Abstract
BackgroundAmino acid adenylation domains (A domains) are critical enzymes that dictate the identity of the amino acid building blocks to be incorporated during nonribosomal peptide (NRP) biosynthesis. NRPs represent a large group of valuable natural products that are widely applied in medicine, agriculture, and biochemical research. Salinispora arenicola CNS-205 is a representative strain of the first discovered obligate marine actinomycete genus, whose genome harbors a large number of cryptic secondary metabolite gene clusters.Methodology/Principal FindingsIn order to investigate cryptic NRP-related metabolites in S. arenicola CNS-205, we cloned and identified the putative gene sare0718 annotated “amino acid adenylation domain”. Firstly, the general features and possible functions of sare0718 were predicted by bioinformatics analysis, which suggested that Sare0718 is a soluble protein with an AMP-binding domain contained in the sequence and its cognate substrate is L-Val. Then, a GST-tagged fusion protein was expressed and purified to further explore the exact adenylation activity of Sare0718 in vitro. By a newly mentioned nonradioactive malachite green colorimetric assay, we found that L-Ala but not L-Val is the actual activated amino acid substrate and the basic kinetic parameters of Sare0718 for it are Km = 0.1164±0.0159 (mM), Vmax = 3.1484±0.1278 (µM/min), kcat = 12.5936±0.5112 (min−1).Conclusions/SignificanceBy revealing the biochemical role of sare0718 gene, we identified an alanine-activating adenylation domain in marine actinomycete Salinispora arenicola CNS-205, which would provide useful information for next isolation and function elucidation of the whole cryptic nonribosomal peptide synthetase (NRPS)-related gene cluster covering Sare0718. And meanwhile, this work also enriched the biochemical data of A domain substrate specificity in newly discovered marine actinomycete NRPS system, which bioinformatics prediction will largely depend on.
Highlights
Actinomycetes are a remarkably prolific source of structurally diverse natural products, including many that possess pharmaceutically relevant biological activities [1]
Among the three essential catalytic domains, A domains are responsible for the selection and activation of cognate substrates; they are critical in dictating the identity of the amino acid building blocks to be incorporated during nonribosomal peptide (NRP) biosynthesis
The composition and structural diversity of NRPs are derived primarily from the building block-activating A domains in each nonribosomal peptide synthetases (NRPSs) module
Summary
Actinomycetes are a remarkably prolific source of structurally diverse natural products, including many that possess pharmaceutically relevant biological activities [1]. Nonribosomal peptides (NRPs), with complicated structures and diverse bioactivities, usually 3–15 amino acids in length, represent a large group of valuable natural products that are widely applied in medicine, agriculture, and biochemical research [6,7]. A minimal elongation module harbors three core catalytic domains — the adenylation (A), peptidyl carrier protein (PCP or T), and condensation (C) domains, necessary for recognition, activation, and covalent binding of a single building block monomer, as well as for peptide-bond formation with the growing chain [14]. Among the three essential catalytic domains, A domains are responsible for the selection and activation of cognate substrates; they are critical in dictating the identity of the amino acid building blocks to be incorporated during nonribosomal peptide (NRP) biosynthesis. Amino acid adenylation domains (A domains) are critical enzymes that dictate the identity of the amino acid building blocks to be incorporated during nonribosomal peptide (NRP) biosynthesis. Salinispora arenicola CNS-205 is a representative strain of the first discovered obligate marine actinomycete genus, whose genome harbors a large number of cryptic secondary metabolite gene clusters
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