A biosynthetic gene cluster for the 24-membered macrolactam antibiotic incednine was identified from the producer strain, Streptomyces sp. ML694-90F3. Among the putative incednine biosynthetic enzymes, a novel pyridoxal 5'-phosphate (PLP)-dependent β-glutamate-β-decarboxylase, IdnL3, was functionally characterized in vitro by demonstrating its (S)-3-aminobutyrate-forming activity with β-glutamate in the presence of PLP. Because (S)-3-aminobutyrate is known for the direct precursor of incednine, this enzyme supplies the unique β-amino acid starter unit. The identified gene cluster encodes five characteristic β-amino acid carrying enzymes, consisting of a pathway-specific ATP-dependent ligase, a discrete acyl carrier protein (ACP), β-aminoacyl-ACP β-amino group-protecting ATP-dependent ligase, dipeptidyl-ACP:PKS-loading ACP dipeptidyltransferase and a terminal amino acid peptidase, which are completely conserved in β-amino acid-containing macrolactam biosynthetic gene clusters. Overall, a plausible biosynthetic pathway for incednine was proposed.

Incednine is a 24-membered macrolactam antibiotic produced by Streptomyces sp. ML694-90F3. A previous study demonstrated that its unique nitrogen-containing starter unit was derived from L-glutamate. To elucidate the missing link between L-glutamate and the starter unit, deuterium labeled amino acid feeding experiments were conducted. These experiments revealed that 3-[3-(2)H]aminobutyrate and β-[2,2,4,4-(2)H(4)]glutamate were incorporated into the starter moiety. The results indicate that a novel decarboxylation of β-glutamate to give 3-aminobutyrate is involved in incednine biosynthesis.

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.

Edited by M. J. Romao, Universidade Nova de Lisboa, Portugal

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