The catalytic domain of human neuropathy target esterase mediates an organophosphate-sensitive ionic conductance across liposome membranes

In humans and other vertebrates, reaction of organophosphates with a neuronal membrane protein, neuropathy target esterase (NTE), initiates events which culminate in axonal degeneration. The initiation process appears to involve modification of a property of the protein distinct from its esterase activity, subsequent to formation of a negatively charged adduct with the active site serine residue. Here, we show that membrane patches from liposomes containing NEST, a recombinant hydrophobic polypeptide comprising the esterase domain of human NTE, display a transmembrane ionic conductance with both stable and high-frequency flickering components. An asymmetric current-voltage relationship suggested that ion flow was favoured in one direction relative to the membrane and its associated NEST molecules. Flow of anions was slightly favoured compared with cations. The flickering current formed a much larger proportion of the overall conductance in patches containing wild-type NEST compared with the catalytically inactive S966A mutant form of the protein. The conductance across patches containing NEST, but not those with the S966A mutant, was significantly reduced after adding neuropathic organophosphates to the bathing medium. By contrast, non-neuropathic covalent inhibitors of the catalytic activity of NEST did not reduce NEST-mediated conductance. Future work may establish whether NTE itself mediates an organophosphate-sensitive ion flux across intracellular membranes within intact cells.     

Unusual catalytic triad of Escherichia coli outer membrane phospholipase A

Escherichia coli outer membrane phospholipase A (OMPLA) is an integral membrane enzyme. OMPLA is active as a homodimer and requires calcium as a cofactor. The crystal structures of the monomeric and the inhibited dimeric enzymes were recently determined [Snijder, H. J., et al. (1999) Nature 401, 717-721] and revealed that OMPLA monomers are folded into a 12-stranded antiparallel beta-barrel. The active site consists of previously identified essential residues Ser144 and His142 in an arrangement resembling the corresponding residues of a serine hydrolase catalytic triad. However, instead of an Asp or Glu that normally is present in the triad of serine hydrolases, a neutral asparagine (Asn156) was found in OMPLA. In this paper, the importance of the catalytic Asn156 is addressed by site-directed mutagenesis studies. All variants were purified at a 30 mg scale, and were shown to be properly folded using SDS-PAGE and circular dichroism spectroscopy. Using chemical cross-linking, it was shown that all variants were not affected in their calcium-dependent dimerization properties. The Asn156Asp variant exhibited a 2-fold lower activity than wild-type OMPLA at neutral pH. Interestingly, the activity of the variant is 1 order of magnitude higher than that of the wild type at pH >10. Modest residual activities (5 and 2.5%, respectively) were obtained for the Asn156Ala and Asn156Gln mutants, showing that the active site of OMPLA is more tolerant toward replacements of this third residue of the catalytic triad than other serine hydrolases, and that the serine and histidine residues are minimally required for catalysis. In the X-ray structure of dimeric OMPLA, the cofactor calcium is coordinating the putative oxyanion via two water molecules. We propose that this may lessen the importance for the asparagine in the catalytic triad of OMPLA.     

Identification of essential acidic residues of outer membrane protease OmpT supports a novel active site

Escherichia coli outer membrane protease OmpT has previously been classified as a serine protease with Ser(99) and His(212) as active site residues. The recently solved X-ray structure of the enzyme was inconsistent with this classification, and the involvement of a nucleophilic water molecule was proposed. Here, we substituted all conserved aspartate and glutamate residues by alanines and measured the residual enzymatic activities of the variants. Our results support the involvement of a nucleophilic water molecule that is activated by the Asp(210)/His(212) catalytic dyad. Activity is also strongly dependent on Asp(83) and Asp(85). Both may function in binding of the water molecule and/or oxyanion stabilization. The proposed mechanism implies a novel proteolytic catalytic site.     

Chlorophyllase as a serine hydrolase: identification of a putative catalytic triad

Chlorophyllases (Chlases), cloned so far, contain a lipase motif with the active serine residue of the catalytic triad of triglyceride lipases. Inhibitors specific for the catalytic serine residue in serine hydrolases, which include lipases effectively inhibited the activity of the recombinant Chenopodium album Chlase (CaCLH). From this evidence we assumed that the catalytic mechanism of hydrolysis by Chlase might be similar to those of serine hydrolases that have a catalytic triad composed of serine, histidine and aspartic acid in their active site. Thus, we introduced mutations into the putative catalytic residue (Ser162) and conserved amino acid residues (histidine, aspartic acid and cysteine) to generate recombinant CaCLH mutants. The three amino acid residues (Ser162, Asp191 and His262) essential for Chlase activity were identified. These results indicate that Chlase is a serine hydrolase and, by analogy with a plausible catalytic mechanism of serine hydrolases, we proposed a mechanism for hydrolysis catalyzed by Chlase.     

Molecular basis of formaldehyde detoxification. Characterization of two S-formylglutathione hydrolases from Escherichia coli, FrmB and YeiG

The Escherichia coli genes frmB (yaiM) and yeiG encode two uncharacterized proteins that share 54% sequence identity and contain a serine esterase motif. We demonstrated that purified FrmB and YeiG have high carboxylesterase activity against the model substrates, p-nitrophenyl esters of fatty acids (C2-C6) and alpha-naphthyl acetate. However, both proteins had the highest hydrolytic activity toward S-formylglutathione, an intermediate of the glutathione-dependent pathway of formaldehyde detoxification. With this substrate, both proteins had similar affinity (Km = 0.41-0.43 mM), but FrmB was almost 5 times more active. Alanine replacement mutagenesis of YeiG demonstrated that Ser145, Asp233, and His256 are absolutely required for activity, indicating that these residues represent a serine hydrolase catalytic triad in this protein and in other S-formylglutathione hydrolases. This was confirmed by inspecting the crystal structure of the Saccharomyces cerevisiae S-formylglutathione hydrolase YJG8 (Protein Data Bank code 1pv1), which has 45% sequence identity to YeiG. The structure revealed a canonical alpha/beta-hydrolase fold and a classical serine hydrolase catalytic triad (Ser161, His276, Asp241). In E. coli cells, the expression of frmB was stimulated 45-75 times by the addition of formaldehyde to the growth medium, whereas YeiG was found to be a constitutive enzyme. The simultaneous deletion of both frmB and yeiG genes was required to increase the sensitivity of the growth of E. coli cells to formaldehyde, suggesting that both FrmB and YeiG contribute to the detoxification of formaldehyde. Thus, FrmB and YeiG are S-formylglutathione hydrolases with a Ser-His-Asp catalytic triad involved in the detoxification of formaldehyde in E. coli.     

Modeling the Tertiary Structure of the Patatin Domain of Neuropathy Target Esterase

Neuropathy target esterase (NTE) is a transmembrane protein of unknown function whose specific chemical modification by certain organophosphorus (OP) compounds leads to distal axonopathy. Therefore, solving the 3D structure of NTE would advance the understanding of its pathogenic and physiologic roles. In this study, the tertiary structures of the patatin (catalytic) domain and the N-terminal transmembrane domain of NTE were modeled using the crystal structures of patatin (PDB ID 1oxw) and moricin (PDB ID 1kv4) as templates. Sequence alignments and secondary structure predictions were obtained from the INUB server (Buffalo, NY). O and PyMol were used to build the PNTE and NTE TMD chains from these sequence alignments. The PNTE model was refined in the presence of water using the crystallography and NMR system, while the NTE TMD model was refined in vacuo using the GROMOS implementation in the Swiss PDB viewer. The modeled active site of NTE was found to consist of a Ser966-Asp1086 catalytic dyad, which is characteristic of phospholipase A2 enzymes. The Ser966 Ogamma was located 2.93 A from the Odelta2 of Asp1086. In addition, our NTE model was found to contain a single N-terminal transmembrane domain. This modeling effort provided structural and mechanistic predictions about the catalytic domain of NTE that are being verified via experimental techniques.     

Influence of lysophospholipid hydrolysis by the catalytic domain of neuropathy target esterase on the fluidity of bilayer lipid membranes

Neuropathy target esterase (NTE) is an integral membrane protein localized in the endoplasmic reticulum in neurons. Irreversible inhibition of NTE by certain organophosphorus compounds produces a paralysis known as organophosphorus compound-induced delayed neuropathy. In vitro, NTE has phospholipase/lysophospholipase activity that hydrolyses exogenously added single-chain lysophospholipids in preference to dual-chain phospholipids, and NTE mutations have been associated with motor neuron disease. NTE's physiological role is not well understood, although recent studies suggest that it may control the cytotoxic accumulation of lysophospholipids in membranes. We used the NTE catalytic domain (NEST) to hydrolyze palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (p-lysoPC) to palmitic acid in bilayer membranes comprising 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and the fluorophore 1-oleoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (NBD-PC). Translational diffusion coefficients (D_L) in supported bilayer membranes were measured by fluorescence recovery after pattern photobleaching (FRAPP). The average D_L for DOPC/p-lysoPC membranes without NEST was 2.44 µm²s^-1 ± 0.09; the D_L for DOPC/p-lysoPC membranes containing NEST and diisopropylphosphorofluoridate, an inhibitor, was nearly identical at 2.45 ± 0.08. By contrast, the D_L for membranes comprising NEST, DOPC, and p-lysoPC was 2.28 ± 0.07, significantly different from the system with inhibited NEST, due to NEST hydrolysis. Likewise, a system without NEST containing the amount of palmitic acid that would have been produced by NEST hydrolysis of p-lysoPC was identical at 2.26 ± 0.06. These results indicate that NTE's catalytic activity can alter membrane fluidity.     

Human neuropathy target esterase catalyzes hydrolysis of membrane lipids

A neuronal membrane protein, neuropathy target esterase (NTE), reacts with those organophosphates that initiate a syndrome of axonal degeneration. NTE has homologues in Drosophila and yeast and is detected in vitro by assays with a non-physiological ester substrate, phenyl valerate. We report that NEST, the recombinant esterase domain of NTE (residues 727-1216) purified from bacterial lysates, can catalyze hydrolysis of several naturally occurring membrane-associated lipids. The active site regions of NEST and calcium-independent phospholipase A(2) (iPLA(2)) share sequence similarity, and the phenyl valerate hydrolase activity of NEST is inhibited by low concentrations of iPLA(2) inhibitors. However, on incubation with NEST, fatty acid was liberated only extremely slowly from the sn-2 position of phospholipids (V(max) approximately 0.01 micromol/min/mg and K(m) approximately 0.4 mm for 1-palmitoyl, 2-oleoylphosphatidylcholine). Comparison of the NEST-mediated generation of (14)C-labeled products from two differentially labeled (14)C-phospholipid substrates suggested that a rate-limiting sn-2 cleavage was followed very rapidly by hydrolysis of the resulting lysophospholipid. Among the various naturally occurring lipids tested with NEST, lysophospholipids were by far the most avidly hydrolyzed substrates (V(max) approximately 20 micromol/min/mg and K(m) approximately 0.05 mm for 1-palmitoyl-lysophosphatidylcholine). NEST also catalyzed the hydrolysis of monoacylglycerols, preferring the 1-acyl to the 2-acyl isomer (V(max) approximately 1 micromol/min/mg and K(m) approximately 0.4 mm for 1-palmitoylglycerol). NEST did not catalyze hydrolysis of di- or triacylglycerols or fatty acid amides. This demonstration that membrane lipids are its putative cellular substrates raises the possibility that NTE and its homologues may be involved in intracellular membrane trafficking.     

Identification and characterisation of the catalytic triad of the alkaliphilic thermotolerant PHA depolymerase PhaZ7 of Paucimonas lemoignei

The recently discovered extracellular poly[(R)-3-hydroxybutyrate] (PHB) depolymerase PhaZ7 of Paucimonas lemoignei represents the first member of a new subgroup (EC 3.1.1.75) of serine hydrolases with no significant amino acid similarities to conventional PHB depolymerases, lipases or other hydrolases except for a potential lipase box-like motif (Ala-His-Ser136-Met-Gly) and potential candidates for catalytic triad and oxyanion pocket amino acids. In order to identify amino acids essential for activity 11 mutants of phaZ7 were generated by site-directed mutagenesis and expressed in recombinant protease-deficient Bacillus subtilis WB800. The wild-type depolymerase and 10 of the 11 mutant proteins (except for Ser136Cys) were expressed and efficiently secreted by B. subtilis as shown by Western blots of cell-free culture fluid proteins. No PHB depolymerase activity was detected in strains harbouring one of the following substitutions: His47Ala, Ser136Ala, Asp242Ala, Asp242Asn, His306Ala, indicating the importance of these amino acids for activity. Replacement of Ser136 by Thr resulted in a decrease of activity to about 20% of the wild-type level and suggested that the hydroxy group of the serine side chain is important for activity but can be partially replaced by the hydroxy function of threonine. Alterations of Asp256 to Ala or Asn or of the putative serine hydrolase pentapeptide motif (Ala-His-Ser136-Met-Gly) to a lipase box consensus sequence (Gly134-His-Ser136-Met-Gly) or to the PHB depolymerase box consensus sequence (Gly134-Leu135-Ser136-Met-Gly) had no significant effect on PHB depolymerase activity, indicating that these amino acids or sequence motifs were not essential for activity. In conclusion, the PHB depolymerase PhaZ7 is a serine hydrolase with a catalytic triad and oxyanion pocket consisting of His47, Ser136, Asp242 and His306.     

The role of OXA-1 beta-lactamase Asp(66) in the stabilization of the active-site carbamate group and in substrate turnover

The OXA-1 beta-lactamase is one of the few class D enzymes that has an aspartate residue at position 66, a position that is proximal to the active-site residue Ser(67). In class A beta-lactamases, such as TEM-1 and SHV-1, residues adjacent to the active-site serine residue play a crucial role in inhibitor resistance and substrate selectivity. To probe the role of Asp(66) in substrate affinity and catalysis, we performed site-saturation mutagenesis at this position. Ampicillin MIC (minimum inhibitory concentration) values for the full set of Asp(66) mutants expressed in Escherichia coli DH10B ranged from < or =8 microg/ml for cysteine, proline and the basic amino acids to > or =256 microg/ml for asparagine, leucine and the wild-type aspartate. Replacement of aspartic acid by asparagine at position 66 also led to a moderate enhancement of extended-spectrum cephalosporin resistance. OXA-1 shares with other class D enzymes a carboxylated residue, Lys(70), that acts as a general base in the catalytic mechanism. The addition of 25 mM bicarbonate to Luria-Bertani-broth agar resulted in a > or =16-fold increase in MICs for most OXA-1 variants with amino acid replacements at position 66 when expressed in E. coli. Because Asp(66) forms hydrogen bonds with several other residues in the OXA-1 active site, we propose that this residue plays a role in stabilizing the CO2 bound to Lys(70) and thereby profoundly affects substrate turnover.     

Cloning and expression of islandisin, a new thermostable subtilisin from Fervidobacterium islandicum, in Escherichia coli

A gene encoding a subtilisin-like protease, designated islandisin, from the extremely thermophilic bacterium Fervidobacterium islandicum (DSMZ 5733) was cloned and actively expressed in Escherichia coli. The gene was identified by PCR using degenerated primers based on conserved regions around two of the three catalytic residues (Asp, His, and Ser) of subtilisin-like serine protease-encoding genes. Using inverse PCR regions flanking the catalytic residues, the gene could be cloned. Sequencing revealed an open reading frame of 2,106 bp. The deduced amino acid sequence indicated that the enzyme is synthesized as a proenzyme with a putative signal sequence of 33 amino acids (aa) in length. The mature protein contains the three catalytic residues (Asp177, His215, and Ser391) and has a length of 668 aa. Amino acid sequence comparison and phylogenetic analysis indicated that this enzyme could be classified as a subtilisin-like serine protease in the subgroup of thermitase. The whole gene was amplified by PCR, ligated into pET-15b, and successfully expressed in E. coli BL21(DE3)pLysS. The recombinant islandisin was purified by heat denaturation, followed by hydroxyapatite chromatography. The enzyme is active at a broad range of temperatures (60 to 80 degrees C) and pHs (pH 6 to 8.5) and shows optimal proteolytic activity at 80 degrees C and pH 8.0. Islandisin is resistant to a number of detergents and solvents and shows high thermostability over a long period of time (up to 32 h) at 80 degrees C with a half-life of 4 h at 90 degrees C and 1.5 h at 100 degrees C.     

Purification and characterization of neuropathy target esterase (NTE) from rat brain

Neuropathy target esterase (NTE) is an integral membrane protein in vertebrate neurons and a member of a novel family of putative serine hydrolases. Neuropathic organophosphates react covalently with the active site serine residue of NTE, causing degeneration of long axons in spinal cord and peripheral nerves which becomes clinically evident 1-3 weeks after exposure to OPs, hence termed as organophosphate induced delayed neuropathy. The present study reports the isolation and characterization of NTE protein from rat brain. Rat brain microsomes were solubilized with phospholipase A2 and they were fractionated by gel filtration chromatography in S-300 column. The sample was eluted in buffer containing polyoxyethylene W1 detergent, which yielded an active fraction of 200 kDa. The most enriched NTE active fraction was further purified by 3-9'-mercaptononylthio-1,1,1-trifluoropropan-2-one bound to sepharose CL4B. The SDS-PAGE confirmed the 155-kDa protein as the most likely candidate for NTE. Database searching of rat N-terminal protein revealed homology with variety of polypeptides from different organisms and suggested that NTE protein has function beyond the nervous system and mediates a biochemical reaction highly conserved through evolution.     

Structural relationship between lipases and peptidases of the prolyl oligopeptidase family.

In prolyl oligopeptidase and its homologues, which constitute a new serine protease family, the order of the catalytic Ser and His residues in the amino acid sequence is the reverse of what is found in the trypsin and subtilisin families. The exact position of the third member of the catalytic triad, an Asp residue, has not yet been identified in the new family. Recent determination of the three-dimensional structures of pancreatic and microbial lipases has shown that the order of their catalytic residues is Ser, Asp, His, and this fits the order Ser, His of prolyl oligopeptidase. However, there is no sequence homology between lipases and peptidases, except for a 10-residue segment, which encompasses the essential Ser, and for the immediate vicinity of the catalytic Asp and His residues. This comparison identifies the catalytic Asp residue in the prolyl oligopeptidase family. The relative positions of the three catalytic residues in peptidases and microbial lipases were the same and this indicated structural and possibly evolutionary relationship between the two families.     

Design of a serine protease-like catalytic triad on an antibody light chain displayed on the yeast cell surface

Lc-WT, the wild-type light chain of antibody, and Lc-Triad, its double mutant with E1D and T27aS designing for the construction of catalytic triad within Asp1, Ser27a, and original His93 residues, were displayed on the cell surface of the protease-deficient yeast strain BJ2168. When each cell suspension was reacted with BODIPY FL casein and seven kinds of peptide-MCA substrates, respectively, a remarkable difference in hydrolytic activities toward Suc-GPLGP-MCA (succinyl-Gly-Pro-Leu-Gly-Pro-MCA), a substrate toward collagenase-like peptidase, was observed between the constructs: Lc-Triad-displaying cells showed higher catalytic activity than Lc-WT-displaying cells. The difference disappeared in the presence of the serine protease inhibitor diisopropylfluorophosphate, suggesting that the three amino acid residues, Ser27a, His93, and Asp1, functioned as a catalytic triad responsible for the proteolytic activity in a similar way to the anti-vasoactive intestinal peptide (VIP) antibody light chain. A serine protease-like catalytic triad (Ser, His, and Asp) is considered to be directly involved in the catalytic mechanism of the anti-VIP antibody light chain, which moderately catalyzes the hydrolysis of VIP. These results suggest the possibility of new approach for the creation of tailor-made proteases beyond limitations of the traditional immunization approach.     

Identification of the catalytic residues of alpha-amino acid ester hydrolase from Acetobacter turbidans by labeling and site-directed mutagenesis

The alpha-amino acid ester hydrolase from Acetobacter turbidans ATCC 9325 is capable of hydrolyzing and synthesizing the side chain peptide bond in beta-lactam antibiotics. Data base searches revealed that the enzyme contains an active site serine consensus sequence Gly-X-Ser-Tyr-X-Gly that is also found in X-prolyl dipeptidyl aminopeptidase. The serine hydrolase inhibitor p-nitrophenyl-p'-guanidino-benzoate appeared to be an active site titrant and was used to label the alpha-amino acid ester hydrolase. Electrospray mass spectrometry and tandem mass spectrometry analysis of peptides from a CNBr digest of the labeled protein showed that Ser(205), situated in the consensus sequence, becomes covalently modified by reaction with the inhibitor. Extended sequence analysis showed alignment of this Ser(205) with the catalytic nucleophile of some alpha/beta-hydrolase fold enzymes, which posses a catalytic triad composed of a nucleophile, an acid, and a base. Based on the alignments, 10 amino acids were selected for site-directed mutagenesis (Arg(85), Asp(86), Tyr(143), Ser(156), Ser(205), Tyr(206), Asp(338), His(370), Asp(509), and His(610)). Mutation of Ser(205), Asp(338,) or His(370) to an alanine almost fully inactivated the enzyme, whereas mutation of the other residues did not seriously affect the enzyme activity. Circular dichroism measurements showed that the inactivation was not caused by drastic changes in the tertiary structure. Therefore, we conclude that the catalytic domain of the alpha-amino acid ester hydrolase has an alpha/beta-hydrolase fold structure with a catalytic triad of Ser(205), Asp(338), and His(370). This distinguishes the alpha-amino acid ester hydrolase from the Ntn-hydrolase family of beta-lactam antibiotic acylases.     

NMR studies of the hydrogen bonds involving the catalytic triad of Escherichia coli thioesterase/protease I

Escherichia coli thioesterase/protease I (TEP-I) is a lipolytic enzyme of the serine protease superfamily with Ser(10), Asp(154) and His(157) as the catalytic triad residues. Based on comparison of the low-field (1)H nuclear magnetic resonance spectra of two mutants (S10G and S12G) and two transition state analogue complexes we have assigned the exchangeable proton resonances at 16.3 ppm, 14.3 ppm, and 12.8 ppm at pH 3.5 to His(157)-N(delta1)H, Ser(10)-O(gamma)H and His(157)-N(epsilon2)H, respectively. Thus, the presence of a strong Asp(154)-His(157) hydrogen bond in free TEP-I was observed. However, Ser(10)-O(gamma)H was shown to form a H-bond with a residue other than His(157)-N(epsilon2).     

Studies of specificity and catalysis in trypsin by structural analysis of site-directed mutants

We are probing the determinants of catalytic function and substrate specificity in serine proteases by kinetic and crystallographic characterization of genetically engineered site-directed mutants of rat trypsin. The role of the aspartyl residue at position 102, common to all members of the serine protease family, has been tested by substitution with asparagine. In the native enzyme, Asp102 accepts a hydrogen bond from the catalytic base His57, which facilitates the transfer of a proton from the enzyme nucleophile Ser195 to the substrate leaving group. At neutral pH, the mutant is four orders of magnitude less active than the naturally occurring enzyme, but its binding affinity for model substrates is virtually undiminished. Crystallographic analysis reveals that Asn102 donates a hydrogen bond to His57, forcing it to act as donor to Ser195. Below pH 6, His57 becomes statistically disordered. Presumably, the di-protonated population of histidyl side chains are unable to hydrogen bond to Asn102. Steric conflict may cause His57 to rotate away from the catalytic site. These results suggest that Asp102 not only provides inductive and orientation effects, but also stabilizes the productive tautomer of His57. Three experiments were carried out to alter the substrate specificity of trypsin. Glycine residues at positions 216 and 226 in the substrate-binding cavity were replaced by alanine residues in order to differentially affect lysine and arginine substrate binding. While the rate of catalysis by the mutant enzymes was reduced in the mutant enzymes, their substrate specificity was enhanced relative to trypsin. The increased specificity was caused by differential effects on the catalytic activity towards arginine and lysine substrates. The Gly----Ala substitution at 226 resulted in an altered conformation of the enzyme which is converted to an active trypsin-like conformation upon binding of a substrate analog. In a third experiment, Lys189, at the bottom of the specificity pocket, was replaced with an aspartate with the expectation that specificity of the enzyme might shift to aspartate. The mutant enzyme is not capable of cleaving at Arg and Lys or Asp, but shows an enhanced chymotrypsin-like specificity. Structural investigations of these mutants are in progress.     

Identification of the amino acid residues essential for proteolytic activity in an archaeal signal peptide peptidase

Signal peptide peptidases (SPPs) are enzymes involved in the initial degradation of signal peptides after they are released from the precursor proteins by signal peptidases. In contrast to the eukaryotic enzymes that are aspartate peptidases, the catalytic mechanisms of prokaryotic SPPs had not been known. In this study on the SPP from the hyperthermophilic archaeon Thermococcus kodakaraensis (SppA(Tk)), we have identified amino acid residues that are essential for the peptidase activity of the enzyme. DeltaN54SppA(Tk), a truncated protein without the N-terminal 54 residues and putative transmembrane domain, exhibits high peptidase activity, and was used as the wild-type protein. Sixteen residues, highly conserved among archaeal SPP homologue sequences, were selected and replaced by alanine residues. The mutations S162A and K214A were found to abolish peptidase activity of the protein, whereas all other mutant proteins displayed activity to various extents. The results indicated the function of Ser(162) as the nucleophilic serine and that of Lys(214) as the general base, comprising a Ser/Lys catalytic dyad in SppA(Tk). Kinetic analyses indicated that Ser(184), His(191) Lys(209), Asp(215), and Arg(221) supported peptidase activity. Intriguingly, a large number of mutations led to an increase in activity levels of the enzyme. In particular, mutations in Ser(128) and Tyr(165) not only increased activity levels but also broadened the substrate specificity of SppA(Tk), suggesting that these residues may be present to prevent the enzyme from cleaving unintended peptide/protein substrates in the cell. A detailed alignment of prokaryotic SPP sequences strongly suggested that the majority of archaeal enzymes, along with the bacterial enzyme from Bacillus subtilis, adopt the same catalytic mechanism for peptide hydrolysis.     

Multiple conformations of catalytic serine and histidine in acetylxylan esterase at 0.90 A

Acetylxylan esterase (AXEII; 207 amino acids) from Penicillium purpurogenum has substrate specificities toward acetate esters of d-xylopyranose residues in xylan and belongs to a new class of alpha/beta hydrolases. The crystal structure of AXEII has been determined by single isomorphous replacement and anomalous scattering, and refined at 0.90- and 1.10-A resolutions with data collected at 85 K and 295 K, respectively. The tertiary structure consists of a doubly wound alpha/beta sandwich, having a central six-stranded parallel beta-sheet flanked by two parallel alpha-helices on each side. The catalytic residues Ser(90), His(187), and Asp(175) are located at the C-terminal end of the sheet, an exposed region of the molecule. The serine and histidine side chains in the 295 K structure show the frequently observed conformations in which Ser(90) is trans and the hydroxyl group is in the plane of the imidazole ring of His(187). However, the structure at 85 K displays an additional conformation in which Ser(90) side-chain hydroxyl is away from the plane of the imidazole ring of His(187). The His(187) side chain forms a hydrogen bond with a sulfate ion and adopts an altered conformation. The only other known hydrolase that has a similar tertiary structure is Fusarium solani cutinase. The exposed nature of the catalytic triad suggests that AXEII is a pure esterase, i.e. an alpha/beta hydrolase with specificity for nonlipidic polar substrates.     

Identification and analysis of a new family of bacterial serine proteinases

A family of hypothetical proteins, identified predominantly from archaeal genomes, has been analyzed in order to understand its functional characteristics. Using extensive sequence similarity searches it is inferred that this family is remotely related (best sequence identity is 19%) to ClpP proteinases that belongs to serine proteinase class. This family of hypothetical proteins is referred to as SDH proteinase family based on conserved sequential order of Ser, Asp and His residues and predicted serine proteinase activity. Results of fold recognition of SDH family sequences confirmed the remote relationship between SDH proteinases and Clp proteinases and revealed similar tertiary location of putative catalytic triad residues critical for serine proteinase function. However, the best sequence alignment we could obtain suggests that while catalytic Ser is conserved across Clp and SDH proteinases the location of the other catalytic triad residues, namely, His and Asp are swapped in their amino acid alignment positions and hence in 3-D structure. The evidence of conserved catalytic triad suggests that SDH could be a new family of serine proteinases with the fold of Clp proteinase, however sharing the catalytic triad order of carboxypeptidase clan. Signal peptide sequence identified at the N-terminus of some of the homologues suggests that these might be secretory serine proteinases involved in cleavage of extracellular proteins while the remote homologues, ClpP proteinases, are known to work in intracellular environment.