Studies on the reactions between human tyrosinase, superoxide anion, hydrogen peroxide and thiols.

Human tyrosinase (5.5 mg) has been purified from a single human melanotic melanoma metastasis (50.5 g). In the presence of dioxygen, L-tyrosine proved to be a very poor substrate for this enzyme with barely detectable activity compared to L-dopa. However, saturating superoxide anion (i.e., greater than 5 x 10(-3) M) enhanced the oxidation rate of L-tyrosine to dopachrome 40-fold. Hydrogen peroxide was shown to be a competitive inhibitor of tyrosinase when L-tyrosine was the substrate. This reversible inhibition is based on a slow pseudocatalase activity for tyrosinase. Monothiols and dithiols inhibit tyrosinase by different mechanisms. Reduced human thioredoxin and 2,3-dithiopropanol are allosteric inhibitors of tyrosinase yielding bis-cysteinate complexes with one of the copper atoms in the enzyme active site. Bis-cysteinate tyrosinase activity is down-regulated to 30% of native enzyme activity in the L-dopa assay; suggesting a true regulatory role for dithiols. Monothiols such as reduced glutathione and beta-mercaptoethanol are much less reactive with tyrosinase although 10(-3) M monothiol totally inhibits enzyme activity. Reduced thioredoxin inhibits tyrosinase 23-fold more than reduced glutathione under the same experimental conditions.     

Expression and characterization of human tyrosinase from a bacterial expression system

To carry out biochemical characterizations of human tyrosinase and to provide an unlimited source of the enzyme for further study, an expression plasmid, pHis-Tyrosinase, which contains the entire coding sequence except the signal sequence of a human tyrosinase was constructed and expressed in Escherichia coli. The expressed enzyme was simply purified by an immobilized metal affinity chromatography. The recombinant enzyme had the same electrophoretic mobility as the native enzyme from human melanoma cell and cross-reacted with the polyclonal antibody raised against the native enzyme. The recombinant enzyme retained its catalytic function with both hydroxylating and oxidative activities. K_m values for l-tyrosine and l-3,4-dihydroxy-phenylalanine of the recombinant enzyme were 0.17 and 0.36 mM, respectively. The activity of the recombinant enzyme was optimal at pH 7.5. Glutathione notably inhibited the enzymatic activity. This work is a further enzymatic characterization of human tyrosinase.     

Folding of the polypeptide chain(s), conformational flexibility and reactivity of the metal active site of hemocyanin and tyrosinase

Summary The comparative accessibility of the active sites of hemocyanin and tyrosinase, two proteins containing a binuclear type-3 copper site, has been investigated. The approaches were: (a) the kinetic study of the reaction of hemocyanin with cyanide in the presence of conformation perturbants; (b) the comparison between the kinetic parameters of the cyanide reaction on hemocyanin and tyrosinase; (c) the study of the efficiency and reaction mechanism of hemocyanin interaction with a typical tyrosinase substrate like catechol. The results indicate that the active site of tyrosinase is much more exposed than that of hemocyanin.     

Purification and properties of fluoroacetate dehalogenase from <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> DSM 8341

The degradation of fluoroacetate by microorganisms has been established for some time, although only a handful of dehalogenases capable of hydrolyzing the stable C–F bond have been studied. Pseudomonas fluorescens DSM 8341 was originally isolated from soil and readily degrades fluoroacetate, thus it was thought that its dehalogenase might have some desirable properties. The enzyme was purified from cell-free extracts and characterised: it is a monomer of 32,500 Da, with a pH optimum of 8 and is stable between pH 4 and 10; its activity is stimulated by some metal ions (Mg²⁺, Mn²⁺ and Fe³⁺), but inhibited by others (Hg²⁺, Ag²⁺). The enzyme is specific for fluoroacetate, and the K _m for this substrate (0.68 mM) is the lowest determined for enzymes of this type that have been investigated to date.     

Expression in <Emphasis Type="Italic">Escherichia coli</Emphasis> of the recombinant <Emphasis Type="Italic">Vibrio anguillarum</Emphasis> metalloprotease and its purification and characterization

The full length empA gene encoding Vibrio anguillarum metalloprotease was amplified by PCR and fused to the expression vector pBAD24. The carboxy-terminal 6xHis-tagged recombinant metalloprotein (rEmpA) was expressed from plasmid pBAD-VAP6his in E. coli TOP10 and purified with affinity chromatography using a Ni-NTA column. SDS-PAGE analysis and Western blotting revealed a molecular mass of the mature rEmpA predicted to be 36 kDa. The optimal temperature and pH for the purified rEmpA were 37°C and 8.0, respectively. The enzyme was stable below 30°C and between pH 5.0 and 8.0, respectively. The results show that Ca²⁺, Na⁺ and Mg²⁺ had an activating effect on the enzyme while Zn²⁺ and Cu²⁺ acted as inhibitors of the enzyme. The purified rEmpA was characterized as a zinc metalloprotease as it was inhibited by zinc- and metal-specific inhibitors, such as 1,10-phenanthroline, EDTA and EGTA. The results indicate that some characteristics of EmpA from marine V. anguillarum had been modified after expression and processing in the engineered E. coli. The purified rEmpA showed degradation activity towards various kinds of proteins, indicating its potential role in pathogenesis.     

Soluble cuticular phenoloxidase in the gypsy moth Lymantria dispar

The soluble enzyme phenoloxidase (tyrosinase) from the larval cuticle of Lymantria dispar has been partially purified using Ultrogel ACA 34, and the activity has been determined using phenolic substrates. The enzyme exhibited more activity toward O-diphenolic substrates and monophenolic substrates. The enzyme is inhibited by diethyl dithiocarbamate, phenylthiourea, and thiourea. The enzyme has been localized in the 7% slab and disc PAGE as an intense band. The enzyme is suggested to be involved in wound healing. © 1992 Wiley-Liss, Inc.     

Repeated-batch fermentation using flocculent hybrid, <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> CHFY0321 for efficient production of bioethanol

A new tyrosinase was isolated from Aeromonas media strain WS and purified to homogeneity. The purified tyrosinase, termed TyrA, had a molecular mass of 58 kDa and an isoelectric point of 4.90. It exhibited optimal monophenol and diphenol oxidase activities under basic conditions (pH > 8.0). TyrA had a relatively higher affinity to diphenol substrate l-dihydroxyphenylalanine (l-dopa) than many other tyrosinases. EDTA or glutathione notably inhibited the enzymatic activities of TyrA, whereas Triton X-100 and SDS activated them. The full-length TyrA gene was cloned, and it encodes a 518 amino acid protein with little similarities to other reported tyrosinases. However, the purified recombinant TyrA expressed in Escherichia coli demonstrated tyrosinase activity. These results suggest that TyrA is the first reported distinct tyrosinase involved in melanin production in the genus Aeromonas.     

The Tyrosinase Gene in Gorillas and the Albinism of ‘Snowflake’

The sequence of the tyrosinase (Tyr) gene coding tracts has been obtained for the gorilla (Gorilla gorilla gorilla). The five exons of the gene were sequenced in three gorillas and in a normally pigmented human. The tyrosinase gene has been found to be a very conserved locus with a very low substitution rate. Some nucleotide and amino acid differences were found between the gorilla and human tyrosinase coding sequences. One of the gorillas included in the study is the only known case of albinism in a gorilla (‘Snowflake’). Mutations of the TYR gene lead to Oculocutaneous Albinism type 1 (OCA1), the most common type of albinism in humans (OMIM accession number 203100). The TYR gene encodes the tyrosinase enzyme (E.C. 1.14.18.1), whose activity was found to be completely lacking in ‘Snowflake’, indicating that a mutation in the Tyr gene is the likely cause of his albinism. Nonetheless, no nucleotide changes were detected that could account for the lack of Tyr product or tyrosinase activity in Snowflake, and explanations of these findings are discussed.     

Inulavosin and its benzo‐derivatives,melanogenesis inhibitors,target the copper loading mechanism to the active site of tyrosinase

Tyrosinase, a melanosomal membrane protein containing copper, is a key enzyme for melanin synthesis in melanocytes. Inulavosin inhibits melanogenesis by enhancing a degradation of tyrosinase in lysosomes. However, the mechanism by which inulavosin redirects tyrosinase to lysosomes is yet unknown. The analyses of structure–activity relationship of inulavosin and its benzo‐derivatives reveal that the hydroxyl and the methyl groups play a critical role in their inhibitory activity. Intriguingly, the docking studies to tyrosinase suggest that the compounds showing inhibitory activity bind through hydrophobic interactions to the cavity of tyrosinase below which the copper‐binding sites are located. This cavity is proposed to be required for the association with a chaperon that assists in copper loading to tyrosinase in Streptomyces antibioticus. Inulavosin and its benzo‐derivatives may compete with the copper chaperon and result in a lysosomal mistargeting of apo‐tyrosinase that has a conformational defect.     

C-terminal processing of tyrosinase is responsible for activation of Pholiota microspora proenzyme

Tyrosinase is expressed as a 67-kDa protein in Pholiota microspora (synonym Pholiota nameko), whereas the same enzyme purified from fruiting bodies of P. microspora is a 42-kDa protein that is cleaved with a C-terminal 25-kDa polypeptide from the 67-kDa protein. To confirm the role of C-terminal processing in enzyme activity, we expressed a recombinant 67-kDa tyrosinase in Escherichia coli cells. To obtain a soluble protein, the recombinant tyrosinase was expressed as a thioredoxin fusion protein with an enterokinase-cleavable site. Enterokinase digestion of the fusion protein produced a recombinant 67-kDa tyrosinase that did not have any catalytic activity. However, chymotrypsin digestion of the fusion protein produced a recombinant 44-kDa tyrosinase that was catalytically active and had a 25-kDa cleaved C-terminal. Kinetic parameters of the 44-kDa tyrosinase were similar to those of the 42-kDa tyrosinase purified from the fruiting bodies. These results suggest that tyrosinase is expressed in P. microspora as a latent 67-kDa proenzyme and is converted to the mature active 42-kDa enzyme by proteolytic processing of the C-terminal.     

Identification of a fibrinolytic enzyme by <Emphasis Type="Italic">Bacillus vallismortis</Emphasis> and its potential as a bacteriolytic enzyme against <Emphasis Type="Italic">Streptococcus mutans</Emphasis>

A potent fibrinolytic enzyme-producing bacterium was isolated from the traditional Korean condiment Chungkook-jang and identified as Bacillus vallismortis Ace02. The extracellular fibrinolytic enzyme was purified with a 18% recovery of activity from supernatant cultures using CM-Sepharose column chromatography and Sephacryl S-200 gel filtration. The specific activity of the purified enzyme was 757 kFU mg⁻¹. Its molecular mass was about 28 kDa and the initial amino acids of the N-terminal sequence were AQSVPYGVSQ. The full amino acid sequence of fibrinolytic enzyme Ace02 corresponded with bacteriolytic enzyme, L27, from Bacillus licheniformis, which has strong lytic activity against Streptococcus mutans, a major causative strain of dental caries. This suggests that the purified enzyme should be used for prevention of dental caries as well as being an effective thrombolytic agent.     

Preparation of Purified Tyrosinase Devoid of Dopachrome Tautomerase From Mammalian Malignant Melanocytes

Although tyrosinase has been considered for a long time the only enzyme involved in mammalian melanosynthesis, it has been shown that mouse melanoma melanosomes contain high levels of dopachrome tautomerase (DCT²), an enzyme catalyzing DC tautomerization to DHICA. At least in B16 mouse melanoma, DCT is present in higher catalytic amounts than tyrosinase. Moreover, it can be anticipated that tyrosinase and DCT should be very difficult to resolve by most conventional biochemical techniques because of the structural similarity between these enzymes, as predicted from the sequence of their corresponding cDNAs. It is shown that the presence of DCT can cause serious artifacts when tyrosinase activity is determined by most of the currently available methods, such as the Dopa oxidase and melanin formation assays. We describe a simple and convenient method for the preparation of tyrosinase devoid of DCT. The method takes advantage of the different thermal stability of both enzymes. Heating of crude melanosomal extracts at 60°C for 1 hr results in a complete denaturation of DCT, while tyrosinase activity is recovered almost quantitatively. The resulting tyrosinase preparation is considerably purified and the electrophoretic, immunologic and kinetic characteristics of the enzyme appear unaltered. Because if its high yield and simplicity, the method can be used for the microscale partial purification of DCT-free tyrosinase from mammalian malignant melanocytes grown in culture.     

The catalytic cycle of tyrosinase: peroxide attack on the phenolate ring followed by O-O bond cleavage

The oxidation of phenols to ortho-quinones, catalyzed by tyrosinase, has been studied using the hybrid DFT method B3LYP. Since no X-ray structure exists for tyrosinase, information from the related enzymes hemocyanin and catechol oxidase were used to set up a chemical model for the calculations. Previous studies have indicated that the direct cleavage of O(2) forming a Cu(2)(III,III) state is energetically very unlikely. The present study therefore followed another mechanism previously suggested. In this mechanism, dioxygen attacks the phenolate ring which is then followed by O[bond]O cleavage. The calculations give a reasonable barrier for the O(2) attack of only 12.3 kcal/mol, provided one of the copper ligands is able to move substantially away from its direct copper coordination. This can be achieved with six histidine ligands even if these ligands are held in their positions by the enzyme, but can also be achieved if one of the coppers only has two histidine ligands and the third ligand is water. The next step of O[bond]O cleavage has a computed barrier of 14.4 kcal/mol, in reasonable agreement with the experimental overall rate for the catalytic cycle. For the other steps of the mechanism, only a preliminary investigation was made, indicating a few problems which require future QM/MM studies.     

Synthesis and theoretical calculations of carbazole substituted chalcone urea derivatives and studies their polyphenol oxidase enzyme activity

Synthesis of carbazole substituted chalcone urea derivatives and their polyphenol oxidase enzyme activity effects on the diphenolase activity of banana tyrosinase were evaluated. Tyrosinase has been purified from banana on an affinity gel comprised of Sepharose 4B-l-tyrosine-p-aminobenzoic acid. The results showed that most of the compounds (3,4,5a,5d–h) inhibited and some of them (5c,5i–l) activated the tyrosinase enzyme activity. The molecular calculations were performed using Gaussian software for the synthesized compounds to explain the experimental results.     

盐水球菌Salinicoccus ventosaetal B2-3-5的代谢产物分析及其抑制酪氨酸酶活性的机制

[背景] 酪氨酸酶是黑色素合成过程中的关键酶,也是引起人体色素障碍性疾病和产生果蔬酶促褐变的主要原因。目前,酪氨酸酶抑制剂的开发已引起广泛关注,但一些酪氨酸酶抑制剂如熊果苷、曲酸等均存在一定的安全隐患。微生物资源丰富且具有许多优点,从微生物中寻找特异性强、高效的酪氨酸酶抑制剂已成为该领域研究的热点。[目的] 通过测定分离自新疆乌鲁木齐达坂城盐湖的盐水球菌Salinicoccus ventosaetal B2-3-5和B6-1-4代谢物提取物对酪氨酸酶活性的影响,比较2株菌发酵过程中代谢物的差异,了解所筛选菌株B2-3-5抑制酪氨酸酶活性的机制。[方法] 以曲酸为阳性对照分别测定B2-3-5和B6-1-4这2个菌株发酵产生的代谢物提取物对蘑菇酪氨酸酶的抑制活性;应用LC-MS代谢组学方法检测2株菌在相同条件下产生的所有代谢物质;采用单变量、多元变量、正交偏最小二乘判别分析（Orthogonal Partial Least Squares-Discrimination Analysis,OPLS-DA）法识别差异代谢物;利用层次聚类分析（Hierarchial Cluster Analysis,HCA）法对识别的差异物进行聚类分析;通过Kyoto Encyclopedia of Genes and Genomes （KEGG）代谢通路对比法分析这些差异代谢物主要参与的代谢途径。[结果] 菌株B2-3-5代谢物提取物对蘑菇酪氨酸酶二酚酶活性的抑制率为67%,其IC₅₀为0.277 mg/mL,同属菌株B6-1-4代谢物提取物则对酪氨酸酶无抑制活性。采用代谢组学的检测方法从2株菌的代谢物中筛选出63个差异代谢物,其中氨基酸类化合物、维生素类化合物和羧酸类化合物的种类及相对含量均是B2-3-5菌株明显高于B6-1-4菌株。通过代谢途径分析发现这些差异代谢物主要参与15个代谢通路,其中维生素B6生物合成通路的影响较为显著。[结论] 推测B2-3-5菌株可能是通过增加一些氨基酸类、维生素类及羧酸类等小分子化合物的含量来抑制酪氨酸酶活性。维生素B6代谢途径的上调也表明菌体细胞可通过产生维生素B6与酪氨酸酶中的必需氨基作用或清除酶催化循环过程中产生的活性氧自由基（reactive oxygen species,ROS）来抑制酪氨酸酶活性。     

Purification and properties of the carbonic anhydrase of Rhodospirillum rubrum

The carbonic anhydrase (EC 4.2.1.1) of Rhodospirillum rubrum has been purified to apparent homogeneity and some of its properties have been determined. The enzyme was cytoplasmic and was found only in photosynthetically grown cells. It had a molecular weight of about 28,000, and was apparently composed of two equal subunits. The amino acid composition was similar to that of other reported carbonic anhydrases except that the R. rubrum enzyme contained no arginine. The isoelectric point of the enzyme was 6.2 and the pH optimum was 7.5. It required Zn(II) for stability and enzymatic activity. The K _m(CO₂) was 80 mM. Typical carbonic anhydrase inhibition patterns were found with the R. rubrum enzyme. Strong acetazolamide and sulfanilamide inhibition confirmed the importance of Zn(II) for enzymatic activity as did the anionic inhibitors iodide, and azide. Other inhibitors indicated that histidine, sulfhydryl, lysine and serine residues were important for enzymatic activity.Abbreviation CA carbonic anhydrase In memory of R. Y. Stanier     

Biochemical characterisation of an allantoate-degrading enzyme from French bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis>): the requirement of phenylhydrazine

In tropical legumes like French bean (Phaseolus vulgaris) or soybean (Glycine max), most of the atmospheric nitrogen fixed in nodules is used for synthesis of the ureides allantoin and allantoic acid, the major long distance transport forms of organic nitrogen in these species. The purpose of this investigation was to characterise the allantoate degradation step in Phaseolus vulgaris. The degradation of allantoin, allantoate and ureidoglycolate was determined “in vivo” using small pieces of chopped seedlings. With allantoate and ureidoglycolate as substrates, the determination of the reaction products required the addition of phenylhydrazine to the assay mixture. The protein associated with the allantoate degradation has been partially purified 22-fold by ultracentrifugation and batch separation with DEAE-Sephacel. This enzyme was specific for allantoate and could not use ureidoglycolate as substrate. The activity was completely dependent on phenylhydrazine, which acts as an activator at low concentrations and decreases the affinity of the enzyme for the substrate at higher concentrations. The optimal pH for the activity of the purified protein was 7.0 and the optimal temperature was 37°C. The activity was completely inhibited by EDTA and only manganese partially restored the activity. The level of activity was lower in extracts obtained from leaves and fruits of French bean grown with nitrate than in plants actively fixing nitrogen and, therefore, relying on ureides as nitrogen supply. This is the first time that an allantoate-degrading activity has been partially purified and characterised from a plant extract. The allosteric regulation of the enzyme suggests a critical role in the regulation of ureide degradation.     

Purification and characterization of laccase from <Emphasis Type="Italic">Pycnoporus sanguineus</Emphasis> and decolorization of an anthraquinone dye by the enzyme

The white rot fungus Pycnoporus sanguineus produced high amount of laccase in the basal liquid medium without induction. Laccase was purified using ultrafiltration, anion-exchange chromatography, and gel filtration. The molecular weight of the purified laccase was estimated as 61.4 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme oxidized typical substrates of laccases including 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonate), 2,6-dimethoxyphenol, and syringaldazine. The optimum pH and temperature for the purified laccase were 3.0 and 65°C, respectively. The enzyme was stable up to 40°C, and high laccase activity was maintained at pH 2.0–5.0. Sodium azide, l-cysteine, and dithiothreitol strongly inhibited the laccase activity. The purified enzyme efficiently decolorized Remazol Brilliant Blue R in the absence of added redox mediators. The high production of P. sanguineus laccase as well as its decolorization ability demonstrated its potential applications in dye decolorization.     

Nucleotides modulate the activity of aspartate racemase of Scapharca broughtonii

The activity of -aspartate racemase purified from Scapharca broughtonii has been found to depend markedly on some nucleotides. Purine nucleoside monophosphates enhanced the enzyme activity, which was, on the contrary, lowered by purine nucleoside triphosphates and not affected by pyrimidine nucleotides. AMP produced the highest increase of seven-fold in the enzyme activity at 6 mM and a half-maximum increase at approximately 3.8 mM. ATP caused a half-maximum decrease in the activity at approximately 1.4 mM and the remaining activity was lower than 7% at saturating ATP concentrations. AMP and ATP both brought about changes in V_max and not in K_m. Analysis of the effect of AMP and ATP suggests that each of them has its own primary binding site, which is different from the substrate-binding site. In view of these effects of the nucleotides, the roles of the racemase and -aspartate in energy metabolism under anoxic conditions are discussed.     

Molecular cloning and characterization of the gene encoding a fibrinolytic enzyme from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Strain A1

A fibrinolytic metalloprotease gene from Bacillus subtilis has been cloned in Escheridria coliXL1-Blue and the bacterial expressed enzyme was purified. The nucleotide sequence of the cloned fibrinolytic enzyme gene revealed a single open reading frame of 1023 bp coding for 341 amino acids (M _r 37708.21 Da). N-terminal amino acid sequencing of the fibrinolytic enzyme excreted from E. coli host cells revealed that the mature fibrinolytic enzyme consists of 288 amino acids (M _r 31391.1 Da). The deduced amino acid sequence showed significant homology with Erwina carotovora neutral metalloprotease and Serratia marcescens minor metalloprotease by 65 and 58% amino acid sequence identity, respectively. The protein showed significant alignments with the conserved domain of catalytic activity and the -helix domain in Bacillus anthracisthermolysis metalloprotease. The biochemical properties of the purified enzyme suggested that the enzyme is a fibrinolytic metalloprotease, which has optimal activity at pH 7.0 and 50 °C.