Engineering subtilisin BPN' for site-specific proteolysis

A combination of protein engineering and substrate optimization was used to create variants of the serine protease, subtilisin BPN', which efficiently and specifically cleave a designed target sequence in a fusion protein. The broad substrate specificity of wild-type subtilisin BPN' is greatly restricted by substitution of the catalytic histidine-containing of the catalytic histidine 64 with alanine (H64A) so that certain histidine-containing substrates are preferentially hydrolysed (Carter, P., Wells, J.A. Science 237:394-399, 1987). The catalytic efficiency, (kcat/Km), of this H64A variant was increased almost 20-fold by judicious choice of substrate and by installing three additional mutations which increase the activity of wild-type subtilisin. The most favorable substrate sequence identified was introduced as a linker in a fusion protein between a synthetic IgG binding domain of Staphylococcus aureus protein A and Escherichia coli alkaline phosphatase. The fusion protein (affinity purified on an IgG column) was cleaved by the prototype H64A enzyme and its improved variant, efficiently and exclusively at the target site, to liberate an alkaline phosphatase product of the expected size and N-terminal sequence. Several features of H64A variants of subtilisin make them attractive for site-specific proteolysis of fusion proteins: they have exquisite substrate specificity on the N-terminal side of the cleavage site and yet are broadly specific on the C-terminal side; they can be produced in large quantities and remain highly active even in the presence of detergents, reductants (modest concentrations), protease inhibitors, at high temperatures, or when specifically immobilized on a solid support.     

Three-dimensional Structure of a Kunitz-type Inhibitor in Complex with an Elastase-like Enzyme

Elastase-like enzymes are involved in important diseases such as acute pancreatitis, chronic inflammatory lung diseases, and cancer. Structural insights into their interaction with specific inhibitors will contribute to the development of novel anti-elastase compounds that resist rapid oxidation and proteolysis. Proteinaceous Kunitz-type inhibitors homologous to the bovine pancreatic trypsin inhibitor (BPTI) provide a suitable scaffold, but the structural aspects of their interaction with elastase-like enzymes have not been elucidated. Here, we increased the selectivity of ShPI-1, a versatile serine protease inhibitor from the sea anemone Stichodactyla helianthus with high biomedical and biotechnological potential, toward elastase-like enzymes by substitution of the P1 residue (Lys¹³) with leucine. The variant (rShPI-1/K13L) exhibits a novel anti-porcine pancreatic elastase (PPE) activity together with a significantly improved inhibition of human neuthrophil elastase and chymotrypsin. The crystal structure of the PPE·rShPI-1/K13L complex determined at 2.0 Å resolution provided the first details of the canonical interaction between a BPTI-Kunitz-type domain and elastase-like enzymes. In addition to the essential impact of the variant P1 residue for complex stability, the interface is improved by increased contributions of the primary and secondary binding loop as compared with similar trypsin and chymotrypsin complexes. A comparison of the interaction network with elastase complexes of canonical inhibitors from the chelonian in family supports a key role of the P3 site in ShPI-1 in directing its selectivity against pancreatic and neutrophil elastases. Our results provide the structural basis for site-specific mutagenesis to further improve the binding affinity and/or direct the selectivity of BPTI-Kunitz-type inhibitors toward elastase-like enzymes.     

Comparative site-directed mutagenesis in the catalytic amino acid triad in calicivirus proteases

Calicivirus proteases cleave the viral precursor polyprotein encoded by open reading frame 1 (ORF1) into multiple intermediate and mature proteins. These proteases have conserved histidine (His), glutamic acid (Glu) or aspartic acid (Asp), and cysteine (Cys) residues that are thought to act as a catalytic triad (i.e. general base, acid and nucleophile, respectively). However, is the triad critical for processing the polyprotein? In the present study, we examined these amino acids in viruses representing the four major genera of Caliciviridae: Norwalk virus (NoV), Rabbit hemorrhagic disease virus (RHDV), Sapporo virus (SaV) and Feline calicivirus (FCV). Using single amino‐acid substitutions, we found that an acidic amino acid (Glu or Asp), as well as the His and Cys in the putative catalytic triad, cannot be replaced by Ala for normal processing activity of the ORF1 polyprotein in vitro. Similarly, normal activity is not retained if the nucleophile Cys is replaced with Ser. These results showed the calicivirus protease is a Cys protease and the catalytic triad formation is important for protease activity. Our study is the first to directly compare the proteases of the four representative calicivirus genera. Interestingly, we found that RHDV and SaV proteases critically need the acidic residues during catalysis, whereas proteolytic cleavage occurs normally at several cleavage sites in the ORF1 polyprotein without a functional acid residue in the NoV and FCV proteases. Thus, the substrate recognition mechanism may be different between the SaV and RHDV proteases and the NoV and FCV proteases.     

Identification of amino acid residues responsible for the alpha5 subunit binding selectivity of L-655,708, a benzodiazepine binding site ligand at the GABA(A) receptor

L-655,708 is a ligand for the benzodiazepine site of the gamma-aminobutyric acid type A (GABA(A)) receptor that exhibits a 100-fold higher affinity for alpha5-containing receptors compared with alpha1-containing receptors. Molecular biology approaches have been used to determine which residues in the alpha5 subunit are responsible for this selectivity. Two amino acids have been identified, alpha5Thr208 and alpha5Ile215, each of which individually confer approximately 10-fold binding selectivity for the ligand and which together account for the 100-fold higher affinity of this ligand at alpha5-containing receptors. L-655,708 is a partial inverse agonist at the GABA(A) receptor which exhibited no functional selectivity between alpha1- and alpha5-containing receptors and showed no change in efficacy at receptors containing alpha1 subunits where amino acids at both of the sites had been altered to their alpha5 counterparts (alpha1Ser205-Thr,Val212-Ile). In addition to determining the binding selectivity of L-655,708, these amino acid residues also influence the binding affinities of a number of other benzodiazepine (BZ) site ligands. They are thus important elements of the BZ site of the GABA(A) receptor, and further delineate a region just N-terminal to the first transmembrane domain of the receptor alpha subunit that contributes to this binding site.     

The crystal structure of three site-directed mutants of Escherichia coli dihydrodipicolinate synthase: further evidence for a catalytic triad

Dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52) catalyses the branchpoint reaction of lysine biosynthesis in plants and microbes: the condensation of (S)-aspartate-beta-semialdehyde and pyruvate. The crystal structure of wild-type DHDPS has been published to 2.5A, revealing a tetrameric molecule comprised of four identical (beta/alpha)(8)-barrels, each containing one active site. Previous workers have hypothesised that the catalytic mechanism of the enzyme involves a catalytic triad of amino acid residues, Tyr133, Thr44 and Tyr107, which provide a proton shuttle to transport protons from the active site to solvent. We have tested this hypothesis using site-directed mutagenesis to produce three mutant enzymes: DHDPS-Y133F, DHDPS-T44V and DHDPS-Y107F. Each of these mutants has substantially reduced activity, consistent with the catalytic triad hypothesis. We have determined each mutant crystal structure to at least 2.35A resolution and compared the structures to the wild-type enzyme. All mutant enzymes crystallised in the same space group as the wild-type form and only minor differences in structure are observed. These results suggest that the catalytic triad is indeed in operation in wild-type DHDPS.     

Thiol protease-like active site found in the enzyme dienelactone hydrolase: localization using biochemical, genetic, and structural tools

The active site of dienelactone hydrolase (DLH), a microbial enzyme of the beta-ketoadipate pathway, has been conclusively located using a combination of crystallographic, biochemical, and genetic techniques. DLH hydrolyzes a dienelactone to maleylacetate and has esterase activity on p-nitrophenyl acetate and trans-cinnamoyl imidazole. The identification of Cys-123 as containing the essential thiol confirms the localization of the active site as suggested by the crystal structure of DLH, and disproves an earlier hypothesis regarding its location. Two mutant proteins have been engineered in which Cys-123 has been converted to a serine (C123S DLH) and an alanine (C123A DLH), respectively. C123S DLH (Km = 9900 +/- 2300 microM; Vmax = 4.4 +/- 0.8 mumol/min-mg) displays burst kinetics with p-nitrophenyl acetate and is 10% as active as DLH (Km = 170 +/- 7 microM; Vmax = 21.1 +/- 0.4 mumol/min-mg). C123A DLH is inactive. The structures of DLH, C123S DLH, and C123A DLH have been refined at 1.8, 2.2, and 2.0 A, respectively. Comparison of the structures of these proteins demonstrates that the only differences between them are centered at residue 123. The structures of the active sites of DLH, papain, and subtilisin are similar and are suggestive of the three enzymes having evolved convergently to similar active sites with similar enzymic mechanisms.     

MD simulation of subtilisin BPN' in a crystal environment.

In this paper we present a molecular dynamics (MD) simulation of subtilisin BPN' in a crystalline environment containing four protein molecules and solvent. Conformational and dynamic properties of the molecules are compared with each other and with respect to the X-ray structure to test the validity of the force field. The agreement between simulated and experimental structure using the GROMOS force field is better than that obtained in the literature using other force fields for protein crystals. The overall shape of the molecule is well preserved, as is the conformation of alpha-helices and beta-strands. Structural differences are mainly found in loop regions. Solvent networks found in the X-ray structure were reproduced by the simulation, which was unbiased with respect to the crystalline hydration structure. These networks seem to play an important role in the stability of the protein; evidence of this is found in the structure of the active site. The weak ion binding site in the X-ray structure of subtilisin BPN' is occupied by a monovalent ion. When a calcium ion is placed in the initial structure, three peptide ligands are replaced by 5 water ligands, whereas a potassium ion retains (in part) its original ligands. Existing force fields yield a reliable method to probe local structure and short-time dynamics of proteins, providing an accuracy of about 0.1 nm.     

The roles of residues Tyr150, Glu272, and His314 in class C β-lactamases

Serine β-lactamases contribute widely to the β-lactam resistance phenomena. Unfortunately, the intimate details of their catalytic mechanism remain elusive and subject to some controversy even though many “natural” and “artificial” mutants of these different enzymes have been isolated. This paper is essentially focused on class C β-lactamases, which contain a Tyr (Tyr150) as the first residue of the second conserved element, in contrast to their class A counterparts, in which a Ser is found in the corresponding position. We have modified this Tyr residue by site-directed mutagenesis. On the basis of the three-dimensional structure of the Enterobacter cloacae P99 enzyme, it seemed that residues Glu272 and His314 might also be important. They were similarly substituted. The modified enzymes were isolated and their catalytic properties determined. Our results indicated that His314 was not required for catalysis and that Glu272 did not play an important role in acylation but was involved to a small extent in the deacylation process. Conversely, Tyr150 was confirmed to be central for catalysis, at least with the best substrates. On the basis of a comparison of data obtained for several class C enzyme mutants and in agreement with recent structural data, we propose that the phenolate anion of Tyr150, in conjunction with the alkyl ammonium of Lys315, acts as the general base responsible for the activation of the active-site Ser64 during the acylation step and for the subsequent activation of a water molecule in the deacylation process. The evolution of the important superfamily of penicillin-recognizing enzymes is further discussed in the light of this proposed mechanism. © 1996 Wiley-Liss, Inc.     

Catalysis by dienelactone hydrolase: A variation on the protease mechanism

Dienelactone hydrolase (DLH), an enzyme from the β-ketoadipate pathway, catalyzes the hydrolysis of dienelactone to maleylacetate. Our inhibitor binding studies suggest that its substrate, dienelactone, is held in the active site by hydrophobic interactions around the lactone ring and by the ion pairs between its carboxylate and Arg-81 and Arg-206. Like the cysteine/serine proteases, DLH has a catalytic triad (Cys-123, His-202, Asp-171) and its mechanism probably involves the formation of covalently bound acyl intermediate via a tetrahedral intermediate. Unlike the proteases, DLH seems to protonate the incipient leaving group only after the collapse of the first tetrahedral intermediate, rendering DLH incapable of hydrolyzing amide analogues of its ester substrate. In addition, the triad His probably does not protonate the leaving group (enolate) or deprotonate the water for deacylation; rather, the enolate anion abstracts a proton from water and, in doing so, supplies the hydroxyl for deacylation. © 1993 Wiley-Liss, Inc.     

phi29 DNA polymerase-terminal protein interaction. Involvement of residues specifically conserved among protein-primed DNA polymerases

By multiple sequence alignments of DNA polymerases from the eukaryotic-type (family B) subgroup of protein-primed DNA polymerases we have identified five positively charged amino acids, specifically conserved, located N-terminally to the (S/T)Lx(2)h motif. Here, we have studied, by site-directed mutagenesis, the functional role of phi29 DNA polymerase residues Arg96, Lys110, Lys112, Arg113 and Lys114 in specific reactions dependent on a protein-priming event. Mutations introduced at residues Arg96, Arg113 and Lys114 and to a lower extent Lys110 and Lys112, showed a defective protein-primed initiation step. Analysis of the interaction with double-stranded DNA and terminal protein (TP) displayed by mutant derivatives R96A, K110A, K112A, R113A and K114A allows us to conclude that phi29 DNA polymerase residue Arg96 is an important DNA/TP-ligand residue, essential to form stable DNA polymerase/DNA(TP) complexes, while residues Lys110, Lys112 and Arg113 could be playing a role in establishing contacts with the TP-DNA template during the first step of DNA replication. The importance of residue Lys114 to make a functionally active DNA polymerase/TP complex is also discussed. These results, together with the high degree of conservation of those residues among protein-primed DNA polymerases, strongly suggest a functional role of those amino acids in establishing the appropriate interactions with DNA polymerase substrates, DNA and TP, to successfully accomplish the first steps of TP-DNA replication.     

Structure‐based replacement of methionine residues at the catalytic domains with serine significantly improves the oxidative stability of alkaline amylase from alkaliphilic Alkalimonas amylolytica

The alkaline amylase requires high resistance towards chemical oxidation for use in the detergent and textile industries. This work aims to improve the oxidative stability of alkaline amylase from alkaliphilic Alkalimonas amylolytica by site‐directed mutagenesis based on the enzyme structure model. Five mutants were created by individually replacing methionine at positions 145, 214, 229, 247, and 317 in the amino acid sequence of alkaline amylase with oxidative‐resistant serine. The pH stability of the mutant enzymes was almost the same as that of the wild‐type (WT) enzyme (pH 7.0–11.0). The stable temperature range of the mutant enzymes M145S and M247S decreased from <50°C of the WT to <40°C, while the thermal stability of the other three mutant enzymes (M214S, M229S, and M317S) was almost the same as that of the WT enzyme. The catalytic efficiency (k_cat/K_m) of all the mutant enzymes decreased when compared to WT enzyme. The mutant enzymes showed increased activity in the presence of surfactants Tween‐60 and sodium dodecyl sulfate. When incubated with 500 mM H₂O₂ at 35°C for 5 h, the WT enzyme retained only 13.3% of its original activity, while the mutant enzymes M145S, M214S, M229S, M247S, and M317S retained 55.6, 70.2, 54.2, 62.5, and 46.4% of the original activities, respectively. The results indicated that the substitution of methionine residues at the catalytic domains with oxidative‐resistant serine can significantly improve the oxidative stability of alkaline amylase. This work provides an effective strategy to improve the oxidative stability of amylase, and the high oxidation resistance of the mutant enzymes shows their potential applications in the detergent and textile industries. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Identification of amino acid residues involved in the binding of Huperzine A to cholinesterases.

Huperzine A, a potential agent for therapy in Alzheimer's disease and for prophylaxis of organophosphate toxicity, has recently been characterized as a reversible inhibitor of cholinesterases. To examine the specificity of this novel compound in more detail, we have examined the interaction of the 2 stereoisomers of Huperzine A with cholinesterases and site-specific mutants that detail the involvement of specific amino acid residues. Inhibition of fetal bovine serum acetylcholinesterase by (-)-Huperzine A was 35-fold more potent than (+)-Huperzine A, with KI values of 6.2 nM and 210 nM, respectively. In addition, (-)-Huperzine A was 88-fold more potent in inhibiting Torpedo acetylcholinesterase than (+)-Huperzine A, with KI values of 0.25 microM and 22 microM, respectively. Far larger KI values that did not differ between the 2 stereoisomers were observed with horse and human serum butyrylcholinesterases. Mammalian acetylcholinesterase, Torpedo acetylcholinesterase, and mammalian butyrylcholinesterase can be distinguished by the amino acid Tyr, Phe, or Ala in the 330 position, respectively. Studies with mouse acetylcholinesterase mutants, Tyr 337 (330) Phe and Tyr 337 (330) Ala yielded a difference in reactivity that closely mimicked the native enzymes. In contrast, mutation of the conserved Glu 199 residue to Gln in Torpedo acetylcholinesterase produced only a 3-fold increase in KI value for the binding of Huperzine A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kunitz 型丝氨酸蛋白酶抑制剂结构与功能研究

蛋白酶抑制剂在酶学及蛋白质的结构与功能关系研究中有重要意义,Kunitz型丝氨酸蛋白酶抑制剂是其中最重要的,也是研究最广泛的蛋白酶抑制剂之一．该类蛋白酶抑制剂三维结构高度保守：由一个明显的疏水核心、三对高度保守的二硫键桥、三链β-折叠和一个N端3 10螺旋及一个C端α-螺旋组成．3对二硫键对分子空间结构的稳定起着非常重要的作用．这一类型抑制剂有5个主要的活性位点：P1、P1’、P3、P3’、P4,它们都位于一个溶剂暴露的环上．P1位点是抑制作用的关键活性位点,抑制剂的专一性由P1位点氨基酸残基的性质决定;P1’位点氨基酸残基的侧链大小对抑制剂．酶的结合常数有很大影响,用大的侧链残基取代会导致结合常数降低;P4位点残基被取代经常产生负效应,会导致活性区域环的构象发生很大改变,从而影响酶与抑制剂的结合．     

定点突变对酰胺水解酶DamH可溶性表达和酶活的影响

摘要:【目的】DamH是一种具有酯酶活性的酰胺水解酶,其非活性中心氨基酸残基的突变对重组酶可溶性表达和比酶活产生一定的影响。拟探索DamH的活性中心氨基酸残基构成,并对其非活性中心氨基酸残基突变对可溶性表达和比酶活的影响进行研究。【方法】通过重叠延伸的方法对DamH可能的活性中心氨基酸S149、E244和H274以及非活性中心氨基酸D165及N192进行定点突变,通过静息细胞测活验证了S149、E244和H274 在催化2-氯-N-(2’-甲基-6’-乙基苯基)乙酰胺(CMEPA)水解反应中的作用,通过Ni2+-NTA亲和层析对D165及N192突变子进行纯化,对突变株和野生型比酶活进行比较。【结果】研究表明S149A使DamH的CMEPA 水解酶活性下降为野生型的5%,E244A和H274A突变导致其失去活性;D165P和N192P突变影响到DamH的可溶性表达,表达量分别为野生型的28.2%和20.8%,突变子N192P、D165P比酶活分别为野生型比酶活的55.5%和49.7%。【结论】DamH催化酯类底物和芳基酰胺类底物可能共用同一活性中心S149、E244和H274,其两个α螺旋的转角处氨基酸侧链极性和刚性结构的改变对可溶性表达以及活性有很大的影响。