Article

Investigation of Streptomyces antibioticus tyrosinase reactivity toward chlorophenols

Department of Biology, University of Padova, 35121 Padova, Italy.
Archives of Biochemistry and Biophysics (Impact Factor: 3.04). 09/2010; 505(1):67-74. DOI: 10.1016/j.abb.2010.09.019
Source: PubMed

ABSTRACT Tyrosinase (Ty) is a copper-containing enzyme ubiquitously distributed in nature. In recent years, Ty has attracted interest as a potential detoxifying agent for xenobiotic compounds with phenolic structure. Among these, chlorophenols are particularly relevant pollutants, commonly found in waste waters. The activity of Streptomyces antibioticus tyrosinase toward isomeric monochlorophenols was studied. Tyrosinase oxidizes both 3- and 4-chlorophenol to the same product, 4-chloro-1,2-ortho-quinone, which subsequently undergoes a nucleophilic substitution reaction at the chlorine atom by excess phenol to give the corresponding phenol-quinone adduct. By contrast, 2-chlorophenol is not reactive and acts as a competitive inhibitor. Docking calculations suggest that the substrates point to one of the copper atoms of the dinuclear center (copper B) and appear to interact preferentially with one of the two coordinated oxygen atoms. The approach of the substrate toward the active site is favored by a π-stacking interaction with one of the copper-coordinated histidines (His194) and by a hydrogen bonding interaction with the O1 oxygen. With this study, we provide the first characterization of the early intermediates in the biotechnologically relevant reaction of Ty with chlorophenols. Additionally, combining experimental evidences with molecular modeling simulations, we propose a detailed reaction scheme for Ty-mediated oxidation of monochlorophenols.

0 Followers
 · 
117 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Tyrosinase is a natural enzyme and is often purified to only a low degree and it is involved in a variety of functions which mainly catalyse the o-hydroxylation of monophenols into their corresponding o-diphenols and the oxidation of o-diphenols to o-quinones using molecular oxygen, which then polymerizes to form brown or black pigments. The synthesis of o-diphenols is a potentially valuable catalytic ability and thus tyrosinase has attracted a lot of attention with respect to industrial applications. In environmental technology it is used for the detoxification of phenol-containing wastewaters and contaminated soils, as biosensors for phenol monitoring, and for the production of L-DOPA in pharmaceutical industries, and is also used in cosmetic and food industries as important catalytic enzyme. Melanin pigment synthesized by tyrosinase has found applications for protection against radiation cation exchangers, drug carriers, antioxidants, antiviral agents, or immunogen. The recombinant V. spinosum tryosinase protein can be used to produce tailor-made melanin and other polyphenolic materials using various phenols and catechols as starting materials. This review compiles the recent data on biochemical and molecular properties of microbial tyrosinases, underlining their importance in the industrial use of these enzymes. After that, their most promising applications in pharmaceutical, food processing, and environmental fields are presented.
    05/2014; 2014:854687. DOI:10.1155/2014/854687
  • [Show abstract] [Hide abstract]
    ABSTRACT: The suicide inactivation kinetics of tyrosinase acting on 3-isopropyl-6-methylcatechol, 3-tert-butyl-6-methylcatechol and 3,6-difluorocatechol was studied. All three substrates act as suicide substrates despite the fact that their 3 and 6 positions are occupied, confirming the mechanism proposed in Biochem. J. (2008) 416, 431-440. Although the most active substrate for the suicide inactivation was 3,6-difluorocatechol, its efficiency was much lower than that of the catechol used as reference. Its r value, the number of turnovers made by one mol of enzyme before its inactivation, is the highest described in the bibliography, highlighting the great difference between the catalytic and inactivation constants. A study of the effect of the pH on the enzymatic activity of tyrosinase showed that both 3-isopropyl-6-methylcatechol and 3-tert-butyl-6-methylcatechol behave as typical substrates of tyrosinase, while 3,6-difluorocatechol behaves differently. The remarkable behavior of 3,6-difluorocatechol when reacts with tyrosinase may be due to the fact that its two hydroxyl groups have very low pK values as a result of the strong electron-withdrawing effect of the fluorine atoms in the ortho positions, so that, at pH 7.0, the substrate would be mainly negatively charged. The apparent Michaelis constant shows a minimum value at pH 6.0, but increases at both high and low pH. However, the values of the catalytic constant and maximum apparent inactivation constant do not vary with the pH, so that the r remains practically constant. Under anaerobic conditions, 3,6-difluorocatechol acts as an irreversible inhibitor of the deoxy- and met-tyrosinase forms. (C) 2011 Elsevier B.V. All rights reserved.
    Journal of Molecular Catalysis B Enzymatic 03/2012; 75:11-19. DOI:10.1016/j.molcatb.2011.11.001 · 2.75 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Tyrosinases with different physico-chemical properties have been identified from various bacterial phyla such as Actinobacteria and Proteobacteria and their production is often inducible by environmental stresses. Tyrosinases are enzymes catalysing the oxidation of mono- and di-phenolic compounds to corresponding quinones with the concomitant reduction of molecular oxygen to water. Since the quinone produced can further react non-enzymatically with other nucleophiles, e.g. amino groups, many tyrosinases have a recorded cross-linking activity on proteins. Various bacterial tyrosinases oxidise tyrosine, catechol, l/d-DOPA, caffeic acid and polyphenolic substrates such as catechins. This substrate specificity has been exploited to engineer biosensors able to detect even minimal amounts of different phenolic compounds. The physiological role of tyrosinases in the biosynthesis of melanins has been used for the production of coloured and dyeing agents. Moreover, the cross-linking activity of tyrosinases has found application in food processing and in the functionalisation of materials. Numerous tyrosinases with varying substrate specificities and stability features have been isolated from bacteria and they can constitute valuable alternatives to the well-studied tyrosinase from common mushroom.
    PROCESS BIOCHEMISTRY 12/2012; 47(12):1749–1760. DOI:10.1016/j.procbio.2012.08.018 · 2.52 Impact Factor