Biochemical characterization and structural insight into aliphatic β-amino acid adenylation enzymes IdnL1 and CmiS6.
Macrolactam antibiotics such as incednine and cremimycin possess an aliphatic β-amino acid as a starter unit of their polyketide chain. In the biosynthesis of incednine and cremimycin, unique stand-alone adenylation enzymes IdnL1 and CmiS6 select and activate the proper aliphatic β-amino acid as a starter unit. In this study, we describe the enzymatic characterization and the structural basis of substrate specificity of IdnL1 and CmiS6. Functional analysis revealed that IdnL1 and CmiS6 recognize 3-aminobutanoic acid and 3-aminononanoic acid, respectively. We solved the X-ray crystal structures of IdnL1 and CmiS6 to understand the recognition mechanism of these aliphatic β-amino acids. These structures revealed that IdnL1 and CmiS6 share a common recognition motif that interacts with the β-amino group of the substrates. However, the hydrophobic side-chains of the substrates are accommodated differently in the two enzymes. IdnL1 has a bulky Leu220 located close to the terminal methyl group of 3-aminobutanoate of the trapped acyl-adenylate intermediate to construct a shallow substrate-binding pocket. In contrast, CmiS6 possesses Gly220 at the corresponding position to accommodate 3-aminononanoic acid. This structural observation was supported by a mutational study. Thus, the size of amino acid residue at the 220 position is critical for the selection of an aliphatic β-amino acid substrate in these adenylation enzymes. Proteins 2017; 85:1238-1247. © 2017 Wiley Periodicals, Inc.
Department of Chemistry and Materials Science, Tokyo Institute of Technology, O-okayama, Meguro-ku, Tokyo, 152-8551, Japan.
Jolanta
J
Cieślak
Department of Chemistry, Tokyo Institute of Technology, O-okayama, Meguro-ku, Tokyo, 152-8551, Japan.
Akimasa
A
Miyanaga
Department of Chemistry and Materials Science, Tokyo Institute of Technology, O-okayama, Meguro-ku, Tokyo, 152-8551, Japan.
Ryoma
R
Takaku
Department of Chemistry and Materials Science, Tokyo Institute of Technology, O-okayama, Meguro-ku, Tokyo, 152-8551, Japan.
Makoto
M
Takaishi
Department of Chemistry and Materials Science, Tokyo Institute of Technology, O-okayama, Meguro-ku, Tokyo, 152-8551, Japan.
Keita
K
Amagai
Department of Chemistry, Tokyo Institute of Technology, O-okayama, Meguro-ku, Tokyo, 152-8551, Japan.
Fumitaka
F
Kudo
Department of Chemistry and Materials Science, Tokyo Institute of Technology, O-okayama, Meguro-ku, Tokyo, 152-8551, Japan.|Department of Chemistry, Tokyo Institute of Technology, O-okayama, Meguro-ku, Tokyo, 152-8551, Japan.
Tadashi
0000-0002-7830-7104
T
Eguchi
D020816:Amino Acid Motifs; D000599:Amino Acids, Diamino / Q000737:chemistry* / Q000378:metabolism; D000613:Aminobutyrates / Q000737:chemistry* / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000096:biosynthesis; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004187:Disaccharides / Q000096:biosynthesis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D007769:Lactams / Q000378:metabolism; D008958:Models, Molecular; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D054730:Protein Interaction Domains and Motifs; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013302:Streptomyces / Q000737:chemistry* / Q000201:enzymology; D040681:Structural Homology, Protein; D013379:Substrate Specificity
D000599:Amino Acids, Diamino; D000613:Aminobutyrates; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004187:Disaccharides; D007769:Lactams; D011994:Recombinant Proteins; C111761:cremimycin; C527896:incednine; D009713:Nucleotidyltransferases
3-aminobutanoic acid; 3-aminononanoic acid; crystal structure; macrolactam antibiotics; polyketide