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Chapter 111

Thermolysin and Related Bacillus Metallopeptidases DATABANKS MEROPS name: thermolysin MEROPS classification: clan MA, subclan MA(E), family M4, peptidase M04.001 IUBMB: EC 3.4.24.27 (BRENDA) Tertiary structure: Available Species distribution: class Bacilli Reference sequence from: Bacillus thermoproteolyticus (UniProt: P00800)

Name and History Thermolysin is the name first given to an extracellular 34.6 kDa metalloendopeptidase secreted by the Grampositive thermophilic bacterium Bacillus thermoproteolyticus [1]. It was the first metalloendopeptidase to be crystallized and to have its structure solved (reviewed in Matthews [2]). There is some confusion over the naming of, and the relationships among, the enzymes in metalloproteinase clan MA, family M4. Many of the enzymes have trivial names and have been treated as separate entries previously. However, it is becoming increasingly clear that many of these enzymes share significant characteristics, in particular with respect to activity, specificity and structural features, justifying that they are treated as one group. This overview concerns enzymes that previously belonged to at least three groups: (1) the thermolysin group, consisting of enzymes from A. acidocaldarius, B. caldolyticus, B. stearothermophilus, B. thermoproteolyticus, and others; (2) thermolysin homologs from other Bacillus sp. and related Gram-positive bacteria, e.g. the enzymes from B. cereus, B. megaterium, B. thuringensis and Lactobacillus species; and (3) enzymes often referred to as bacillolysins, found in, e.g. B. amyloliquefaciens, B. cereus and B. subtilis. All these enzymes share significant sequence similarities that indicate similar threedimensional structures, catalytic mechanisms, and even, most probably, substrate specificities [3]. A CLUSTALW-based alignment of the mature enzyme

sequences yields a family that does not divide strongly into separate groups (Figures 111.1 and 111.2). Several of the metalloproteases described in other chapters show significant sequence (and structural) similarity to thermolysin. However, this chapter only deals with the metalloproteases described above, that is, enzymes with MEROPS IDs M04.001, M04.014 and M04.015. This selection was made partly for historical reasons and partly on the basis of phylogenetic analyses (e.g. de Kreij et al. [3]). It seems worth considering a further review of the subdivision of these metalloproteases in the near future. While many aspects of thermolysin have been studied in large detail, information on most related proteases (‘TLPs’ for Thermolysin-Like Proteases) is scarce and scattered. Therefore, this chapter primarily deals with results and conclusions derived from work conducted with thermolysin and its most closely related homologs. Published work on related enzymes such as TLPs from B. subtilis [4], B. stearothermophilus CU21 [5], and B. cereus [6], as well as our own unpublished observations indicate that thermolysin and TLPs are generally quite similar in terms of enzymological properties (e.g. de Kreij et al. [3]). Thermolysin is widely used in protein chemistry as a nonspecific protease to obtain sequence or conformational data [7,8]. The enzyme can also act as a peptide and ester synthetase [9
14] and has been used for the production of a precursor of the artificial sweetener, aspartame [15,16]. Thermolysin has also been used to generate an active-site model for the design of inhibitors for mammalian zinc endopeptidases such as neprilysin [17].

Activity and Specificity The pH
activity profiles of thermolysin and TLPs follow a bell-shaped curve with maximal activity at or near pH 7.0 for both proteolysis [18
22] and esterolysis [20]. The enzyme is inhibited by phosphate buffers [23] and CaCl2 in the range 1
10 mM is usually included in the buffers to minimize autolysis. High concentrations of neutral salts

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GBT:U25629 PR:JC4113 GBT:U25630 SW:P23384 SW:P06874 SW:P00800 SW:P43133 PR:S41312 GBT:M21663 GBT:D29673 PR:A24306 SW:Q00891 SW:P43263 SW:P29148 SW:P39899 SW:P06142 PR:JQ2129 SW:P06832

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(B. caldolyticus YPT) (B. acidocaldarius) (Bacillus sp. EA1) (B. caldolyticus) (B. stearothermophilus CU21) (B. thermoproteolyticus Rokko) (B. stearothermophilus TELNE) (B. thermoproteolyticus Rokko) (B. stearothermophilus MK232) (Lactobacillus spp) (B. cereus) (B. megaterium) (B. brevis) (B. polymyxa) (B. subtilis) (B. subtilis var. amylosacchariticus) (B. subtilis var. amylosacchariticus) (B. subtilis var. amyloliquifaciens)

FIGURE 111.1 Evolutionary tree for the thermolysin/bacillolysin group. The tree was produced from a CLUSTAL alignment (DNASTAR). There is no clear segregation of a bacillolysin and thermolysin family, although a group of enzymes known most commonly as ‘thermolysins’ are most tightly clustered. Key to enzymes: U25629, Bacillus caldolyticus neutral proteinase (npr) gene; JC4113, neutral protease from Bacillus sp.; U25630, Bacillus sp. neutral proteinase (npr) gene, complete CDS; P23384, bacillolysin precursor (gene npr) from Bacillus caldolyticus; P06874, thermolysin precursor, Bacillus stearothermophilus; S41312, thermolysin from Bacillus thermoproteolyticus; X76986, Thermolysin (gene npr) from Bacillus thermoproteolyticus; P43133, bacillolysin precursor from Bacillus stearothermophilus strain TELNE; D29673, hydrolase from Lactobacillus sp.; A24306, bacillolysin precursor from Bacillus cereus; D5DEH5, bacillolysin precursor from Bacillus megaterium; P43263, bacillolysin precursor from Bacillus brevis; P29148, bacillolysin precursor from Bacillus polymyxa; P39899, neutral protease precursor from Bacillus subtilis; P68734, bacillolysin precursor from Bacillus subtilis var. amylosacchariticus and Bacillus mesentericus; JQ2129, bacillolysin (EC 3.4.24.28) precursor
Bacillus subtilis var. amylosacchariticus; P06832, bacillolysin precursor from Bacillus amyloliquefaciens. PR, PIR; SW, SwissProt; GB, Genbank (translated).

activate the hydrolysis of certain thermolysin substrates [20,24
27]. Thermolysin specificity has been investigated using a variety of different peptide and model peptide substrates [18,28
35]. The major specificity site of the enzyme is at S10 , which accepts large hydrophobic residues. Thermolysin therefore preferentially cleaves peptides and proteins at the N-terminal side of Leu, Phe, Ile and Val, although hydrolysis of bonds with Met, His, Tyr, Ala, Asn, Ser, Thr, Gly, Lys, Glu or Asp at P10 has been observed [33]. Substrates can also interact with residues in the S1, S2 and S20 subsites, although these interactions are less important for specificity. A hydrophobic residue is preferred in the P1 position, Ala or Phe is preferred to Gly in P2 and in P20 , the order of preference is Leu . Ala . Phe . Gly. Amino acids in the P3 and P30 positions can also affect activity, with Ala or Phe promoting catalysis as compared to Gly in both cases. Studies with FRETS-libraries have shown that thermolysin has a preference for basic residues in P30 [36]. The quenched fluorescent substrate Dabcyl-Ser-Phe-EDANS has been developed for thermolysin and dispase an unsequenced metallopeptidase from Paenibacillus polymyxa); cleavage occurs at the Ser-Phe bond [37]. Available information for TLPs indicates that these enzymes have similar overall catalytic properties as thermolysin. However, the enzymes do display variation in their S10 subsites, with considerable effects on the preferences

for various peptide substrates [3,38
41]. De Kreij et al. [3] showed that these differences are due to a very limited number of sequence variations. It is important to note that TLPs seem to have a truly broad substrate specificity. For example, it has been stated that, for a proteinaceous substrate, conformational features rather than sequence characteristics determine the sites of proteolytic attack [42]. TLP activity can be measured using nonspecific protease substrates such as casein [5] but more specific substrates are commercially available, of which the furylacryloyl dipeptide FA-Gly-Leu-NH2 or FAGLA, is the most commonly used. Hydrolysis of the Gly-Leu bond can be continuously monitored by measuring the decrease in A345 [23]. FA-Gly-Leu-NH2 has also been used for other enzymes of the group, but does not appear to be hydrolyzed by thermolysin from Bacillus sp. var. EA1 [43]. FA-Gly-Leu-NH2 has the disadvantage of a high Km (30 mM) relative to its solubility (2 mM in 10% DMSO). The tripeptide FA-Gly-Phe-Leu has more favorable kinetic properties [32,41]. A two-step assay for thermolysin uses 3-carboxypropanoyl-Ala-AlaLeu-NHPhNO2 as a substrate, which is cleaved at the Ala-Leu bond. Addition of an aminopeptidase releases 4-nitroaniline, which can be followed by the increase in A405 [44]. Fluorescent substrates developed include the internally quenched Abz-Ala-Gly-Leu-Ala-Nba (Nba: nitrobenzylamide) [45]. The radioactive pentapeptide [3H] Tyr-GlyGly-Phe-Leu is hydrolyzed at the Gly-Phe bond and the

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FIGURE 111.2 Alignment of the thermolysin/bacillolysin family. The alignment was generated using CLUSTALW and the output was displayed using the program MALPLOT (S.J. Hubbard, personal communication, http://sjh.bi.umist.ac.uk) to include the secondary structure cartoon of thermolysin PDB: 6TMN. Key to enzymes: See Figure 111.1.

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product, [3H] Tyr-Gly-Gly, can be isolated using hydrophobic beads [46,47]. Other chromogenic [48] and fluorogenic [49,50] substrates have been synthesized for thermolysin. Thermolysin is reversibly inhibited by millimolar concentrations of zinc-chelating agents such as 1,10-phenanthroline [51]. Inhibition by EDTA is irreversible, as EDTA preferentially chelates the calcium ions, leading to autolysis [42]. The enzyme is also inhibited by high concentrations of zinc .10 μM [51] which binds to an active-site histidine residue [52]. More specific thermolysin inhibitors are generally modified di- or tripeptide substrate analogs, with a hydrophobic residue to fit the S10 subsite and containing a strong zinc-binding agent such as a phosphoramidate, phosphonamidate, sulfhydryl, hydroxamate or carboxylate group. The development and synthesis of some of these molecules is discussed in the following references: [53
63]. The collection of available (potential) TLP inhibitors is still expanding, partly as a side product of the search for effective inhibitors of mammalian zinc endopeptidases. One recent development concerns the use of beta-amino acids which yielded the potent but unstable inhibitor N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-amino benzoate (CFP; [64]). Another recent development concerns the synthesis of short peptides with a C-terminal thiol group, which were supposed to coordinate the catalytic zinc and interact with the major substrate binding site in concert [65]. Various potent inhibitors were obtained by these means, the most efficient one being Pro-Leu-CA-SH (Ki 5 30 μM).

Phosphorus-Containing Inhibitors The commercially available inhibitor phosphoramidon [N-(α-L-rhamnopyranosyl-oxyhydroxyphosphinyl)- L-leucyl-L-tryptophan] is a naturally occurring phosphoramidate from Streptomyces tanashiensis which inhibits thermolysin with a Ki of 30 nM at neutral pH [66]. Phosphoramidon is a slow binding inhibitor [54,67] and its Ki value is highly pH dependent, varying from 1.4 nM at pH5.0 to 8.5 μM at pH8.5 [67]. The structure of the thermolysin
phosphoramidon complex has been studied by X-ray crystallography [68] and NMR spectroscopy [69]. The unsubstituted phosphoramidate, N-phosphoryl Leu-amide (Ki 1.9 μM), has also been co-crystallized with the enzyme [68]. Studies of a series of phosphonamidate inhibitors and corresponding substrate analogs showed a strong correlation between the Ki values of the inhibitors and the kcat/Km values for the corresponding substrate analogs, leading to the conclusion that the phosphoamidates are transition state analogs [58,60]. One of these, the slow-binding Z-PheP*-L-Leu-L-Ala (PheP* denotes that the trigonal carbon of the peptide linkage is replaced

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by the tetrahedral phosphorus of a phosphonamidate group), is the most potent thermolysin inhibitor so far described, with a Ki value of 68 pM, and its binding has been studied by X-ray crystallography (4TMN) [70] and NMR spectroscopy [71]. Thermolysin has also been cocrystallized with two cyclic phosphonamidate inhibitors [63]. Phosphonate ester inhibitors have a 1000-fold lower affinity for thermolysin than their phosphonamidate analogs and the possible reasons for this have been the subject of crystallographic [72], mechanistic and molecular dynamics studies [73
79].

Hydroxamate-, Sulfhydryl- and CarboxylateContaining Inhibitors Several hydroxamate-containing thermolysin inhibitors have been synthesized and the enzyme has been co-crystallized with L-Leu-HNOH (Ki 5 190 μM, Brookhaven PDB: 4TLN) and HONH-benzylmalonyl-L-Ala-GlyNHPhNO2 (Ki 5 0.43 μM, Brookhaven PDB: 5TLN) [80]. The binding of a hydroxamate inhibitor to thermolysin has also been the subject of a mechanistic study [81]. Thermolysin has been co-crystallized with three β-thiol inhibitors: (2-benzyl-3-mercaptopropanoyl)-L-alanylglycinamide (Ki 5 0.75 μM) [82], N-(S)-2-(mercaptomethyl)-1oxo-3-phenylpropylgly-cine (thiorphan) (Ki 5 1.8 μM) and ([(R)-1-(mercaptomethyl)-2-phenylethyl]amino)-3oxopropanoic acid (retrothiorphan) (Ki 5 2.3 μM) [83] and with the carboxylate-containing inhibitors N(S)-(1-carboxy-3-phenylpropyl)-(S)-leucyl-(S)-tryptophan (Ki 5 50 nM, Brookhaven PDB: 1TMN) [84], N-[1-(2(R, S)-carboxy-4-phenylbutyl)-cyclopentylcarbonyl]-(S)-tryptophan [85] and benzylsuccinic acid (Brookhaven PDB: 1HYT) [86].

Irreversible Inhibition Thermolysin is irreversibly inhibited by the D-enantiomer of the active site-directed inhibitor N-chloroacetyl-Nhydroxyleucine methyl ester [87,88], which alkylates the active-site glutamate residue [89]. Molecular dynamics studies of the binding of inhibitors to thermolysin can be found in, e.g. Bohacek & McMartin [62], Merz & Kollman [76], Ghosh & Edholm [90], Giessner-Prettre & Jacob [91], Wasserman & Hodge [92], Garmer et al. [93], Murray et al. [94] and Bohm & Klebe [95].

Structural Chemistry The first crystallographic structure of thermolysin was reported in 1972 [96,97] and the structure has been refined [98] and analyzed with bound inhibitors since then [2,99].

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Glu143

His142

His146

Zn Glu166

His231

FIGURE 111.4 The thermolysin active site. The catalytic residues, referred to in the text, are highlighted (image produced by Setor [187]).

FIGURE 111.3 The structure of thermolysin. (A) The active-site residues, catalytic zinc atom, and the four calcium atoms are highlighted (image produced by Setor [187]). (B) A GRASP (Nichols, [188]) image of thermolysin, in approximately the same orientation, highlights the active-site cleft.

The initial structures reported were thought to be those of the free enzyme, but it is now clear that a dipeptide was present in the active site [100]. Hausrath & Matthews [99] reported a structure of thermolysin without a ligand bound in the active site (Brookhaven PDB: 1L3F). Mature thermolysin comprises 316 residues, forming a bilobal structure in which the N-terminal region is predominantly β-pleated sheet and the C-terminal domain is predominantly α-helical (Figure 111.3). The catalytically essential zinc atom is located in a deep active-site cleft that lies between the two lobes. A central helical segment

contains the His-Glu-Leu-Thr-His (HELTH) zinc-binding motif (residues 142
146) that is common to this group of enzymes and in which the two histidines are zinc ligands. A Glu166 residue C-terminal to the motif and a water molecule provide the third and fourth ligands for the zinc atom, which is thus bound in an approximate tetrahedral geometry (Figure 111.4). Activity can be restored to the zinc-free enzyme by stoichiometric addition of Zn21 (100%), Co21 (200%) and Mn21 (10%), or a high molar excess of Fe21 (60%) [51], and the structural changes occurring on zinc replacement have been examined by X-ray crystallography [52]. Thermolysin also binds four calcium atoms, Ca(1) and Ca(2) at a double binding site near the active-site cleft and Ca(3) and Ca(4) at exposed loops in the N- and C-terminal lobes respectively. The calcium ions have no catalytic role but contribute to the enzyme’s stability by protecting it from autolysis [42,101] (see below); they can also be replaced by a variety of other ions [52,102]. Stark et al. [103] published the crystal structure of the TLP from B. cereus, showing that the structure of this enzyme was highly similar to that of thermolysin (which was to be expected considering the 73% sequence identity between the two enzymes). The two available TLP crystal structures have been used as templates for building structural models of other TLPs such as those from B. stearothermophilus CU21 (86% sequence identity with thermolysin) and from B. subtilis (48% sequence identity with thermolysin) [104,105]. Considering their sequence similarities, all TLPs discussed here are expected to have highly similar three-dimensional structures. One major difference seems to be the number of bound calcium ions since sequence comparisons indicate that the less stable TLPs lack binding sites Ca3 and Ca4 (see below).

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Mechanism of Action The crystallographic studies on thermolysin, coupled with parallel studies on the zinc endopeptidase carboxypeptidase A (Chapter 289), have led to a general base mechanism of action being proposed for the zinc peptidase family, with the glutamate of the HELTH sequence polarizing the zinc-bound water molecule which subsequently attacks the scissile bond. Another (protonated) histidine residue (His231 in thermolysin) is proposed to contribute by stabilizing the transition state. This is more fully discussed by Hangauer et al. [106], Matthews [2] and Christianson & Lipscomb [107]. An alternative mechanism has recently been proposed in which an active-site histidine acts as the general base instead of the glutamate residue [48,108]. Several mutagenesis studies have been performed to confirm the role of the various residues in the active site. Beaumont et al. [109] showed that mutation of His231 in thermolysin resulted in a drastic decrease in activity and a greatly reduced pH dependence of activity in the alkaline range. Kubo et al. [110] replaced the Glu143 in NprM from B. stearothermophilus (this enzyme is identical to thermolysin at the amino acid level) by Gln and Asp, which resulted in inactive enzyme variants. Toma et al. [111] confirmed the importance of Glu143 and His231 (thermolysin numbering) in the B. subtilis enzyme by showing that mutation of these residues resulted in drastic reduction of activity. Pelmenschikov et al. [112] conducted a theoretical quantum chemical study of peptide hydrolysis by thermolysin. The results from the latter study appear to favor the peptide hydrolysis mechanism initially proposed by Matthews and co-workers. Comparison of the crystal structures of thermolysinligand complexes with the structure of the free enzyme [52,99,100] strongly indicates that thermolysin undergoes a conformational change upon substrate or inhibitor binding (the active site becomes more closed). Several workers have proposed that conserved glycine residues at positions 78, 135, and 136 to be involved in the hingebending motion that leads to closure of the active-site cleft upon ligand-binding [52,100,103,113]. Indeed, Veltman et al. [114] found that mutation of these glycines in a TLP from B. stearothermophilus (86% overall sequence identity with thermolysin) impaired catalytic activity. On the other hand, the recent comparison of the ‘open’ and ‘closed’ structures of thermolysin does neither preclude nor confirm the importance of the 78 and 135
136 regions in hinge bending, as these regions can not completely account for the 5 rotation difference between the open and closed forms. Apparently, another region is involved as well [99]. The existence of open and closed forms of thermolysin has been investigated by hydrogen/deuterium exchange and electrospray ionization

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mass spectrometry, which shows that elements in the periphery of the two lobes are most mobile, and that the active-site residues and zinc ion are most rigid [115].

Site-Directed Mutagenesis Studies Probing Activity and Specificity Hangauer et al. [106] identified four major substrate binding pockets in thermolysin, of which the hydrophobic S10 subsite is considered the major determinant of substrate specificity. Crystallographic studies of thermolysin have indicated that the S10 subsite allows efficient binding of hydrophobic residues, in particular Leu [2,106,116,117]. Modeling and experimental studies with the TLP from B. stearothermophilus CU21 (TLP-ste) suggested that its S10 subsite was more suited for Phe [3,41,81]. This was attributed to the substitution of Leu133 (thermolysin) by Phe133 (TLP-ste). Indeed, mutational substitution of Phe133 by Leu in TLP-ste resulted in a more thermolysinlike substrate specificity [3]. The S10 subsite of TLP-ste was further adapted by mutation of Leu202. A correlation was observed between increasing the size of the pocket by replacing Leu202 by the smaller Val, Ala and Gly residues and increasing the activity towards substrates with a Phe at the P10 position [41]. Furthermore, it was shown that thermolysin and TLP-ste in which the S10 pockets were made identical with respect to the amino acids composing the pockets had virtually similar substrate specificities towards the di- and tripeptide substrates tested [3,41]. The contribution of several residues in and near the active site to activity and specificity have been probed by site-directed mutagenesis of thermolysin and TLP-ste (86% overall sequence identity). Some results from studies that have resulted in obtaining more active variants are listed in Table 111.1 (residue numbering of thermolysin). Several of the mutations listed in Table 111.1 have been combined and in several cases the mutational effects were additive [22,118]. Kidokoro [118] showed that the combination of three mutations (Leu144Ser-Asp150TrpAsn227His) resulted in a thermolysin variant that was almost 10 times more active. De Kreij et al. [22] combined 4 amino acid substitutions (Asn116Asp-Gln119ArgAsp150Asn-Gln225Arg) in TLP-ste, thus increasing activity by a factor four.

Environmental Effects on Activity Inouye et al. [24,119] observed that thermolysin was significantly activated in the presence of neutral salts. In the presence of 4 M NaCl activity was enhanced 13
15 times at pH 7 and 25 C. The activity was even further increased in the presence of 4 M NaCl when the active-site zinc was replaced by cobalt [120]. The effects of NaCl

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TABLE 111.1 Mutations in thermolysin and related proteases that lead to increased activity Residue Replacement Proposed mechanism of activation

Reference

Tyr110

Trp

exposed residue near active site

[110]

Phe114

Ala

S2 subsite

[110]

Trp115

Tyr/Phe

S2 subsite

[27]

Asn116

Asp

charge distribution active site

[22]

Gln119

Arg/Glu/Gln/ Asp/His/

active site flexibility

[144]

Met/Ser/Gly/ Ala/Arg

charge distribution active site

[22]

Phe133

Leu

S10 subsite

[3]

Leu144

Ser

active site dynamics

[118]

Asp150

Asn/His/Trp

active site electrostatics

[118]

0

Leu202

Phe/Tyr/Gly/ Ala/Val

S1 subsite

[41]

Tyr211

Trp

exposed residue near active site

[110]

Gln225

Arg

active site electrostatics

[22]

Asn227

His

active site electrostatics

[118]

addition and the cobalt substitution were shown to be independent. Whereas the activity of the cobalt-substituted thermolysin was pH-dependent, the relevant pKa values remain constant in the presence of increasing NaCl concentrations. From these results it was concluded that the electrostatic environment of the cobalt-substituted thermolysin is more stable than that of the native enzyme. Salt activation was also demonstrated by lyophilizing thermolysin in the presence of KCl [121]. The lyophilized preparations were analyzed for the condensation of Nfurylacryloylglycine and L-Leu-NH2 in tert-amyl alcohol. The thermolysin samples containing 98% (w/w) KCl were almost 1700 times more reactive than the 0% KCl lyophilized preparations. It was suggested that the mechanism involves the stabilization of a charge separation in the enzyme transition state. Modulation of thermolysin activity by polyhydroxylic additives like polyols and simple sugars has been demonstrated [122,123]. By the addition of several small sugars, e.g. sucrose, glucose and malitol, up to 4-fold enhancement of activity could be obtained. These effects on catalytic activity were supposedly obtained mainly through changes of the activation free energy.

Thermolysin activity can also be enhanced by increasing pressure. However, above 2
3 bar the enzyme is inactivated by the dissociation of the zinc ion [124]. Some mutants, in which the Gln119 was replaced, showed altered pressure-activity dependencies [125].

Preparation Commercial preparations of thermolysin can be repurified by crystallization [126] or by affinity chromatography using ligands such as D-Phe-Gly coupled to cyanogenbromide-activated Sepharose [127], which has also been used to purify the enzyme directly from culture supernatants [47,109]. Bacitracin coupled to silica has also been used to purify large quantities of several Bacillus TLPs from culture supernatants [128,129]. Bacillus enzymes are generally expressed in strains of B. subtilis from which other proteases have been deleted. In B. subtilis DB104 an alkaline protease gene has been deleted and the TLP (‘neutral protease’) gene is functionally inactivated by two point mutations [130]. The most frequently used, B. subtilis DB117, is a derivative of DB104, where both protease genes have been deleted [131]. Recombinant thermolysin has been produced in Escherichia coli by co-expressing the mature sequence and the propeptide sequence as independent polypeptides [132].

Biological Aspects Maximal synthesis of the Bacillus enzymes occurs in the late exponential and early stationary phase, before sporulation, when nutrients become limiting [133]. The extracellular proteases therefore appear to have a scavenging nutritional role for their bacteria. A TLP-specific transcriptional activator gene, prtA, has been identified in B. stearothermophilus strain TELNE, upstream from the protease gene, which enhances protease synthesis 5-fold [134]. PrtA is produced in the late stage of logarithmic growth and may bind to the upstream region of the protease promoter. TLPs are synthesized as preproproteins [135
139] and the pro sequences, of around 200 residues, are removed autocatalytically [6,109,110,140]. It has been shown that the pro sequence of thermolysin acts as an intramolecular chaperone (facilitating folding) and acts as a noncompetitive inhibitor of activity [138,141]. It is important to note the analogy between TLPs and other proteases that are produced as preproproteins, such as α-lytic protease (Chapter 567). For the latter, the role of the pro protein has been described in great detail (e.g. Jaswal et al. [142]).

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Distinguishing Features Thermal Stability and Activity at High Temperatures Different members of the TLP family show large variation in thermal stability, which has made the TLP family an attractive target for the study of structural features of enzyme stability. Thermolysin was originally identified as a thermostable enzyme [1]. When the structure of thermolysin was published, the presence of four bound calcium ions was the only notable feature that could be linked to the high stability of the enzyme. This idea was subsequently strengthened by the observation that TLPs with lower thermal stability tend to lack the residues that make up calcium sites 3 and 4. Thermal inactivation of TLPs is caused by local unfolding followed by rapid autolysis, unfolding being the rate-limiting step [104,143
145]. It has been shown that one or two calcium atoms, presumably Ca3 and/or Ca4, are released from thermolysin during thermal denaturation, depending on the conditions used [42,101,143]. Mutagenesis studies have confirmed that the ‘extra’ calcium ions Ca3 and Ca4 in thermolysin and other stable TLPs do contribute to stability. However, these studies have also made clear that the contribution of calcium binding in sites 3 and 4 to stability differences between TLPs is not particularly big and certainly not predominant over the contributions of amino acid substitutions that do not relate to calcium binding [146
149]. Four autodegradation sites in thermolysin have been identified, and substitution of one of these increases thermostability [150]. The structural causes of (differences in) thermal stability have been studied in much detail, mainly using the TLPs from B. subtilis (reviewed by Vriend & Eijsink [104]; see also Frigerio et al. [105,151]) and B. stearothermophilus (TLP-ste; see Imanaka et al. [152] and Nakamura et al. [153] and below). It was shown that only a very limited number of the 44 sequence differences between TLP-ste and thermolysin account for the differences in stability [137,154]; the temperatures at which the wild-type lose 50% of their activity in 30 min, T50, are 73.4 C and 86.9 C, respectively). It was also shown that stability-determining mutations cluster in a surfacelocated part of the N-terminal domain (residues 4
69) and that combining five of the natural mutations in this region yielded a TLP-ste variant that was more stable than thermolysin itself (T50 5 93.3 C [154]). The clustering of stability-determining mutations in one region indicates that this region is essential in the local unfolding processes that determine the thermal inactivation process [155]. One of the ‘extra’ calcium ions (Ca3) in the more stable TLPs (such as thermolysin and

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TLP-ste) binds in this region and was shown to contribute to stability and to determine the calcium-dependency of stability [148,149]. The importance of this region was further illustrated by the fact that TLP-ste could be effectively stabilized by designed mutations in this region (e.g. Veltman et al. [147] and Mansfeld et al. [156]). Immobilization of TLP-ste through engineered cysteine residues only had stabilizing effects if the cysteines were introduced in the stability-determining region [157,158]. By combining stabilizing mutations identified in comparative studies with designed mutations, van den Burg et al. [159] were able to create a hyperstable variant of TLP-ste with a half-life of 170 minutes at 100 C. This 8-fold mutant retained full activity at 37 C, but was also capable of degrading proteinaceous substrates at 100 C (see van den Burg et al. [159
161] for more details). Other studies that have generated a thermolysin with greater activity and thermal stability include Menach et al. [162] and Kusano et al. [163,164].

Application of Thermolysin-Like Proteases as Synthetases The industrial scale application of thermolysin for the synthesis of the artificial sweetener aspartame is welldocumented (e.g. [165
167]). The process to synthesize aspartame is based on the fact that the unreacted enantiomer D-Phe-OMe forms a salt with the product that precipitates from aqueous solution, and therefore enables 95% conversion yields [167]. An increasing number of reports describe the application of thermolysin-like proteases for the condensation of peptides, including the incorporation of non-natural amino acids [168
174]. Although some of these examples have been performed in aqueous media the application of organic media, including tert-amyl alcohol [121,175] and DMSO [176] have been shown to be applicable and efficient as well. The study of Pedersen et al. [176] demonstrates the application of thermolysin for the transesterification of sucrose and vinyl laurate in dimethylsulfoxide, resulting in the formation of 2-O-lauroyl-sucrose. Solid-to-solid peptide synthesis by thermolysin has been described [12,14,177
179]. Different peptide synthesis procedures have been compared by Ulijn et al. [180]. Immobilization of thermolysin-like proteases is an attractive method for improving performance in (organic) synthesis [180]. Thermolysin has been successfully used in immobilized form, either in the form of CLECs (crosslinked enzyme crystals; e.g. [181,182] or immobilized to carriers such as Sepharose, celite, silica, Amberlite XAD-7, or polypropylene [157,179]. Immobilization often

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leads to higher enzyme stability [157,158,182
184], whereas the enzyme can be recovered and reused rather easily.

Further Reading Beaumont & Beynon [185] give a thorough overview of this family of proteases. The broader topic of bacterial metalloproteases is covered by Ha¨se & Finkelstein [186]. Details of the X-ray structures may be found in Holmes & Matthews [98] and Matthews [2]. The stability of TLPs is discussed in detail in van den Burg et al. [159] and in Eijsink et al. [145].

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Bertus van den Burg Bioengineering BV, PO Box 14, 9750 AA Haren, The Netherlands. Email: [email protected]

Vincent Eijsink Agricultural University of Norway, Department of Chemistry and Biotechnology, PO Box 5040, N-1432 A˚s, Norway. Email: [email protected] This article is reproduced from the previous edition, volume 1, pp. 374
387, r 2004, Elsevier Ltd., with revisions made by the Editors. Handbook of Proteolytic Enzymes, 3rd Edn ISBN: 978-0-12-382219-2

© 2013 Elsevier Ltd. All rights reserved. DOI: http://dx.doi.org/10.1016/B978-0-12-382219-2.00111-3