Structures of Bacterial Tyrosinases

Structures of Bacterial Tyrosinases Tyrosinases oxidise phenolic hydroxyl groups of small molecules or large polymeric substrates such as proteins. Tyrosinases catalyse first the ortho-hydroxylation of the phenolic substrate and second its subsequent oxidation to quinone (Figure 1) with the concomitant reduction of oxygen to water. The reaction is chromogenic as the quinones produced can undergo further non-enzymatic polymerisation to form black eu-melanins and, when reacting with thiol groups, brownish pheo-melanins [14]. This process can be inhibited by antioxidants such as ascorbic acid, for example to prevent the browning reaction in food preparations [15]. Tyrosinase activity is generally measured by either determining the consumption of oxygen during the reaction or spectrophotometrically by following the increase of absorbance at 475 nm due to dopachrome formation. The cross-linking activity of tyrosinase on proteins is usually analysed by SDS PAGE, size-exclusion chromatography, UV spectroscopy or mass spectrom etry [16]. The active site of tyrosinases interacts with both the phenolic substrates and the co-substrate oxygen and it alternates among three different oxidation states. When in the oxy state, tyrosinase binds oxygen and is able to catalyse the hydroxylation of monophenols to diphenols, thus changing into the met form. The met form of tyrosinase is responsible for the oxidation of diphenols to quinones and the reaction turns the enzyme into the deoxy form that, upon binding molecular oxygen, returns to the oxy form. The met form is the resting state of the enzyme and it has been calculated that up to 85% of the enzyme is in this state when in solution [17, 18]. The inability of most of the enzymes in an enzyme population to act on monophenols explains why a significant lag phase is detected in the activity when monophenols are the substrate of the reaction. Bacterial tyrosinases have been divided in five types according to the organisation of domains and the possible requirement of a caddie protein for enzyme activity [12]. The necessity of a secondary helper protein (caddie protein) for secretion, correct folding, assembly of the copper atoms and activity of the enzyme is common to tyrosinases of type I, e.g. the enzyme from S. castaneoglobisporus and S. antibioticus [19, 20]. Type II tyrosinases are small, monomeric enzymes containing only the catalytic domain, which do not require additional helper proteins and are possibly secreted. An example is the tyrosinase from B. megaterium [6]. Type III tyrosinases are represented by the enzyme from Verrucomicrobium spinosum. Like the fungal tyrosinases it carries a C-terminal domain whose removal led to about 100-fold higher activity [21]. This supports the theory that the role of the C-terminal extension in plant and fungal tyrosinases is to keep the enzyme in an inactive form inside the ce ll [22-24]. Among the smallest bacterial tyrosinases reported (Type IV) are the ones produced by Streptomyces nigrifaciens (18 kDa) and Bacillus thuringiensis (14 kDa) [25, 26]. However, it is debated whether these proteins are true tyrosinases [12]. Type V tyrosinases include enzymes that do not carry the sequence features of tyrosinases but show features typical of laccase and have only marginal activity on tyrosine. For example, a membrane-bound tyrosinase active on the typical laccase substrate ABTS (NCBI ID: AAF75831.2) has been isolated from Marinomonas mediterranea. A tyrosinase with a classical substrate specificity that is activated by SDS (NCBI ID: AAV49996.1) has also been reported from the same organism [27]. Similar to catechol oxidases and the oxygen carrying haemocyanins, tyrosinases are type-3 copper proteins, containing two copper atoms in the active site. The absorbance spectrum of oxy-tyrosinases has a characteristic maximum in the UV region (330-345 nm). As reported for the structurally similar catechol oxidases, a fluorescence intensity maximum at 330 nm upon excitation at 280 nm is also detected [28, 29]. Copper is essential for the catalytic activity of tyrosinases. The crystal structure of these enzymes has demonstrated the presence of two copper ions in the catalytic core (Table 1). In all tyrosinases of different origins and in the haemocyanins each of the copper ions is coordinated by three histidine residues that are found in a characteristic pattern in the primary structure (Figure 2). In the tyrosinase from Streptomyces glaucescens, for example, the key role of histidines at position 37, 53, 62, 189, 193 and 215 in the coordination of copper, and thus in catalytic activi ty, was confirmed by the decrease of activity upon their substitution with other amino acids [30, 31]. Various additional residues have been identified to have a function in fungal and bacterial tyrosinases, either being essential for or modulating tyrosinase activity. Sequence analysis and various mutagenesis studies have been performed in order to identify the residues necessary for the activity of the enzyme. In tyrosinase sequences from plants and fungi, the N-terminal signal peptide, when present, is followed by a conserved arginine residue that marks the beginning of the central catalytic domain and that forms a pi-cation interaction with a conserved C-terminal Y/FXY tyrosine motif, where X is any amino acid [32]. These residues are conserved also in bacterial tyrosinases (Supplementary file 1). Substitution of the N-terminal conserved arginine (R40) has been reported to abolish the production of tyrosinase from V. spinosum [21]. Two single-amino acid substitutions have been reported to improve the catalytic activity of the tyrosinase from Rhizobium etli CFN42. The independent r eplacement of proline at position 334 and of aspartic acid at position 535 (Supplementary file 1) with a smaller residue such as serine (P334S) or glycine (D535G), respectively, led to a significant enhancement of the catalytic activity and melanin formation [33-35]. In the tyrosinase from B. megaterium, a single substitution of arginine by histidine within the copper B binding region (R209H) has been sufficient for a 1.7-fold improvement of the activity towards tyrosine (monophenolase) and for a 1.5-fold reduction of activity on L-DOPA (diphenolase), whereby the overall protein stability was not affected [36]. The crystal structure of the tyrosinase from B. megaterium showed that this arginine is positioned at the entrance of the active site in a flexible position and plays a role in the docking of the substrate [6]. However, the conservative substitution of the corresponding residue asparagine 190 to glutamine (N190Q) in S. glaucescens tyrosinase abolished the catalytic activity, indicating that this residue was possibly involved in hydrogen bonding at the active site [30]. Moreover, the conservative substitution of the residue aspartic acid 209 (D209E) has been reported to stabilise the oxy-form of the same enzyme [37]. To our knowledge, no study has investigated the role of the oxygen binding motif PYWDW [38] with regards to the affinity for oxygen in tyrosinase. The affinity for the co-substrate oxygen has been evaluated for the tyrosinase from Streptomyces antibioticus that carries the PYWDW motif. It was found that this enzyme had a three-fold lower dissociation constant (kD) for oxygen than the A. bisporus tyrosinase [39, 40] that carries a PFWDW motif, i.e. 16.5 ÃŽ ¼M compared to 46.6 ÃŽ ¼M. The analysis of the characterised bacterial tyrosinases evidenced the presence of functionally active variants of this motif (Supplementary file 1 and 2), e.g. PYWNY in the tyrosinase from M. mediterranea, PFWDW in tyrosinase from R. etli, PYWEW in the tyrosinase from B. megaterium, PYWRF and PYWNW in the tyrosinases from Ralstonia solanacearum. Mutational studies have also addressed the interaction of tyrosinases from streptomycetes and their caddie protein. In S. antibioticus, the two histidine residues at positions 102 and 117 of the caddie protein MelC1 have been found to be crucial for the biosynthesis of active tyrosinase [41]. The available crystal structures of bacterial tyrosinases and their mutant forms have been obtained from Gram-positive S. castaneoglobisporus and B. megaterium (Table 1). While the B. megaterium tyrosinase formed crystals containing only the enzyme, the S. castaneoglobisporus tyrosinase required the presence of a second protein, referred to as caddie protein, to stabilise its structure [4]. Moreover, the structure of the Streptomyces tyrosinase has been solved in different states of oxidation. Aiming at understanding the interaction between tyrosinase and caddie protein, tyrosinase has been crystallised in the presence of mutant forms of the caddie protein (Table 1). Likewise, the fungal tyrosinase from A. bisporus was crystallised as a tetramer in a complex with a second protein, a lectin-like protein [7]. Both intracellular and secreted bacterial tyrosinases have been isolated and characterised. For example, the tyrosinases from Streptomyces nigrifaciens, Bacillus thuringiensis, M. mediterranea, R. solanacearum and Thermomicrobium roseum were isolated from cell biomass and the ones from S. antibioticus, S. glaucescens, S. castaneoglobisporus, Streptomyces albus, B. megaterium, Sinorhizobium meliloti, Aeromonas media, R. etli and V. spinosum were either isolated from the culture medium or predicted to be secreted [19, 21, 25, 26,42-51]. The twin-arginine signal peptide is often found in cofactor-binding oxidoreductases that undergo complete folding in the cytoplasm prior to secretion to the periplasmic or extracellular space. Twin-arginine type signal peptides [52] could be identified in the N-terminal region of tyrosinases from R. solanacearum (34-amino acid long) and V. spinosum (33-amino acid long). A more detailed analysis of the sequence retrieved for the tyrosinase from R. etli a nd the alignment with the other sequences of tyrosinases (Supplementary File 1) suggests the possibility of incorrect open reading frame prediction. The true N-terminal methionine may be M112 (underlined in Supplementary file 1) as it aligns with the initial residue of the tyrosinase from R. solanacearum (number 15 in Supplementary file 1) and is followed by a predicted twin-arginine signal peptide of 31 amino acids [51]. Thus, we suggest that these proteins purified from the cell biomass but carrying a signal peptide for secretion are localised in the periplasm. Tyrosinases, also from bacteria, and their caddie proteins generally lack conserved cysteine residues (for comments see [11, 12]). The paucity of cysteine residues, and thus disulphide bonds, allowed, however, the isolation of tyrosinases with significant thermal stability, e.g. the enzyme from B. megaterium had an optimum temperature of 50Â °C [48]. A single cysteine residue is conserved in proximity of the second histidine residue of the copper A binding motif in the characterised tyrosinases from M. mediterranea, R. solanacearum, S. meliloti, R. etli and V. spinosum (Supplementary file 1). A cysteine residue at this position has been found to be covalently bound to a histidine residue two positions forward in, for example, the fungal tyrosinase from Neurospora crassa [53], the plant catechol oxidase from I. batata [8] and haemocyanins from the snail Helix pomatia [54]. The function of this unusual cysteine-histidine bond is not established, but it could confer structural rigidity to the copper-binding region and affect the redox potential [8]. Replacement of this cysteine residue (C84) with serine abolished the production of the tyrosinase from V. spinosum [21]. Type-3 copper proteins carrying six conserved cysteines (forming three in silico predicted disulphide bonds) and characterised by significant thermal stability have been reported in fungi [26]. No mutagenesis study has addressed a possible improvement of the thermal stability of bacterial tyrosinases by introducing disulphide bonds. However, in silico analysis revealed the possible presence of one disulphide bond in the tyrosinases from R. solanacearum and S. meliloti and two in the enzymes from M. mediterranea and R. etli (Dianna software, http://clavius.bc.edu/~clotelab/DiANNA). The tyrosinase from S. castaneoglobisporus and the one from B. megaterium share approximately 30% sequence similarity with a catechol oxidase from Aspergillus oryzae that showed a melting temperature above 70Â °C and a ha lf-life of 20 hours when incubated at 50Â °C [29]. It should be noted that the tyrosinase from A. media exhibits different sequence features when compared to the other enzymes. The sequence alignment with bacterial tyrosinases shows that none of the typical signature motifs (copper A and B regions, oxygen binding motif and tyrosine motif) are present (see Supplementary file 1). Moreover, this enzyme has a predicted 23-amino acid long signal peptide [50] and shows strong sequence similarity to bacterial periplasmic proteins that are responsible for the uptake of peptides and involved in nutrition and sensing of the environment [55].

War and Memory in Irene Zabytko?s ?Home Soil?, Bruce Weigl?s ?Song of N

Bullets flying through the air right over me, my knees are shaking, and my feet are numb. I see familiar faces all around me dodging the explosives illuminating the air like lightning. Unfortunately, numerous familiar faces seem to disappear into the trenches. I try to run from the noise, but my mind keeps causing me to re-illustrate the painful memories left behind. The three narratives â€Å"Home Soil† by Irene Zabytko, â€Å"Song of Napalm† by Bruce Weigl, and â€Å"Dulce et Decorum Est† by Wilfred Owen all have the same feelings of war and memory, although not everyone experiences the same war. Zabytko, Weigl, and Owen used shifting beats, dramatic descriptions, and intense, painful images, to convince us that the horror of war far outweighs the devoted awareness of those who fantasize war and the memories that support it. In the story â€Å"Home Soil† by Irene Zabytko, the reader is enlightened about a boy who was mentally and emotionally drained from the horrifying experiences of war. The father in the story knows exactly what the boy is going through, but he cannot help him, because everyone encounters his or her own recollection of war. â€Å"When their faces are contorted from sucking the cigarette, there is an unmistakable shadow of vulnerability and fear of living. That gesture and stance are more eloquent than the blood and guts war stories men spew over their beers† (Zabytko 492). The father, as a young man, was forced to reenact some of the same obligations, yet the father has learne...

Human Relations Case Study

Human Relations Human relations can be defined as a study of group behavior for the purpose of improving interpersonal and social relationships in work environment. In order to improve work productivity, achieve successful teamwork and understand the importance of managing people, it is necessary for managers to develop appropriate ways how to do it. Human relations management has become a concern of many companies. To increase work productivity each company must create a way how to motivate their employees, see them as individuals and not machines, and provide them with good working conditions.Since human being is the main key factor of the organization, managers should always be encouraged to seek for new opportunities for the company growth. Human relations movement approach was started and established by Elton Mayo – a Harvard professor who is most famous for the well-known Hawthorne studies experiment at the Hawthorne plant of Western Electric Company in Chicago,USA. Mayo conducted and analyzed the experiment for several years. The intention of the experiment was to investigate the relationship between work conditions and productivity by examining environmental influences at workplace.After a while experiment moved on to the psychological aspects as well. During this research, new approaches to motivation, social relations and employee care were developed in order to increase work performance and productivity. Experiment gave an opportunity to understand human behavior in groups and their needs in work environment. The experiment had four stages- illumination studies, relay assembly test room, interviewing process and bank wiring observation room. For the first stage, there were selected workers from the Hawthorne plant. Workers were separated in groups and placed under the eye of supervisor in separate test rooms.The idea was to research and determine relationship between illumination in workplace and employee work efficiency, by increasing or decr easing lighting in the test rooms. The final results were confusing and everyone thought that the experiment has failed, because despite of the increase or decrease of lighting-productivity has been increased or stayed the same at all times. Researchers tried to figure out different theories, why it has happened. The general conclusion was that illumination is not the most important factor of output. After this stage change in employee’s behavior was discovered, workers felt important and knew that the work erformance will be researched. Researchers decided to call this phenomenon as Hawthorne effect. This unusual effect is still noticeable in nowadays and it has a big role in human relations management. For example, clinical research organizations- when patients are randomized for the trial, organization is subordinating patients for the Hawthorne effect. Patient health is studied for a long period of time and special attention is received from many employees. Clinical trial organizations usually choose two randomization options for patients-giving the actual trial medicine or the placebo medicine, which is not a real treatment.In most of the cases, clinical trial measures can result either in true or false improvement, which is caused by Hawthorne effect. In clinical research industry important factor is not only scientific, but also psychological effect, because without investigating human factors, finding necessary answers would be much harder, if the theory would be based only on scientific and medical impacts. The second stage of the experiment was relay assembly test room. Again there were selected people from the Hawthorn plant and divided in groups.This stage was conducted to investigate importance of working hours, lunch breaks, rest periods, wages etc. There were numerous questions that needed to be answered in order to understand how to manage efficient working day. That is why researchers kept changing rest periods, work weeks, wages, super visory practices etc. This stage had the same effect as the illumination stage- no matter what change researchers introduced; productivity either increased or stayed the same. After this stage the most important conclusion was that material gains are not as important as the social processes at the workplace.Every company must pay attention to employee well being, in order to establish positive working conditions and improve employee motivation scheme. One of the most popular motivation programs in nowadays is the â€Å"Employee of the month† title. Organizations make effort to analyze employee’s accomplishments every month. That is a simple way how to say â€Å"thank you† for the effort that has been made and motivate for even more successful results by granting workers with the special title and, in some cases, even with material bonus. The third stage at the Hawthorne plant was interview process.Over 21,000 of employees were interviewed by the time of three ye ars to find out the reasons for the increased productivity in previous two stages. Questionnaires were about everything what affect employees working conditions. Workers were allowed to talk about anything, but not always the answers were truthful or useful for the experiment. During the interviews Mayo thought that he had spread his research too wide and too far, because many of the answers were too vague. At the end of the interviews, researchers were surprised by the unpredictable results again.Investigators came to revelation that employees started to reveal more of personal facts, by the time of interviewing. Rather than giving direct answers, workers chose to speak about their personal life details and issues. After this stage became clear, that each person has their own individuality and important fact for the employers is to create certain trainings and interview processes-applicable for every employee. Also it is important to create surveys for employees and clients as well , to make sure that the company is moving forward, not backwards.For example, worldwide hotel â€Å"Premier Inn† has created survey called â€Å"Your say† (Premier Inn, 2011), which is a short questionnaire for employees and guests. By asking questions about the guest satisfactory level, â€Å"Premier Inn† can summarize information and create a plan how to avoid from biggest mistakes and also consider guest recommendations. Survey is also for employees-to make sure every single employee is satisfied and after the survey there is possibility to organize meetings and discuss negative or positive outcomes of their responsibilities.Most of the companies use these surveys in nowadays, to strengthen and improve their business. The last stage at the bank wiring room in Chicago was conducted to study behavior of the workers in informal groups, and research the social structure and its importance at the workplace. Overall conclusion for this stage: when humans interact w ith each other for a long period of time, various social structures are being created. Work productivity is affected by social processes at workplace, which is why important factor in every organization is communication.Many companies use informal meetings to strengthen employees, by creating different team building programs, so employees doesn’t feel like that is a compulsory task to attend. Overall, the main characteristics of the Hawthorne experiment theory are still noticeable in nowadays. Motivation, social relations, employee care/needs in work environment and behavior of groups are the basics of the human relations movement. Elton Mayo’s experiment was a major historical event and even though Hawthorne studies received a lot of criticism, it was a huge step in human relations management.While Scientific Management looked at technology and processes, Mayo found that the real key to high productivity lies within the people and â€Å"work units,† or groups, in the organization (Odportal 2011). Human satisfaction will always lead to productivity, which is why communication at workplace is important factor to achieve positive results. Also motivated workers are always more committed to their job-this was one of the most important characteristics in Hawthorne experiment. Without human relations theory, working conditions would be slightly different nowadays.Every person has their own individuality and needs, why it is important for organizations to take care of employees, make their own researches and seek for different options how to keep employees interested and motivated at all times. References 1. A Fast Learner’s Guide to Leadership. The Human Relations Movement. http://www. odportal. com/leadership/fastlearner/humanrelations. html -accessed 01/11/2011 [i. p. 1] 2. Guest Recommend Survey http://www. premierinn. com/en/why/contact_us/guest-recommend-survey. html – accessed 01/11/2011 [i. p. 1]

Gender And Technology Has Become A Important Factor Within...

Gender and technology has become a very important factor within today’s society, as it reflects upon the overlaps between gender, digital technologies and democracy. Technology has presented both genders in provocative ways that tends to lead to sexual harassment and then for political action to end gender based violence. This then becomes essential and fundamental to the working of and towards a healthy democracy. The term Cyberfeminism has been developed throughout the previous years as feminists who are interested in exploiting with technologies, such as the Internet and cyber-space think it is a vehicle of freedom from the dissolution of sex and gender. An example of online digital media that illustrates the case of gender of Cyberfeminism is the online media campaign called â€Å"Free The Nipple.† This essay will firstly describe this cyberfeminism campaign and how it is relevant towards gender and technology. Then it will examine how digital media can be used as a form of resistance against stereotypes and ideological assumptions in relation to the role of women in contemporary society. Thus the difference between men and women become relevant to these media phenomena and is of importance for gender patterns when considering it in relation to the Internet. (Bimber, 3) On December 12th, 2014, was the initial release of â€Å"Free The Nipple† campaign, which is and/or was targeting all kinds of women from around the world to empower them and to be apart of the equality movement.Show MoreRelatedBusiness Comm Final Paper1535 Words   |  7 PagesWilson December 7, 2014 Final Paper Gender â€Å"Ideals† and the Workplace Introduction For several decades now, women have struggled with equality in the workplace. â€Å"Men are told to think like a woman and women are told to act like a man† (Valian, 1998). 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