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Drosophila Ebony: a novel type of nonribosomal peptide synthetase related enzyme with unusually fast peptide bond formation kinetics
Author(s) -
Hartwig Silvia,
Dovengerds Christine,
Herrmann Christian,
Hovemann Bernhard T.
Publication year - 2014
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.13054
Subject(s) - nonribosomal peptide , thioester , peptide , peptide bond , adenylylation , biochemistry , enzyme kinetics , amino acid , chemistry , biogenic amine , biology , stereochemistry , enzyme , biosynthesis , active site , receptor , neurotransmitter
Drosophila Ebony is a β‐alanyl biogenic amine synthetase with proven function in cuticle and in glia of the nervous system. It is closely related to nonribosomal peptide synthetases ( NRPS s), which typically consist of at least an adenylation, a peptidyl carrier protein and a peptide bond forming condensation domain. Besides its role in cuticle formation, Ebony is in most glia of the brain thought to convert biogenic amines to β‐alanyl conjugates. If the metabolization of the neurotransmitter histamine to β‐alanyl histamine requires a fast reaction in visual signal transduction, Ebony must be able to fulfill this requirement. Since NRPS s are in general slowly acting multi‐modular protein machineries, the enigma of how Ebony quickly facilitates this inactivation remains a key question for understanding its role in vision. To quantitatively analyze the reaction kinetics, we used phosphopantetheinylated holo‐Ebony prepared from Baculovirus infected Sf9 cells. Kinetic parameters for the loading reaction, e.g. the formation of β‐alanyl‐Ebony thioester, complied with those of slow NRPS s. In contrast, single‐turnover analysis of the last reaction step, peptide bond formation between pre‐activated β‐alanyl Ebony thioester and histamine, revealed a very rapid conjugation reaction. This biphasic nature of activity identifies Ebony as a novel type of NRPS related molecule that combines a slow amino acid activation phase with a very fast product formation step.