Substrate specificity of a peptidyl-aminoacyl-<Emphasis Type="SmallCaps">l</Emphasis>/<Emphasis Type="SmallCaps">d</Emphasis>-isomerase from frog skin

In the skin of fire-bellied toads (Bombina species), an aminoacyl-l/d-isomerase activity is present which catalyses the post-translational isomerization of the l- to the d-form of the second residue of its substrate peptides. Previously, this new type of enzyme was studied in some detail and genes potentially coding for similar polypeptides were found to exist in several vertebrate species including man. Here, we present our studies to the substrate specificity of this isomerase using fluorescence-labeled variants of the natural substrate bombinin H with different amino acids at positions 1, 2 or 3. Surprisingly, this enzyme has a rather low selectivity for residues at position 2 where the change of chirality at the alpha-carbon takes place. In contrast, a hydrophobic amino acid at position 1 and a small one at position 3 of the substrate are essential. Interestingly, some peptides containing a Phe at position 3 also were substrates. Furthermore, we investigated the role of the amino-terminus for substrate recognition. In view of the rather broad specificity of the frog isomerase, we made a databank search for potential substrates of such an enzyme. Indeed, numerous peptides of amphibia and mammals were found which fulfill the requirements determined in this study. Expression of isomerases with similar characteristics in other species can therefore be expected to catalyze the formation of peptides containing d-amino acids.     

High‐performance variants of plant diacylglycerol acyltransferase 1 generated by directed evolution provide insights into structure function

Diacylglycerol acyltransferase 1 (DGAT1) catalyzes the acyl‐CoA‐dependent biosynthesis of triacylglycerol, the predominant component of seed oil. In some oil crops, including Brassica napus, the level of DGAT1 activity can have a substantial effect on triacylglycerol production. Structure–function insights into DGAT1, however, remain limited because of the lack of a three‐dimensional detailed structure for this membrane‐bound enzyme. In this study, the amino acid residues governing B. napus DGAT1 (BnaDGAT1) activity were investigated via directed evolution, targeted mutagenesis, in vitro enzymatic assay, topological analysis, and transient expression of cDNA encoding selected enzyme variants in Nicotiana benthamiana. Directed evolution revealed that numerous amino acid residues were associated with increased BnaDGAT1 activity, and 67% of these residues were conserved among plant DGAT1s. The identified amino acid residue substitution sites occur throughout the BnaDGAT1 polypeptide, with 89% of the substitutions located outside the putative substrate binding or active sites. In addition, cDNAs encoding variants I447F or L441P were transiently overexpressed in N. benthamiana leaves, resulting in 33.2 or 70.5% higher triacylglycerol content, respectively, compared with native BnaDGAT1. Overall, the results provide novel insights into amino acid residues underlying plant DGAT1 function and performance‐enhanced BnaDGAT1 variants for increasing vegetable oil production.     

Functional Constraints of 6-Phosphogluconate Dehydrogenase (6-PGD) Based on Sequence and Structural Information

The pentose phosphate cycle is considered as a major source of NADPH and pentose needed for nucleic acid biosynthesis. 6-Phosphogluconate dehydrogenase (6PGD), an enzyme participating in this cycle, catalyzes the oxidative decarboxylation of 6PGD to ribulose 5-phosphate with the subsequent release of CO₂ and the reduction of NADP. We have determined the amino acid sequence of 6PGD of Bactrocera oleae and constructed a three-dimensional model based on the homologous known sheep structure. In a comparative study of 6PGD sequences from numerous species, all the conserved and variable regions of the enzyme were analyzed and the regions of functional importance were localized, in an attempt promoted also by the direct involvement of the enzyme in various human diseases. Thus, analysis of amino acid variability of 37 6PGD sequences revealed that all regions important for the catalytic activity, such as those forming the substrate and coenzyme binding sites, are highly conserved in all species examined. Moreover, several amino acid residues responsible for substrate and coenzyme specificity were also found to be identical in all species examined. The higher percentage of protein divergence is observed at two regions that accumulate mutations, located at the distant parts of the two domains of the enzyme with respect to their interface. These peripheral regions of nonfunctional importance are highly variable and are predicted as antigenic, thus reflecting possible regions for antibody recognition. Furthermore, locating the differences between diptera 6PGD sequences on the three-dimensional model suggests probable positions of different amino acid residues appearing at B. oleae fast, intermediate, and slow allozymic variants. Abbreviations used: 6Pgd, 6-Phosphogluconate dehydrogenase gene; 6PGD, 6-Phosphogluconate dehydrogenase enzyme; NADPH, nicotinamide adenine dinucleotide phosphate; ADP, adenine dinucleotide phosphate; TNBS, 2,4,6 trinitrobenzensulfonic acid.     

Purification and partial characterization of chymotrypsin-like proteases from the digestive gland of the scallop Pecten maximus

Three variants of a chymotrypsin-like protease were purified from scallop digestive glands successively by ion-exchange, gel filtration and high-performance liquid chromatographies. Enzyme activity was detected using succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as a specific synthetic substrate for chymotrypsin. This proteinase was inhibited by chymostatin, diisopropylfluorophosphate and phenylmethylsulfonyl fluoride. Estimated molecular mass of the purified enzyme is around 32 kDa. These isoenzymes exhibit very low activities in hydrolyzing small synthetic specific substrates used for trypsic, elastolytic and collagenolytic measurements and referred mainly to a chymotrypsin-like proteinase. Very few differences were measured concerning pH profiles among the three isoenzymes. Stability is higher at low temperature for two variants. An N-terminal analysis was performed on one variant (B) among the three isoenzymes. The alignment of the N-terminal amino acid sequence indicates some homologies with abalone chymotrypsin-like protein and arthropod chymotrypsin proteases as well as with vertebrate serine protease counterparts (trypsin, chymotrypsin and elastase).     

快速展示糖苷水解酶活性架构单点突变导致结合力变化的电泳技术

酶分子在长期进化过程中形成一系列氨基酸残基组成的活性架构,参与底物的识别、结合与催化过程,而活性架构中相应氨基酸残基是如何影响酶分子结合底物的能力,进而影响酶分子的催化效率,一直是酶分子理性改造研究的热点.利用亲和电泳技术,可以快速展示内切纤维素酶Tr Cel12A和木聚糖酶Tl Xyn A活性架构中不同突变体的催化活性及其迁移率的变化,进而通过在不同底物浓度凝胶中蛋白质相对迁移率变化程度的定量回归分析,发现由氨基酸单点突变导致蛋白质迁移率的相对变化,可以定量表征酶分子突变前后结合底物能力的变化.亲和电泳测定的有效阻滞常数Kb值与等温滴定量热法和荧光光谱法测定的相关参数比较具有明显相关性.由于亲和电泳技术在测定酶分子与底物的结合能力时具有简便、快速、灵敏的特点,因而可作为常规生化实验室常规普筛技术来检测突变文库中系列突变体导致结合力的变化.     

Chemical Modification of Neutral Protease from Bacillus subtilis var. amylosacchariticus: Assignment of Tyrosyl Residues Iodinated

The neutral protease of Bacillus subtilis var. amylosacchariticus (B. amylosacchariticus) was iodinated with a 25-fold molar excess of iodine at pH 9.4 for 3 min at 0°C, by which treatment the proteolytic activity toward casein was markedly reduced, while the hydrolytic activity toward an N-blocked peptide substrate was rather increased. The modified enzyme was digested with Staphylococcus aureus V8 protease at pH 8.0 and the amino acid sequences of resultant peptides were compared with those obtained from the native enzyme. One of the peptides was found to have an amino acid sequence of Thr-Ala-Asn-Leu-Ile-Tyr-Glu, which corresponds to residue Nos. 153—159 of the enzyme, where Tyr-158 was identified to be mono-iodotyrosine. The other two peptides were those containing Tyr-21 which was mono- and di-iodinated, respectively. Referring to nitration experiments on the neutral protease and the active site structure of thermolysin, it was concluded that the iodination of Tyr-158 is mainly responsible for the activity changes of B. amylosacchariticus neutral protease.     

Catalytic improvement and structural analysis of atrazine chlorohydrolase by site-saturation mutagenesis

To improve the catalytic activity of atrazine chlorohydrolase (AtzA), amino acid residues involved in substrate binding (Gln71) and catalytic efficiency (Val12, Ile393, and Leu395) were targeted to generate site-saturation mutagenesis libraries. Seventeen variants were obtained through Haematococcus pluvialis-based screening, and their specific activities were 1.2–5.2-fold higher than that of the wild type. For these variants, Gln71 tended to be substituted by hydrophobic amino acids, Ile393 and Leu395 by polar ones, especially arginine, and Val12 by alanine, respectively. Q71R and Q71M significantly decreased the K_m by enlarging the substrate-entry channel and affecting N-ethyl binding. Mutations at sites 393 and 395 significantly increased the k_cat/K_m, probably by improving the stability of the dual β-sheet domain and the whole enzyme, owing to hydrogen bond formation. In addition, the contradictory relationship between the substrate affinity improvement by Gln71 mutation and the catalytic efficiency improvement by the dual β-sheet domain modification was discussed.     

Gain of function conferred by selenocysteine: catalytic enhancement of one‐electron transfer reactions by thioredoxin reductase

Selenocysteine (Sec) is the 21st amino acid in the genetic code and it is present in a small number of proteins where it replaces the much more commonly used amino acid cysteine (Cys). It is both more complicated and bioenergetically costly to insert Sec into a protein in comparison to Cys, and this cost is most likely compensated by a gain of function to the enzyme/protein in which it is incorporated. Here we investigate one such gain of function, the enhancement of one‐electron transfer catalysis. We compared the ability of Sec‐containing mouse mitochondrial thioredoxin reductase (mTrxR2) to catalyze the reduction of bovine cytochrome c, ascorbyl radical, and dehydroascorbate in comparison to Cys‐containing thioredoxin reductases from D. melanogaster (DmTrxR) and P. falciparum (PfTrxR). The Sec‐containing mTrxR2 was able to reduce all three substrates, while the Cys‐containing enzymes had little or no activity. In addition, we constructed Cys?Sec mutants of DmTrxR and PfTrxR and found that this substitution resulted in a gain of function, as these mutant enzymes were now able to catalyze the reduction of these substrates. We also found that in the case of PfTrxR, reduction of cytochrome c was enhanced five‐fold in a truncated PfTrxR in which the C‐terminal redox center was removed. This shows that some of the ability of thioredoxin reductase to reduce this substrate comes from the flavin coenzyme. We also discuss a possible mechanism by which Sec‐containing thioredoxin reductase reduces dehydroascorbate to ascorbate by two sequential, one‐electron reductions, in part catalyzed by Sec.     

An extracellular halophilic protease SptA from a halophilic archaeon Natrinema sp. J7: gene cloning, expression and characterization

A gene encoding an extracellular protease, sptA, was cloned from the halophilic archaeon Natrinema sp. J7. It encoded a polypeptide of 565 amino acids containing a putative 49-amino acid signal peptide, a 103-amino acid propeptide, as well as a mature region and C-terminal extension, with a high proportion of acidic amino acid residues. The sptA gene was expressed in Haloferax volcanii WFD11, and the recombinant enzyme could be secreted into the medium as an active mature form. The N-terminal amino acid sequencing and MALDI-TOF mass spectrometry analysis of the purified SptA protease indicated that the 152-amino acid prepropeptide was cleaved and the C-terminal extension was not processed after secretion. The SptA protease was optimally active at 50°C in 2.5 M NaCl at pH 8.0. The NaCl removed enzyme retained 20% of its activity, and 60% of the activity could be restored by reintroducing 2.5 M NaCl into the NaCl removed enzyme. When the twin-arginine motif in the signal peptide of SptA protease was replaced with a twin-lysine motif, the enzyme was not exported from Hfx. volcanii WFD11, suggesting that the SptA protease was a Tat-dependent substrate.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Production of 7, 10-dihydroxy-8(<Emphasis Type="Italic">E</Emphasis>)-octadecenoic acid from triolein via lipase induction by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PR3

Hydroxy fatty acids (HFA) have gained importance because of their special properties such as higher viscosity and reactivity compared with other non-hydroxy fatty acids. The bacterial isolate Pseudomonas aeruginosa (PR3) was reported to produce mono-, di-, and trihydroxy fatty acids from different unsaturated fatty acids. Of those, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) was produced with high yield from oleic acid by PR3. Up to now, the substrates used for microbial HFA production were free fatty acids. However, it is possible to utilize triacylglycerides, specifically triolein containing three oleic groups, as a substrate by microbial enzyme system involved in HFA production from oleic acid. In this study we used triolein as a substrate and firstly report that triolein could be efficiently utilized by PR3 to produce DOD. Triolein was first hydrolyzed into oleic acid by the triolein-induced lipase and then the released oleic acid was converted to DOD by PR3. Results from this study demonstrated that natural vegetable oils, without being intentionally hydrolyzed, could be used as efficient substrates for the microbial production of value-added hydroxy fatty acids.     

Studies on the Metabolism of D-amino acid in Microorganisms

Several strains of bacteria belonging to genus Aerobacter were found to oxidize D-glutamate rapidly, while tbey show feeble oxidative activity toward the L-form even when they were grown in the medium containing DL-glutamate.

The isolation of L-glutamate, a natural amino acid, from its DL-form was achieved by the degradation of D-glutamic acid using one of these strains.

This may be the first observation on a natural amino acid obtained from the racemic one by the metabolic action of the organism.

A new enzyme, D-glutamic acid oxidase, which is responsible for D-glutamate degradation in this organism and differs from Krebs’ D-amino acid oxidase, has been postulated.     

Position effect variegation of an acid phosphatase gene in Drosophila melanogaster

Summary X-ray mutagenesis has produced a series of deficiencies in a duplication of part of the third chromosome containing the acid phosphatase gene (Acph-1) in Drosophila melanogaster. In one of these deficiencies, Acph-1 is shown to be undergoing position effect variegation. Naturally occurring electrophoretic variants of the enzyme were used to visualize and determine quantitatively the extent of variegation of the allele which is cis to the heterochromatic breakpoint. Alteration of genotypic background and temperature provided further evidence for position effect. Rocket immunoelectrophoresis was used to correlate the levels of acid phosphatase activity and protein in flies containing the deficiency. A novel result indicates that the variegation is not the consequence of an averaging of active and inactive cells, but rather due to a quantitative alteration of gene activity within at least some individual cells.     

Directed Evolution of the Actinomycete Cytochrome P450 MoxA (CYP105) for Enhanced Activity

Actinomycete cytochrome P450 from Nonomuraea recticatena NBRC 14525 (P450moxA) catalyzes the hydroxylation of a broad range of substrates, including fatty acids, steroids, and various aromatic compounds. Hence, the enzyme is potentially useful in medicinal applications, but the activity is insufficient for practical use. Here we applied directed evolution to enhance the activity. A random mutagenesis library was screened using 7-ethoxycoumarin as a substrate to retrieve 17 variants showing >2-fold activities. Twenty-five amino acid substitutions were found in the variants, of which five mutations were identified to have the largest effects (Q87W, T115A, H132L, R191W, and G294D). These mutations additively increased the activity; the quintet mutant had 20-times the activity of the wildtype. These five single mutations also increased in activity toward structurally distinct substrates (diclofenac and naringenin). Based on the three-dimensional structure of the enzyme, we discerned that mutations in the substrate recognition site improved the activity, which was substrate dependent; mutations apart from the active site improved the activity as well as the substrates did.     

Genetic structure, isolation and characterization of a Bacillus licheniformis cell wall hydrolase.

Summary A DNA fragment containing the gene for a cell wall hydrolase of Bacillus licheniformis was cloned into Escherichia coli. Sequencing of the fragment showed the presence of an open reading frame which encodes a polypeptide of 253 amino acids with a molecular mass of 27 513. The gene was designated as cwlM, for cell wall lysis. The deduced amino acid sequence indicated that there is a repeated sequence consisting of 33 amino acid residues in the C-terminal region. Deletion of the C-terminal region did not lead to any loss of cell wall lytic activity. The gene product purified from E. coli cells harboring a cwlM-bearing plasmid exhibited a M _r value of 29 kDa on SDS-polyacrylamide gels, and characterization of the specific substrate bond cleaved by CWLM indicated that the enzyme is an N-acetylmuramoyl-l-alanine amidase (EC 3.5.1.28). The enzyme hydrolyzed the cell wall of Micrococcus luteus more efficiently than those of B. licheniformis and B. subtilis, but the truncated CWLM (lacking the C-terminal region) had lost this preference. CWLM prepared from B. subtilis cells harboring a plasmid containing cwlM had a similar M _r value to that from E. coli. Amino acid sequence homologies between CWLM and other amidases, and their protein structures are discussed.     

Flexible glycine rich motif of Escherichia coli deoxyuridine triphosphate nucleotidohydrolase is important for functional but not for structural integrity of the enzyme

Deoxyuridine triphosphate nucleotidohydrolase (dUTPase), a ubiquitous enzyme of DNA metabolism, has been implicated as a novel target of anticancer and antiviral drug design. This task is most efficiently accomplished by X-ray crystallography of the relevant protein–inhibitor complexes. However, the topic of the present investigation, a glycine-rich strictly conserved structural motif of dUTPases, could not be located in the crystal structure of the Escherichia coli enzyme, probably due to its increased flexibility. The present work shows that removal of a C-terminal 11-residue fragment, including this motif, by limited trypsinolysis strongly impairs catalytic activity. Kinetic analysis of the intact and digested variants showed that k_cat decreases 40-fold, while K_M increases less than twofold upon digestion. The tryptic site was identified by mass spectrometry, amino acid analysis and N-terminal sequencing. The shortened enzyme variant retains the secondary, tertiary, and quaternary (trimeric) structure of the intact species as suggested by UV absorption, fluorescence and circular dichroism spectroscopy, and analytical gel filtration. Moreover, binding affinity of the shortened variant toward the substrate analogue MgdUDP is identical to the one displayed by the intact enzyme. I conclude that the glycine-rich motif is functionally relevant for E. coli dUTPase. It may play a role in enzymatic catalysis by contributing to the formation of the catalytically potent enzyme–substrate complex. Proteins 28:568–579, 1997. © 1997 Wiley-Liss, Inc.     

Simple high-performance liquid chromatographic method for assaying cysteinesulfinic acid decarboxylase activity in rat tissue

A simple method is described for determining cysteinesulfinic acid decarboxylase activity in rat tissue. Enzyme preparations from the liver, kidney and brain were incubated with cysteinesulfinic acid substrate in the presence of pyridoxal 5-phosphate. The enzyme product, hypotaurine, was derivatized with o-phthalaldehyde and separated by reversed-phase high-performance liquid chromatography (Capsel Pack AG 120A C₁₈ column) using a mobile phase of acetonitrile–water (20:80, v/v) containing 50 mM sodium phosphate buffer (pH 6.0) and detected using a fluorometer (excitation at 360 nm and emission at 455 nm). The method described is reproducible and sensitive enough to determine the activity of cysteinesulfinic acid decarboxylase activity in the liver, kidney and brain. This assay was subsequently used to evaluate the effect of dietary proteins whose sulfur amino acid contents differ. Consistent with reported data, compared to casein and whole egg protein, a dietary protein low in sulfur amino acid (soybean protein) increased cysteinesulfinic acid decarboxylase activity in the liver and kidney. This method is therefore applicable to studies on the dietary regulation of cysteinesulfinic acid decarboxylase in rat tissue.     

A novel Pseudomonas putida strain with high levels of hydantoin-converting activity, producing -amino acids

Optically pure chiral amino acids and their derivatives can be efficiently synthesised by the biocatalytic conversion of 5-substituted hydantoins in reactions catalysed by stereo-selective microbial enzymes: initially a hydantoinase catalyses the cleavage of the hydantoin producing an N-carbamyl amino acid. In certain bacteria where an N-carbamyl amino acid amidohydrolase (NCAAH) is present, the N-carbamyl amino acid intermediate is further converted to amino acid, ammonia and CO₂. In this study we report on a novel Pseudomonas putida strain which exhibits high levels of hydantoin-converting activity, yielding -amino acid products including alanine, valine, and norleucine, with bioconversion yields between 60% and 100%. The preferred substrates are generally aliphatic, but not necessarily short chain, 5-alkylhydantoins. In characterizing the enzymes from this microorganism, we have found that the NCAAH has -selectivity, while the hydantoinase is non-stereoselective. In addition, resting cell reactions under varying conditions showed that the hydantoinase is highly active, and is not subject to substrate inhibition, or product inhibition by ammonia. The rate-limiting reaction appears to be the NCAAH-catalysed conversion of the intermediate. Metal-dependence studies suggest that the hydantoinase is dependent on the presence of magnesium and cobalt ions, and is strongly inhibited by the presence of copper ions. The relative paucity of -selective hydantoin-hydrolysing enzyme systems, together with the high level of hydantoinase activity and the unusual substrate selectivity of this P. putida isolate, suggest that is has significant potential in industrial applications.     

拟南芥谷胱甘肽S-转移酶Zeta类进化酶的获得及其特性分析

拟南芥谷胱甘肽S-转移酶Zeta类(AtGSTZ)是一种与细胞代谢和环境净化密切相关的多功能酶.应用易错PCR和多轮DNA洗牌技术构建了AtGSTZ随机突变文库;再利用pH指示剂颜色改变法对突变文库进行筛选,获得了9个二氯乙酸脱氯活性提高的突变子.其中,NN23含25个氨基酸突变,比活力提高120%,NN20含24个氨基酸突变,比活力提高102%,EC1含2个氨基酸突变,比活力提高47%,其他6个为单点突变,比活力分别提高9%～60%.酶学分析显示,所有进化酶对底物二氯乙酸的催化效率和对谷胱甘肽的亲和力以及个别进化酶的复性能力都得到不同程度的提高,但热稳定性均没有明显改善.同时,对一系列与AtGSTZ空间折叠及催化活性相关位点进行了讨论.     

Some Physicochemical Properties and Substrate Specificity of Acid Protease of Rhodotorula glutinis K-24

Some physicochemical properties, amino acid composition, and substrate specificity of the acid protease of Rhodotorula glutinis K-24 were determined. The molecular weight was estimated to be about 30,000 by the sedimentation equilibrium method. This value also coincided with the data obtained from Andrews’s method.

The isoelectric point was pH 4.5, and the amino terminal amino acid was identified to be alanine. The enzyme contained 14.5% of nitrogen and was composed of 285 residues of amino acid. Substrate specificity toward synthetic peptides was similar to that of pepsin, but its activity was considerably weak.

The enzyme was inactivated by diazoacetyl glycine ethylester, p-bromophenacyl bromide, et al., which attacked the active center of pepsin.     

Engineering active lysostaphin variants that incorporate noncanonical amino acids and characterizing the effects of site-specific PEGylation

We describe a facile strategy to identify sites for the incorporation of noncanonical amino acids into lysostaphin—an enzyme that degrades the cell wall of Staphylococcus aureus—while retaining stapholytic activity. We used this strategy to generate active variants of lysostaphin incorporating para-azidophenylalanine. The incorporation of this “reactive handle” enabled the orthogonal site-specific modification of the enzyme variants with polyethylene glycol (PEG) using copper-free click cycloaddition. PEGylated lysostaphin variants could retain their stapholytic activity, with the extent of retention depending on the site of modification and the PEG molecular weight. The site-specific modification of lysostaphin could be useful not only for PEGylation to improve biocompatibility but also for the incorporation of the enzyme into hydrogels and other biomaterials and for studies of protein structure and dynamics. Moreover, the approach described herein could be readily applied to identify suitable sites for the incorporation of reactive handles into other proteins of interest.