Identification of Sare0718 As an Alanine-Activating Adenylation Domain in Marine Actinomycete Salinispora arenicola CNS-205

BACKGROUND: 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. 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 FINDINGS: In 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 K(m) = 0.1164±0.0159 (mM), V(max) = 3.1484±0.1278 (µM/min), k(cat) = 12.5936±0.5112 (min(-1)). CONCLUSIONS/SIGNIFICANCE: By 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

15 predicted A domain loci and corresponding NRPS gene cluster in <i>Salinispora arenicola</i> CNS-205.

*<p>Data from K Penn <i>et al</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037487#pone.0037487-Mincer1" target="_blank">[21]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037487#pone.0037487-Penn1" target="_blank">[22]</a>.</p

Protein homology analysis by sequence alignment.

<p>Protein homology analysis by sequence alignment.</p

Determination of Sare0718 substrate specificity among the 20 proteinogenic amino acids.

<p>(A) Schematic representation of the substrate screening by malachite green-ammonium molybdate colorimetric assay. Control: the reaction system without Sare0718 (compared with the below Ala sample well); Well 1–19: reaction systems with the substrate of Gly, Val, Leu, Ile, Ser, Thr, Cys, Met, Asp, Glu, Arg, Lys, His, Pro, Phe, Trp, Asn, Gln and Tyr, respectively. (B) Relative activities (substrate specificity) of recombinant Sare0718 for 20 proteinogenic amino acids. Individual substrate activities are presented by bars and the highest activity defined as 100%.</p

Selection of optimum incubation time and enzyme concentration for kinetic parameter assay.

<p>To determine the scope of initial reaction velocity, enzymatic reactions (100 µl) containing 0.5 mM alanine, 0.5 mM ATP and different concentrations of purified Sare0718 were performed at 25°C in 96-well plates for various times (2 min, 5 min, 10 min, and 15 min). (A) Enzyme concentration curve: reaction velocity versus concentration of Sare0718. (B) Time course curve: concentration of production PPi versus reaction time. Sare0718 concentrations in curve 1–6 were 0.0625 µM, 0.125 µM, 0.25 µM, 0.375 µM, 0.5 µM and 0.625 µM, respectively.</p

Determination of kinetic parameters of Sare0718 for L-Ala by Michaelis-Menten analysis using nonlinear regression.

<p>Determination of kinetic parameters of Sare0718 for L-Ala by Michaelis-Menten analysis using nonlinear regression.</p