alkoxide. This is associated with a barrier of 5.7 kcal mol−1 (νIMAG = 114i cm−1), yielding the total activation free energy for the complete acylation reaction of ΔG⧧ENZ = 17.9 kcal mol−1 (Figure 5), making it the rate limiting step of the overall transformation. This gives p-NO2phenol and the acyl enzyme, and the whole process is exergonic, ΔGR = −0.3 kcal mol−1.

Deacylation of the enzyme begins with the nucleophilic addition of the active site water molecule to the substrate carbonyl center, which is facilitated with a concerted deprotonation to His216 acting as a base (Figures 5 and 6). The concerted character of this process is again in agreement with previous reports related to a deacylation reaction of a methanethiolester by water, where a concerted mechanism involving a neutral nucleophile assisted by an imidazole base was suggested with a barrier of 20 kcal mol−1.42,44 According to our calculations, in SrLip this process of water addition is, together with the preceding phenol departure from the active site, associated with a barrier of 17.6 kcal mol−1 (νIMAG = 799i cm−1), being only 0.3 kcal mol−1 lower than that for the acylation step (Figure 5). The close values of the activation parameters of acylation and deacylation reactions have also been previously established in acetylcholinesterase45 and butyrylcholinesterase-catalyzed46 reactions. Accordingly, the system relaxes to the second tetrahedral intermediate, having the charge on the substrate carbonyl/alkoxide oxygen of −0.92 |e|. The subsequent reinforcement of the substrate carbon− oxygen double bond is achieved by the His216−Ser10 proton transfer, which requires only 0.6 kcal mol−1 (νIMAG = 323i cm−1) to give the regenerated SrLip enzyme and a carboxylic acid, anchored in the active site through hydrogen bonds to Asn82 and Gly54 (3.147 and 3.009 Å, respectively). Liberation of the carboxylic acid is accompanied by the gain in the free energy of 9.2 kcal mol−1 (Figure 5), which is in agreement with the demonstrated hydrophobic nature of the SrLip active site. The calculated total reaction free energy of ΔGR = −4.8 kcal mol−1 quantitatively agrees with the reported experimental free energies of hydrolysis of p-nitrophenyl-acetate and methyl-p1933


ACS Chemical Biology nitrophenyl-sulfate in water of −3.147 and −7.8 kcal mol−1,48 respectively. The proposed mechanism in Figure 6 is fully in line with a previously established catalytic regime for a range of serine and cysteine hydrolases.49,50 The complete reaction proceeds downhill in energy, occurring through stationary points at relative energies of 0.0, −0.3, and −2.1 kcal mol−1, which makes it feasible, where acylation represents the rate-limiting step associated with the activation free energy of ΔG⧧ENZ = 17.9 kcal mol−1 (Figure 5). This is found in very good agreement with the experimental value of 14.5 kcal mol−1 determined previously for the p-nitrophenyl caprylate substrate,14 thus providing strong support in favor of the proposed catalytic mechanism based on a dyad. It has to be emphasized that the difference of 3.4 kcal mol−1 is found well within the estimated error of the applied QM−cluster approach of 5 kcal mol−1, assessed by Siegbahn and co-workers on the basis of extensive calculations for a large number of enzymes.51,52 The latter value became an acceptable threshold of the QM−cluster calculations, while this approach turned out to be very useful in determining the catalytic mechanism of a variety of different enzymes.38,40,51,52 Taken all together, a combination of MD simulations and QM calculations presented here offer strong evidence that SrLip is able to operate as a dyad, without the need for a third active site residue. In addition, by now, several families of classical serine protease triads have been discovered differing in the identity of the third catalytic residue being Asp, Glu, His, Ser, or Lys.53,54 Nevertheless, all studies are consistent in the underlying the role of the third residue in increasing the nucleophilicity of the active site serine through (1) stabilizing the ionic transition state through hydrogen bonding without a change in the protonation state, (2) accepting a proton from His in the transition state, thus creating a double proton transfer-type mechanism, or (3) orienting His in a proper position to interact with the substrate, thus contributing entropically to the rate acceleration. Since in SrLip, His216 is directly hydrogen bonded only to the Ser214 backbone carbonyl oxygen, none of these effects have a significant contribution to the SrLip catalysis. This results in the obtained kinetic and thermodynamic parameters that might turn out to be less favorable than those in analogous classical triads, where these effects are operational, which might be a general feature as suggested by Uritsky and co-workers.34 Concluding Remarks. The crystal structure of a bacterial enzyme SrLip of the SGNH-superfamily together with a combination of MD simulations and QM calculations convincingly revealed the active site of the SrLip enzyme operating on a Ser10-His216 catalytic dyad, without the need for a third active site residue as in classical hydrolytic triads. Mechanistic calculations at the DFT level, within a carefully selected cluster model of the SrLip enzyme, demonstrated that the overall transformation is a two-step process with the initial acylation being the rate-limiting step associated with the calculated free energy barrier of ΔG⧧ = 17.9 kcal mol−1. The latter is found in very good agreement with the experimental value of 14.5 kcal mol−1, thus providing strong support in favor of the proposed catalytic mechanism based on a dyad. The obtained results are consistent with the mechanism of some other dyads reported earlier and particularly with the fact that various unconventional “serine-only” and “threonine-only” hydrolases have been described as well.53,54

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ASSOCIATED CONTENT

Data Collection and Processing. The protein crystallized in the monoclinic space group P21, with two molecules in an asymmetric unit and unit-cell parameters a = 38.1, b = 78.7, c = 56.6 Å, and β = 104.5°. The structure was solved by molecular replacement using Phaser55 employing the structure predicted by Phyre2 server.56 The data was subsequently refined using Phenix.57 The summary of data collection and refinement statistics are given in Table 1. An asymmetric unit comprises two molecules in the unit cell. In the solution, the enzyme is monomeric as revealed by comparison of molecular mass determined by size-exclusion chromatography and SDS−PAGE (27.6 and 27.5 kDa, respectively).58 As an extracellular enzyme, it includes a signal peptide of 34 amino acids, whereas a mature enzyme comprises 234 residues, which were traced in the electron density map and amino acid sequence. However, the presence of inhibitor, 3,4-dichloroisocoumarin (DCI), which had been added during the preparation of crystallization sets, was not observed in a difference Fourier map. This is not a total surprise, considering the already observed effect of reactivation of SrLip after prolonged incubation of the completely DCI-inhibited enzyme.59 From the preparation of the SrLip-DCI complex to crystallization and further to synchrotron data collection, 89 days passed, which is enough for the hydrolysis of (already opened) isocoumarin in the enzyme active site, mediated by labile acyl moieties.59 S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.6b01140. Sequence alignment of SrLip (Q93MW7) with two homologous enzymes (K0J3J2 and P22266), figure of nucleophilic elbow in α/β-hydrolases compared with a similar structure in SGNH-hydrolases, representation of the SrLip binding site, titration curves from MD simulations at constant pH, snapshot of the Ser214Asp mutant, scheme of first tetrahedral intermediate formed in SrLip catalysis, geometric parameters and hydrogen bond occupancies during MD simulations, experimental details on protein purification and crystallization, and computational details of molecular dynamics simulations and quantum mechanical calculations (PDF) Accession Codes

The atomic coordinates of SrLip lipase have been deposited in the PDB under accession code PDB: 5MAL.

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AUTHOR INFORMATION

Corresponding Authors

*(Z.Š.) E-mail: [email protected]. *(R.V.) E-mail: [email protected]. ORCID

Aleksandra Maršavelski: 0000-0003-1139-7173 Robert Vianello: 0000-0003-1779-4524 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge Dušan Turk, who kindly offered us the use of the crystallization robot at the Jožef Stefan Institute, Ljubljana, Slovenia, and the Elettra synchrotron, Trieste, Italy for the provision of synchrotron facilities. This work was supported by the Ministry of Science, Education and Sports of the Republic of Croatia, grant no. 098-1191344-2943. R.V. gratefully 1934


Articles


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acknowledges the European Commission for an individual FP7 Marie Curie Career Integration Grant (contract number PCIG12-GA-2012-334493). A.M. wishes to thank the Croatian Science Foundation for a doctoral stipend through the Career Development Project for Young Researchers (Contract No. I3376-2014).

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Catalytic Dyad in the SGNH Hydrolase Superfamily: In-depth Insight

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