Identification of Sare0718 as an alanine-activating adenylation domain in marine actinomycete Salinispora arenicola CNS-205.

Publisher: Public Library of Science Country of Publication: United States NLM ID: 101285081 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1932-6203 (Electronic) Linking ISSN: 19326203 NLM ISO Abbreviation: PLoS One Subsets: MEDLINE

Original Publication: San Francisco, CA : Public Library of Science

Peptide Biosynthesis, Nucleic Acid-Independent*; Actinobacteria/*enzymology ; Actinobacteria/*genetics ; Alanine/*metabolism ; Bacterial Proteins/*metabolism; Actinobacteria/metabolism ; Alanine/genetics ; Bacterial Proteins/genetics ; Bacterial Proteins/isolation & purification ; Cloning, Molecular ; Computational Biology ; Gene Expression ; Genes, Bacterial ; Recombinant Fusion Proteins/genetics ; Recombinant Fusion Proteins/isolation & purification ; Recombinant Fusion Proteins/metabolism ; Substrate Specificity

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.

Nucleic Acids Res. 2012 Jan;40(Database issue):D302-5. (PMID: 22053084)
J Am Chem Soc. 2008 Apr 2;130(13):4507-16. (PMID: 18331040)
Antimicrob Agents Chemother. 2000 May;44(5):1214-22. (PMID: 10770754)
Int J Syst Evol Microbiol. 2005 Sep;55(Pt 5):1759-1766. (PMID: 16166663)
J Mol Biol. 2001 Jan 19;305(3):567-80. (PMID: 11152613)
Methods Enzymol. 1974;29:620-7. (PMID: 4368856)
Chem Biol. 1998 Mar;5(3):155-62. (PMID: 9545426)
J Antibiot (Tokyo). 2005 Jan;58(1):1-26. (PMID: 15813176)
Biochemistry. 2000 Dec 19;39(50):15513-21. (PMID: 11112537)
Biochemistry. 1998 Feb 24;37(8):2648-59. (PMID: 9485415)
Curr Opin Chem Biol. 2001 Apr;5(2):159-64. (PMID: 11282342)
Chembiochem. 2002 Jun 3;3(6):490-504. (PMID: 12325005)
Nat Methods. 2011 Sep 29;8(10):785-6. (PMID: 21959131)
Nucleic Acids Res. 2005 Oct 12;33(18):5799-808. (PMID: 16221976)
Chem Biol. 1999 Aug;6(8):493-505. (PMID: 10421756)
J Biol Chem. 2000 Jul 14;275(28):21754-60. (PMID: 10801869)
J Biol Chem. 2005 Nov 25;280(47):39363-72. (PMID: 16169849)
J Biol Chem. 1998 Dec 4;273(49):32857-63. (PMID: 9830033)
Appl Environ Microbiol. 2007 Feb;73(4):1146-52. (PMID: 17158611)
Structure. 2001 Jan 10;9(1):R3-9. (PMID: 11342140)
Mol Microbiol. 1993 May;8(5):821-31. (PMID: 8355609)
Microbiology (Reading). 2008 Jun;154(Pt 6):1555-1569. (PMID: 18524911)
Anal Biochem. 2005 Aug 15;343(2):341-3. (PMID: 15992757)
Chem Biol. 2000 Aug;7(8):623-42. (PMID: 11048953)
Anal Biochem. 2009 Mar 15;386(2):244-50. (PMID: 19135023)
EMBO J. 1997 Jul 16;16(14):4174-83. (PMID: 9250661)
Methods Enzymol. 2009;458:181-217. (PMID: 19374984)
Chem Rev. 2005 Feb;105(2):715-38. (PMID: 15700962)
Curr Genet. 1994 Aug;26(2):120-5. (PMID: 8001164)
Proc Natl Acad Sci U S A. 2010 Feb 9;107(6):2646-51. (PMID: 20133795)
Curr Opin Microbiol. 2006 Jun;9(3):245-51. (PMID: 16675289)
Curr Opin Microbiol. 2009 Jun;12(3):252-60. (PMID: 19481972)
ISME J. 2009 Oct;3(10):1193-203. (PMID: 19474814)
Methods Mol Biol. 1999;112:531-52. (PMID: 10027275)
Angew Chem Int Ed Engl. 2003 Jan 20;42(3):355-7. (PMID: 12548698)
Nat Chem Biol. 2006 Dec;2(12):666-73. (PMID: 17108984)
Nucleic Acids Res. 2004 Jul 1;32(Web Server issue):W405-13. (PMID: 15215420)
Microbiol Mol Biol Rev. 2006 Mar;70(1):121-46. (PMID: 16524919)
Appl Environ Microbiol. 2002 Oct;68(10):5005-11. (PMID: 12324350)
J Nat Prod. 2003 Jul;66(7):1022-37. (PMID: 12880330)
Chem Biol. 2008 Feb;15(2):118-27. (PMID: 18291316)
Chembiochem. 2006 Feb;7(2):366-76. (PMID: 16408310)

0 (Bacterial Proteins)
0 (Recombinant Fusion Proteins)
OF5P57N2ZX (Alanine)

Date Created: 20120602 Date Completed: 20120927 Latest Revision: 20211021

20250114

PMC3360062

10.1371/journal.pone.0037487

22655051