1887

Abstract

Exogenous addition of copper stimulates cellular differentiation in spp. Several lines of evidence suggested a parallel correlation between the stimulatory effect of copper and phenol-oxidizing enzyme activities in . Here a novel extracytoplasmic phenol oxidase (EpoA) associated with cellular development of this organism was identified and characterized. EpoA activity, examined by an in-gel stain procedure with ,′-dimethyl--phenylenediamine sulfate as a substrate, was repressed by glucose and induced by copper supplied in the medium. The enzyme activity was abolished and markedly reduced in the mutants forA-factor biosynthesis and , respectively, which suggested that the activity of the enzyme depends on those essential regulators for morphogenesis in . EpoA protein was purified to homogeneity and the N-terminal amino acid sequence was determined. A homologous sequence identified in the genomic database of A3(2) was used as a probe to clone the complete gene of . The deduced amino acid sequence of EpoA revealed that the mature protein with a molecular mass of 34 kDa was preceded by a signal peptide consisting of 34 aa, consistent with EpoA being a secreted enzyme. EpoA was predicted to be a laccase-type oxidase by not only the sequence similarity, but its substrate selectivity, oxidizing not tyrosine but dihydroxyphenylalanine (DOPA) to generate melanin pigment. Introduction of on a plasmid partially restored both the EpoA activity and aerial mycelium productivity in an A-factor-deficient mutant. Exogenous supplementation of a substance synthesized by purified EpoA from DOPA stimulated cellular differentiation in and several other species. Ultrafiltration indicated that the molecular mass of the putative stimulant synthesized by EpoA is between 500 and 1000 Da.

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2002-06-01
2019-10-18
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