A highly active and oxidation-resistant subtilisin-like enzyme produced by a combination of site-directed mutagenesis and chemical modification

The subtilisins are known to be susceptible to chemical oxidation due to the conversion of Met222 into the corresponding sulfoxide. A number of derivatives with resistance towards oxidation have previously been prepared by replacement of this group with the other 19 amino acid residues. Unfortunately, the activities of these enzymes were of the order of 1-10% of that obtained with the wild-type enzyme. In contrast, the oxidation-labile cysteine mutant exhibited much higher activity, suggesting that this is associated with the presence of a sulphur atom in the amino acid at position 222. It is shown here that it is possible to maintain a sulphur atom in the amino acid at position 222 without the enzyme becoming labile towards oxidation. A subtilisin from Bacillus lentus, subtilisin 309, in which Met222 was replaced with a cysteinyl residue by site-directed mutagenesis was modified with thioalkylating reagents. Treatment of such enzyme derivatives with H2O2 revealed that their stabilities towards oxidation had increased significantly compared to both wild-type and unmodified [Cys222]subtilisin. One of the chemically modified enzyme derivatives, [Me-S-Cys222]subtilisin, exhibited a kcat/Km value of 56% of that obtained with the wild-type enzyme when assayed against the substrate Suc-Ala-Ala-Pro-Phe-NH-Ph-NO2 (Suc, succinyl) and it exhibited 89% activity when tested in an assay with dimethyl casein as a substrate. The corresponding values obtained for unmodified [Cys222]subtilisin were lower, i.e. 39% for the dimethyl casein activity and 46% for the kcat/Km for the hydrolysis of Suc-Ala-Ala-Pro-Phe-NH-Ph-NO2. This demonstrates the feasibility of replacing the oxidation-labile methionyl residue group in a subtilisin enzyme with a group stable towards oxidation without substantially reducing the activity.     

Studies on the reaction mechanism of a microbial lipase/acyltransferase using chemical modification and site-directed mutagenesis

Aeromonas hydrophila releases a protein which is a member of the lipase superfamily, similar in reaction mechanism to the important mammalian plasma enzyme lecithin-cholesterol acyltransferase. We have used chemical modification and site-directed mutagenesis of the protein to identify amino acids which may be involved in catalysis. The enzyme was unaffected by phenylmethylsulfonyl fluoride, but it was almost completely inhibited by another serine-reactive compound, diethyl p-nitrophenyl phosphate. A serine selectively modified by this reagent was identified by sequencing the amino-terminal region of the protein. It was located at position 16, in the short consensus sequence shared by the enzyme with other lipases. When this serine was changed to asparagine the product was an enzymatically inert protein which nevertheless retained the surface activity of the wild-type enzyme, suggesting its ability to bind to substrate was not changed. Diethylpyrocarbonate treatment drastically reduced the rate of acyl transfer by the native enzyme, but this did not appear to be due to modification of an essential histidine, since inhibition was not reversed by addition of hydroxylamine. We have shown that only two of the histidines in the enzyme can be involved in catalysis (Hilton, S., McCubbin, W. D., Kay, C.M., and Buckley, J. T. (1990) Biochemistry, 29, 9072-9078). Replacing both of these with asparagines had little or no effect on enzyme activity. These results indicate that, in apparent contrast to other lipases, histidine does not participate in the reaction catalyzed by the microbial enzyme. Since catalysis was not inhibited by sulfhydryl reagents, we conclude that a free cysteine is also not required for activity. This may distinguish the microbial enzyme from the mammalian acyltransferase.     

Role of lysines in human angiogenin: chemical modification and site-directed mutagenesis

The role of lysines in the ribonucleolytic and angiogenic activities of human angiogenin has been examined by chemical modification and site-directed mutagenesis. It was demonstrated previously [Shapiro, R., Weremowicz, S., Riordan, J.F., & Vallee, B.L. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8783-8787] that extensive treatment with lysine reagents markedly decreases the ribonucleolytic activity of angiogenin. In the present study, limited chemical modification with 1-fluoro-2,4-dinitrobenzene followed by C18 high-performance liquid chromatography yielded several (dinitrophenyl)angiogenin derivaties. The major derivative formed had slightly increased enzymatic activity compared with the unmodified protein. Tryptic peptide mapping demonstrated the site of modification to be Lys-50. A second derivative, modified at Lys-60, was 34% active. Analysis of a third derivative indicated that modification of Lys-82 did not decrease activity. Thus, Lys-50 and Lys-82 are unessential for enzymatic activity while Lys-60 may play a minor role. No pure derivative modified at Lys-40, corresponding to the active-site residue Lys-41 of the homologous protein ribonuclease A, could be obtained by chemical procedures. Therefore, we employed oligonucleotide-directed mutagenesis to replace this lysine with glutamine or arginine. The Gln-40 derivative had less than 0.05% enzymatic activity compared with the unmodified protein and substantially reduced angiogenic activity when examined with the chick embryo chorioallantoic membrane assay. These results suggest that the angiogenic activity of the protein is dependent on an intact enzymatic active site. The Arg-40 derivative had 2.2% ribonucleolytic activity compared with unmodified angiogenin. The effects of reductive methylation of this derivative indicate that no lysines other than Lys-40 are critical.(ABSTRACT TRUNCATED AT 250 WORDS)     

产碱假单胞菌脂肪酶的克隆表达及酶学性质研究

【目的】克隆产碱假单胞菌的脂肪酶基因,实现其在大肠杆菌中异源表达并进行酶学性质研究。【方法】通过基因文库构建和PCR,获得脂肪酶基因,并以pET30a(+)为表达载体、E.coli BL21(DE3)为宿主菌,在大肠杆菌中进行异源表达,表达产物经HisTrapTM亲和层析柱纯化后进行酶学性质研究。【结果】从产碱假单胞菌中克隆得到一个脂肪酶基因,大小为1 575 bp(GenBank登录号为JN674069)。该酶分子量为55 kD,最适底物为p-NPO,最适反应温度和pH分别为35°C、pH 9.0。重组酶经1 mmol/L的Cu2+处理30 min可使酶活提高至156%。在最适反应条件下重组酶的比活力为275 U/mg,Km和Vmax分别为80μmol/L和290 mmol/(min.g protein)。【结论】产碱假单胞菌脂肪酶基因的克隆与表达不仅积累了脂肪酶基因的资源,并为其在手性拆分中的应用奠定基础。     

Study of the fast-reacting cysteines in phosphoglycerate kinase using chemical modification and site-directed mutagenesis

Horse muscle phosphoglycerate kinase, like other mammalian phosphoglycerate kinases, contains seven cysteine residues of which two react rapidly with 5,5'-dithio-bis(2-nitrobenzoate) (Nbs2) following second-order kinetics (k = 640 M-1.s-1). Selective cyanylation of the fast-reacting cysteines, followed by chemical cleavage and subsequent sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis of the resulting polypeptides, suggested that these cysteines are at positions 378 and 379. Cysteine residues were introduced into yeast phosphoglycerate kinase by site-directed mutagenesis. Mutant enzymes, each containing only one cysteine residue at position 364, 376, or 377, were constructed from a mutant devoid of cysteine (Cys97----Ala). In the last two mutants, the cysteines were at positions corresponding to Cys378 and Cys379, respectively, in the horse muscle enzyme. The chemical reactivity of the cysteine groups in these latter two yeast mutant enzymes was similar to that of the fast-reacting cysteines in the horse muscle enzyme. Furthermore, they were similarly modified upon substrate binding. All these data demonstrate unambiguously that the fast-reacting cysteines in the horse muscle enzyme are Cys378 and Cys379.     

Enhanced activity of an immobilized lipase promoted by site-directed chemical modification with polymers

The activity of a lipase from Geobacillus thermocatenulatus (BTL2) can be greatly improved by site-directed chemical modification of a single external Cys64. This residue is placed in the proximity of the region where the lid is allocated when the lipase exhibits its open and active form. Thiol group of Cys64 was modified by thiol-disulfide exchange with pyridyldisulfide poly-aminated-dextrans or mono-carboxylated-polyethyleneglycol. The modification was performed on the covalently immobilized lipase on CNBr-agarose or glyoxyl-agarose. The activity of modified derivatives was strongly dependent on the immobilized preparation, the polymer used and the substrate assayed. For example, the modification with PEG-COOH of BTL2 immobilized on glyoxyl-agarose increased 5-fold the enzyme activity towards the hydrolysis of 2-O-butyryl-2-phenylacetic acid. However, the modification with 3-(2-pyridyldithio)-propionyl-dextran-NH₂ reduced the activity to 40%.The fact that the modified enzymes can be inhibited by an irreversible inhibitor much more rapidly than the unmodified ones suggested that the main effect of the modification is to somehow stabilize the open form of the lipase.     

Analysis of essential histidine residues of maize branching enzymes by chemical modification and site-directed mutagenesis

Incubation of maize branching enzyme, mBEI and mBEII, with 100 μM diethylpyrocarbonate (DEPC) rapidly inactivated the enzymes. Treatment of the DEPC-inactivated enzymes with 100–500 mM hydroxylamine restored the enzyme activities. Spectroscopic data indicated that the inactivation of BE with DEPC was the result of histidine modification. The addition of the substrate amylose or amylopectin retarded the enzyme inactivation by DEPC, suggesting that the histidine residues are important for substrate binding. In maize BEII, conserved histidine residues are in catalytic regions 1 (His320) and 4 (His508). His320 and His508 were individually replaced by Ala via site-directed mutagenesis to probe their role in catalysis. Expression of these mutants inE. coli showed a significant decrease of the activity and the mutant enzymes hadK _m values 10 times higher than the wild type. Therefore, residues His320 and His508 do play an important role in substrate binding.     

Analysis of essential histidine residues of maize branching enzymes by chemical modification and site-directed mutagenesis

Incubation of maize branching enzyme, mBEI and mBEII, with 100 μM diethylpyrocarbonate (DEPC) rapidly inactivated the enzymes. Treatment of the DEPC-inactivated enzymes with 100–500 mM hydroxylamine restored the enzyme activities. Spectroscopic data indicated that the inactivation of BE with DEPC was the result of histidine modification. The addition of the substrate amylose or amylopectin retarded the enzyme inactivation by DEPC, suggesting that the histidine residues are important for substrate binding. In maize BEII, conserved histidine residues are in catalytic regions 1 (His320) and 4 (His508). His320 and His508 were individually replaced by Ala via site-directed mutagenesis to probe their role in catalysis. Expression of these mutants inE. coli showed a significant decrease of the activity and the mutant enzymes hadK _m values 10 times higher than the wild type. Therefore, residues His320 and His508 do play an important role in substrate binding.     

Structure-function studies of Escherichia coli biotin synthase via a chemical modification and site-directed mutagenesis approach.

In Escherichia coli, biotin synthase (bioB gene product) catalyzes the key step in the biotin biosynthetic pathway, converting dethiobiotin (DTB) to biotin. Previous studies have demonstrated that BioB is a homodimer and that each monomer contains an iron-sulfur cluster. The purified BioB protein, however, does not catalyze the formation of biotin in a conventional fashion. The sulfur atom in the iron-sulfur cluster or from the cysteine residues in BioB have been suggested to act as the sulfur donor to form the biotin molecule, and yet unidentified factors were also proposed to be required to regenerate the active enzyme. In order to understand the catalytic mechanism of BioB, we employed an approach involving chemical modification and site-directed mutagenesis. The properties of the modified and mutated BioB species were examined, including DTB binding capability, biotin converting activity, and Fe(2+) content. From our studies, four cysteine residues (Cys 53, 57, 60, and 97) were assigned as the ligands of the iron-sulfur cluster, and Cys to Ala mutations completely abolished biotin formation activity. Two other cysteine residues (Cys 128 and 188) were found to be involved mainly in DTB binding. The tryptophan and histidine residues were suggested to be involved in DTB binding and dimer formation, respectively. The present study also reveals that the iron-sulfur cluster with its ligands are the key components in the formation of the DTB binding site. Based on the current results, a refined model for the reaction mechanism of biotin synthase is proposed.     

Chemical modification and site-directed mutagenesis of tyrosine residues in cephalosporin C acylase from Pseudomonas strain N176

Cephalosporin C (CC) acylase from Pseudomonas strain N176 was chemically modified by tetranitromethane (TNM), causing complete loss of activity. Modification using molar excesses of TNM up to 10 resulted in complete inactivation when 1.4 mol tyrosines/mol enzyme were modified. Digestion of native and TNM-modified acylase with Achromobacter protease I (API), separation by high performance liquid phase chromatography (HPLC) and amino terminal sequencing of the resultant peptides were used to identify the modified tyrosine residues. The major difference in HPLC profile between these API digests was shown to be the peak corresponding to the peptide Ser²³⁹-Lys³⁰¹ of native acylase. A portion of the peak for the peptide Ala⁴⁵-Lys⁷³ was also shifted in HPLC analysis of TNM-modified acylase. The peptides isolated from the modified acylase were shown to contain nitrated tyrosines (3-nitrotyrosine) at positions 270 and 52, respectively. These findings indicate that Tyr²⁷⁰ is completely modified, and Tyr⁵² is partially modified in the inactivated acylase. Each of the fifteen tyrosines in the acylase was altered to leucine by site-directed mutagenesis to complement the chemical modification with TNM. At pH 8.7, the mutant acylase in which tyrosine at position 270 is changed to leucine showed GL-7ACA and CC acylase activities reduced to 28.0 and 32.2% of native acylase, respectively. The results correspond to those obtained from TNM-modification. A similar reduction in activity was also obtained in the case of Tyr⁴⁹¹ mutant, although nitration of this residue was not confirmed by chemical modification. Therefore Tyr²⁷⁰ and Tyr⁴⁹¹ are important for exerting the maximum activity of the enzyme, but are not essential for catalysis. However, mutation of Tyr⁵² to Leu produced little change in acylase activity. The mutant acylase in which Tyr⁷⁰⁵ is changed to leucine has a lowered pH optimum for GL-7ACA, which may be useful for further improvement of the acylase.     

The purification and chemical composition of the lipopolysaccharide of Pseudomonas alcaligenes

1. A method for obtaining lipopolysaccharide free from glycosaminopeptide from isolated cell walls of Pseudomonas alcaligenes is discussed. 2. About 70-75% of the lipopolysaccharide and 86-90% of the isolated lipid A have been accounted for in terms of identifiable components. 3. Hydrolysates of lipid A contain mainly inorganic phosphate, glucosamine, O-phosphorylglucosamine and fatty acids (dodecanoic acid, dodec-2-enoic acid, 3-hydroxydecanoic acid and 3-hydroxydodecanoic acid), of which the last is the main N-acylating acid of the glucosamine backbone. 4. Material corresponding to the polysaccharide moiety of the lipopolysaccharide is extensively degraded. 5. Solubilization of the lipopolysaccharide by using sodium deoxycholate appreciably affects the chemical composition of the material.     

定点突变提高土曲霉Aspergillus terreus脂肪酶的催化活性

本研究旨在利用理性设计的方法来提高来源于土曲霉Aspergillus terreus的酸性脂肪酶ATL的催化活力。通过同源比对,选择脂肪酶盖子区域和底物结合口袋域中的位点进行定点突变,得到8种ATL的突变脂肪酶。结果发现,盖子区域突变酶ATLLid与底物结合口袋域突变酶ATLV218W的催化活性显著提高。ATLLid和ATLV218W对底物对硝基苯酚月桂酸酯p-nitrophenyl laurate(p-NPL)的催化活性最高,k_(cat)值较ATL分别提高了39.37倍和50.79倍,k_(cat)/K_m值较ATL分别提高了2.85倍和8.48倍。与ATL相比,ATLLid和ATLV218W的热稳定性略有下降,最适p H为5.0,p H 4.0–8.0具有较好的稳定性,说明突变未对ATL的嗜酸耐酸特性产生影响。通过同源建模模拟及分子对接技术分析底物p-NPL与酶分子间的相互作用,解析了ATLLid和ATLV218W催化活性提高的机理。     

Complete cysteine-scanning mutagenesis and site-directed chemical modification of the Tn10-encoded metal-tetracycline/H+ antiporter

Bacterial Tn10-encoded metal-tetracycline/H(+) antiporter was the first found drug exporter and has been studied as a paradigm of antiporter-type major facilitator superfamily transporters. Here the 400 amino acid residues of this protein were individually replaced by cysteine except for the initial methionine. As a result, we could obtain a complete map of the functionally or structurally important residues. In addition, we could determine the precise boundaries of all the transmembrane segments on the basis of the reactivity with N-ethylmaleimide (NEM). The NEM binding results indicated the presence of a transmembrane water-filled channel in the transporter. The twelve transmembrane segments can be divided into three groups; four are totally embedded in the hydrophobic interior, four face a putative water-filled channel along their full length, and the remaining four face the channel for half their length, the other halves being embedded in the hydrophobic interior. These three types of transmembrane segments are mutually arranged with a 4-fold symmetry. The competitive binding of membrane-permeable and -impermeable SH reagents in intact cells indicates that the transmembrane water-filled channel has a thin barrier against hydrophilic molecules in the middle of the transmembrane region. Inhibition and stimulation of NEM binding in the presence of tetracycline reflects the substrate-induced protection or conformational change of the Tn10-encoded metal-tetracycline/H(+) antiporter. The mutations protected from NEM binding by tetracycline were mainly located around the permeability barrier in the N-terminal half, suggesting the location of the substrate binding site.     

Analysis of the psychrotolerant property of hormone-sensitive lipase through site-directed mutagenesis

Mammalian hormone-sensitive lipase (HSL) has given its name to a family of primarily prokaryotic proteins which are structurally related to type B carboxylesterases. In many of these alpha/beta hydrolases, a conserved HG-dipeptide flanks the catalytic pocket. In HSL this dipeptide is followed by two additional glycine residues. Through site-directed mutagenesis, we have investigated the importance of this motif for enzyme activity. Since the presence of multiple glycine residues in a critical region could contribute to cold adaptation by providing local flexibility, we studied the effect of mutating these residues on the psychrotolerant property of HSL. Any double mutation rendered the enzyme completely inactive, without any major effect on the enzyme stability. The partially active single mutants retained the same proportion of activity at reduced temperatures as the wild-type enzyme. These results do not support a role for the HGGG motif in catalysis at low temperatures, but provide further validation of the current three-dimensional model of HSL. Rat HSL was found to be relatively more active than human HSL at low temperatures. This difference was, however, not due to the 12 amino acids which are present in the regulatory module of the rat enzyme but absent in human HSL.     

Engineering an enzyme by site-directed mutagenesis to be resistant to chemical oxidation

Site-directed mutagenesis can be employed to alter activity critical residues in proteins which are susceptible to chemical oxidation. Previous studies have implicated methionine 222 as a primary site for oxidative inactivation of subtilisin (Stauffer, C. E., and Etson, D. (1969) J. Biol. Chem. 244, 5333-5338). Because of uncertainties in predicting which amino acid would be the optimal substitute for methionine 222, we prepared all 19 amino acid substitutions at this site in the cloned subtilisin gene using a cassette mutagenesis method (Wells, J. A., Vasser, M., and Powers, D. P. (1985) Gene (Amst.), in press). Mutant enzymes were expressed in Bacillus subtilis and were found to vary widely in specific activity. Mutants containing nonoxidizable amino acids (i.e. Ser, Ala, and Leu) were resistant to inactivation by 1 M H2O2, whereas methionine and cysteine enzymes were rapidly inactivated. These studies demonstrate the feasibility of improving oxidative stability in proteins by site-directed mutagenesis.     

Oxidative modification of a cephalosporin C acylase from Pseudomonas strain N176 and site-directed mutagenesis of the gene.

A cephalosporanic acid acylase from Pseudomonas strain N176 catalyzes hydrolysis of both glutarylcephalosporanic acid and cephalosporin C to 7-amino-cephalosporanic acid. Chemical modification of the enzyme with acidic hydrogen peroxide was performed to investigate residues which play important roles in enzymatic activity. The activity of the enzyme was reduced to 76% of the original by oxidation. From protein chemical analysis combined with site-directed point mutagenesis, modification of Met-164 was found to correspond to the reduction in activity. To study the effect of Met-164 on the enzymatic character, we prepared mutant acylases in which Met-164 was replaced with several other amino acids and obtained the following data: (i) there existed a trend of mutation to noncharged hydrophilic residues, resulting in an increase of activity against glutarylcephalosporanic acid; (ii) the mutation of Met-164 to Gly and Ala resulted in the lowering of both Km values and the optimal pHs against glutarylcephalosporanic acid; (iii) the mutation to Leu enhanced cephalosporin C acylase activity; and (iv) the mutation to Gln improved the k(cat) value for glutarylcephalosporanic acid. In particular, the mutation to Gln resulted in a high rate of conversion of glutarylcephalosporanic acid to 7-amino-cephalosporanic acid under conditions similar to those of a bioreactor system. These results may indicate that Met-164 is located in or near the cephalosporin compound binding pocket on the enzyme.     

Improving the carboligase activity of benzoylformate decarboxylase from Pseudomonas putida by a combination of directed evolution and site-directed mutagenesis

Benzoylformate decarboxylase (BFD) from Pseudomonas putida was subjected to directed molecular evolution to generate mutants with increased carboligase activity which is a side reaction of the enzyme. After a single round of random mutagenesis mutants were isolated which exhibited a 5-fold increased carboligase activity in aqueous buffer compared to the wild-type enzyme with a high enantiomeric excess of the product (S)-2-hydroxy-1-phenyl-propanone. From the same library, mutants with enhanced carboligase activity in water-miscible organic solvents have been isolated. The selected mutants have been characterized by sequencing, revealing that all mutants carry a mutation at Leu476, which is close to the active site but does not directly interact with the active center. BFD-L476Q has a 5-fold higher carboligase activity than the wild-type enzyme. L476 was subjected to saturation mutagenesis yielding eight different mutants with up to 5-fold increased carboligase activity. Surprisingly, all L476 mutants catalyze the formation of 2-hydroxy-1-phenyl-propanone with significantly higher enantioselectivity than the wild-type enzyme although enantioselectivity was not a selection parameter. Leu476 potentially plays the role of a gatekeeper of the active site of BFD, possibly by controlling the release of the product. The biocatalyst could be significantly improved for its side reaction, the C-C bond formation and for application under conditions that are not optimized in nature.     

Subtle alteration of the active site of ribulose bisphosphate carboxylase/oxygenase by concerted site-directed mutagenesis and chemical modification

Both activities of ribulose bisphosphate carboxylase/oxygenase are dependent on carbamylation by CO2 of a specific lysyl epsilon-amino group (Lys-191 of the enzyme from Rhodospirillum rubrum). To examine the stringency of the requirement for this lysyl side chain, Lys-191 was converted to an aminoethylcysteinyl residue (net replacement of a gamma-methylene group by a sulfur atom) by a combination of site-directed mutagenesis and subsequent chemical modification. The purified Cys-191 mutant was totally devoid of both carboxylase and oxygenase activities. However, this mutant protein exhibited tight-binding of the transition-state analogue, 2-carboxyarabinitol bisphosphate, a property heretofore ascribed solely to the carbamylated form of the carboxylase. Treatment of the mutant protein with ethylene imine restored catalytic activity to 4-7% of the wild-type level. The carboxylase:oxygenase activity ratio of the aminoethylated protein was unperturbed relative to that of wild-type enzyme.