Catecholamine-β-alanyl ligase in the medfly Ceratitis capitata

Abstract

Dopamine (DA) and norepinephrine (NE) derivatives play an important role in the sclerotization and pigmentation of insect cuticles by serving as precursors for cuticular cross-linking. Protein preparations from prepupae of the medfly, Ceratitis capitata, were able to conjugate beta-alanine with DA producing N-beta-alanyldopamine (NBAD) or with NE, synthesizing N-beta-alanylnorepinephrine (NBANE). The latter reaction has been demonstrated for the first time. Apparent kinetic parameters were obtained for both substrates, DA (V(max)=30.7+/-6.0 pmol min(-1) mg(-1); K(m)=29.5+/-3.5 microM) and NE (V(max)=16.1+/-6.6 pmol min(-1)mg(-1); K(m)=89.0+/-8.3 microM). The same protein seems to be responsible for both enzymatic activities, judging from several criteria like identical behavior under heat inactivation as well as identical Mg2+ and Mn2+ dependent stimulation and Co2+ inhibition. Furthermore, the melanic mutants niger of C. capitata and ebony(4) of D. melanogaster, known to be defective for NBAD synthase, were also unable to synthesize NBANE. The protein preparation acylated tyrosine with much less efficiency, to produce sarcophagine (beta-alanyltyrosine). Strikingly, extracts from the melanic mutants were unable to synthesize sarcophagine. Our results strongly suggest that the enzymatic activity previously known as NBAD synthase is in fact a novel catalytic protein showing broad substrate specificity. We propose to identify it as catecholamine-beta-alanyl ligase.