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.


RESULTS
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
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.
Hubei Key Laboratory of Genetic Regulation & Integrative Biology, College of Life Science, Central China Normal University, Wuhan, China.
Sisi
S
Xia
Yanlin
Y
Ma
Wei
W
Zhang
Yi
Y
Yang
Shaowen
S
Wu
Minzhe
M
Zhu
Lingfu
L
Deng
Bing
B
Li
Zhonglai
Z
Liu
Chao
C
Qi
D039903:Actinobacteria / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D000409:Alanine / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D019295:Computational Biology; D015870:Gene Expression; D005798:Genes, Bacterial; D046908:Peptide Biosynthesis, Nucleic Acid-Independent; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D000409:Alanine