Histidine Residue 102 and 117 of MELC1 Play Different Roles in the Chaperone Function for Streptomyces Apotyrosinase

doi:10.1006/bbrc.1995.2307

Biochemical and Biophysical Research Communications

Volume 214, Issue 2, 14 September 1995, Pages 447-453

https://doi.org/10.1006/bbrc.1995.2307 Get rights and content

Abstract

MelC1 regulates the copper incorporation and secretion of Streptomyces apotyrosinase (MelC2) via a transient, competent complex formation. His-102 and His-117 of the chaperone-like MelC1 are known to play important roles in this trans-activation activity of MelC1. In this study, we studied the side chain requirement at these two residues for MelC1 function. Substitution of His-117 with polar, charged, or hydrophobic amino acid resulted in complete abolishment of the tyrosinase activity but no effect on its secretion. When similar amino acid substitutions were introduced at His-102, both tile secretion and the enzymatic activity of tyrosinase were blocked to different extents. Furthermore, the tyrosinase activity of the His-117 mutants but not the His-102 mutants could be partially reactivated by in vitro copper reconstitution. Notably, the defects in the MelC1 mutant protein did not impair the formation of the MelC1·MelC2 binary complex, but rather produced an incompetent complex. In summary, our results reveal that His-102 and His-117 of MelC1 play different roles in MelC1 functions. In particular, His-117 of MelC1 plays a pivotal role in regulation of the copper incorporation but not the secretion of apotyrosinase, while His-102 plays a dual role in both the secretion and the activation of apotyrosinase.

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Cited by (9)

Bacterial tyrosinases and their applications
2012, Process Biochemistry
Citation Excerpt :
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).
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.
Bacterial tyrosinases: Old enzymes with new relevance to biotechnology
2012, New Biotechnology
Citation Excerpt :
The dimer consists of an apo tyrosinase bound to a caddy protein (the melC1 gene product). The caddy protein has several functions including being responsible for the secretion (only it has the TAT signal peptide) of the heterodimer complex to the external medium, the taking up of external copper atoms and the subsequent injection of them into the tyrosinase to activate the protein [48,49,65]. The precise mechanism by which this activation occurs is not fully understood, although the recent crystal structure solved by Matoba et al. of the complex has provided some hints [66–68].
Tyrosinases are copper-containing dioxygen activating enzymes found in many species of bacteria and are usually associated with melanin production. These proteins have a strong preference for phenolic and diphenolic substrates and are somewhat limited in their reaction scope, always producing an activated quinone as product. Despite this fact they have potential in several biotechnological applications, including the production of novel mixed melanins, protein cross-linking, phenolic biosensors, production of l-DOPA, phenol and dye removal and biocatalysis. Although most studies have used Streptomyces sp. enzymes, there are several other examples of these proteins that are also of potential interest. For instance a solvent tolerant enzyme has been described, as well as an enzyme with both tyrosinase and laccase activities, enzymes with altered substrate preferences, an enzyme produced as an inactive zymogen as well as examples which do not require auxiliary proteins for copper insertion (unlike the Streptomyces sp. enzymes which do require such a protein). This article will summarise the reports on the biotechnological applications of bacterial tyrosinases as well as the current information available on the different types of this enzyme.
Roles of copper ligands in the activation and secretion of Streptomyces tyrosinase
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The expression of the melanin operon (melC) of Streptomyces antibioticus requires the chaperone-like protein MelC1 for the incorporation of two copper ions (designated as Cu_A and Cu_B) and the secretion of the apotyrosinase (MelC2) via a transient binary complex formation between these two proteins. To investigate whether the copper ligand of tyrosinase is involved in this MelC1·MelC2 binary complex function, six single substitution mutations were introduced into the Cu_A and Cu_B sites. These mutations led to differential effects on the stability, copper content, and export function of binary complexes but a complete abolishment of tyrosinase activity. The defects in the tyrosinase activity in mutants were not because of the impairment of the formation of MelC1·MelC2 complex but rather the failure of MelC2 to be discharged from the copper-activated binary complex. Moreover, the impairments on the discharge of the mutant MelC2 from all the mutant binary complexes appeared to result from the structural changes in their apoforms or copper-activated forms of the complexes, as evidenced by the fluorescence emission and circular dichroism spectral analysis. Therefore, each of six copper ligands in Streptomyces tyrosinase binuclear copper sites plays a pivotal role in the final maturation and the discharge of tyrosinase from the binary complex but has a less significant role in its secretion.
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Regular ArticleHistidine Residue 102 and 117 of MELC1 Play Different Roles in the Chaperone Function for Streptomyces Apotyrosinase

Abstract

Regular Article
Histidine Residue 102 and 117 of MELC1 Play Different Roles in the Chaperone Function for Streptomyces Apotyrosinase