Biochemical characterisation of a novel Actinobacterial tyrosinase

Abstract

Tyrosinases are ubiquitously distributed melanogenic polyphenol oxidases that have applications in protein cross-linking, polyphenolic wastewater treatment and bioelectrode development. There are four groups of functional bacterial tyrosinases, three of which belong to the tyrosinase protein superfamily. The fourth group of functional bacterial tyrosinases exhibits structural similarities to multicopper oxidases. Tyrosinases are usually distinguished from other polyphenol oxidases (such as laccases) by their monophenolase activity. In this study, a Zambian hot spring isolate, denoted strain 8S1#2, was identified by a partial polyphasic approach as a possibly novel Dietzia species. Strain 8S1#2 forms orange-red colonies on agar plates. In addition, strain 8S1#2 is aerobic, Gram-positive, non-spore forming and non-motile. This thermotolerant haloalkalophile is the first confirmed tyrosinaseactive non-Streptomyces actinobacterium. Tyrosinase expression in strain 8S1#2 was shown to be induced. A multifaceted PCR approach for the amplification of the gene encoding the tyrosinase activity in 881#2 was used. Primers were designed based on a putative multicopper oxidoreductase, laccase gene sequence identified in the recently annotated genome of Dietzia cinnamea P4 (the closest phylogenetic relative of strain 881#2). The amplicon generated with the strain 8S1#2 genomic DNA as a template showed 100 % sequence similarity to the D. cinnamea P4 MPOL gene. The amplicon of the 8S1#2 MPOL gene was cloned and functionally expressed. The 8S1#2 MPOL protein was purified from the insoluble cell fraction and shown to be a 53 kDa homodimer with moderate stability after 3 hr incubation at 60 "C. The optimal tyrosinase activity was observed at pH 7, and the monophenolase-diphenolase ratio was determined as 0.06. The kcat / KM ratio of 8S1#2 MPOL, at 644.1 mM-1s-1, was the highest for tyrosinases using L-tyrosine as a substrate. The kcat / KM ratio for A8TS, a laccase substrate, was 0.9 mM-1s-1• A homology model of the BS1#2 MPOL protein was generated with Neisseria meningitides protein Nmb 0706 chain A (POB code 1 rv9) as the template. Topology analysis of 1 rv9 identified 24 cavities, with the largest cavity selected as a possible active site. 17 constituent residues were selected and correlated with those in BS1#2 MPOL. Of the four copper ligand binding sites (LBS) identified in MPOLs, three were conclusively identified in BS1#2 MPOL protein following a multiple sequence alignment based approach, with two LBSs located in the putative active site. This study showed that the BS1#2 MPOL protein, a member of the OUF152 protein superfamily, exhibited the functional properties of tyrosinases. However, the BS1#2 MPOL protein did not share any significant structural similarities with members of the tyrosinase protein superfamily. These observations provide evidence for tyrosinase functional convergence in strain BS1#2, a member of the suborder Corynebacterineae. Based on this study, MPOLs may be considered as the second structurally dissimilar group of functional bacterial tyrosinases. As such, the overall number of functional bacterial tyrosinase groups may be amended from four to five.