Molecular and Mechanistic Characterization of PddB, the First PLP-Independent 2,4-Diaminobutyric Acid Racemase Discovered in an Actinobacterial D-Amino Acid Homopolymer Biosynthesis

We recently disclosed that the biosynthesis of antiviral γ-poly-D-2,4-diaminobutyric acid (poly-D-Dab) in Streptoalloteichus hindustanus involves an unprecedented cofactor independent stereoinversion of Dab catalyzed by PddB, which shows weak homology to diaminopimelate epimerase (DapF). Enzymological properties and mechanistic details of this enzyme, however, had remained to be elucidated. Here, through a series of biochemical characterizations, structural modeling, and site-directed mutageneses, we fully illustrate the first Dab-specific PLP-independent racemase PddB and further provide an insight into its evolution. The activity of the recombinant PddB was shown to be optimal around pH 8.5, and its other fundamental properties resembled those of typical PLP-independent racemases/epimerases. The enzyme catalyzed Dab specific stereoinversion with a calculated equilibrium constant of nearly unity, demonstrating that the reaction catalyzed by PddB is indeed racemization. Its activity was inhibited upon incubation with sulfhydryl reagents, and the site-directed substitution of two putative catalytic Cys residues led to the abolishment of the activity. These observations provided critical evidence that PddB employs the thiolate-thiol pair to catalyze interconversion of Dab isomers. Despite the low levels of sequence similarity, a phylogenetic analysis of PddB indicated its particular relevance to DapF among PLP-independent racemases/epimerases. Secondary structure prediction and 3D structural modeling of PddB revealed its remarkable conformational analogy to DapF, which in turn allowed us to predict amino acid residues potentially responsible for the discrimination of structural difference between diaminopimelate and its specific substrate, Dab. Further, PddB homologs which seemed to be narrowly distributed only in actinobacterial kingdom were constantly encoded adjacent to the putative poly-D-Dab synthetase gene. These observations strongly suggested that PddB could have evolved from the primary metabolic DapF in order to organize the biosynthesis pathway for the particular secondary metabolite, poly-D-Dab. The present study is on the first molecular characterization of PLP-independent Dab racemase and provides insights that could contribute to further discovery of unprecedented PLP-independent racemases

Substrate specificity of tRNA-dependent amide bond-forming enzyme

Streptothricins (STs) produced by Streptomyces strains are broad-spectrum antibiotics and are characterized by a streptothrisamine core structure with the L-β-lysine (β-Lys) residue and its oligomeric side chains [oligo(β-Lys)]. In addition to the STs, Streptomyces strains has been known to produce ST-related compounds, BD-12, citromycin, glycinothricin, etc., which possess a glycine-derived side chain rather than the β-Lys residue. We have reported that the amide bonds connecting the amino-acid side chains in ST and BD-12 are formed via NRPS (1) and tRNA-dependent (2) pathways, respectively (Figure 1). Please click Additional Files below to see the full abstract

In vitro characterization of MitE and MitB: Formation of N-acetylglucosaminyl-3-amino-5-hydroxybenzoyl-MmcB as a key intermediate in the biosynthesis of antitumor antibiotic mitomycins

Mitomycins, produced by several Streptomyces strains, are potent anticancer antibiotics that comprise an aziridine ring fused to a tricyclic mitosane core. Mitomycins have remarkable ability to crosslink DNA with high efficiency. Despite long clinical history of mitomycin C, the biosynthesis of mitomycins, especially mitosane core formation, remains unknown. Here, we report in vitro characterization of three proteins, MmcB (acyl carrier protein), MitE (acyl AMP ligase), and MitB (glycosyltransferase) involved in mitosane core formation. We show that 3-amino-5-hydroxybenzoic acid (AHBA) is first loaded onto MmcB by MitE at the expense of ATP. MitB then catalyzes glycosylation of AHBA-MmcB with uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) to generate a key intermediate, GlcNAc-AHBA-MmcB, which contains all carbon and nitrogen atoms of the mitosane core. These results provide important insight into mitomycin biosynthesis

Analysis of the Lactobacillus Metabolic Pathway▿ †

We performed analyses of the phenotypic and genotypic relationships focusing on biosyntheses of amino acids, purine/pyrimidines, and cofactors in three Lactobacillus strains. We found that Lactobacillus fermentum IFO 3956 perhaps synthesized para-aminobenzoate (PABA), an intermediate of folic acid biosynthesis, by an alternative pathway

Antimicrobial Activity of ε‑Poly‑l‑lysine after Forming a Water-Insoluble Complex with an Anionic Surfactant

ε-Poly-l-lysine (ε-PL) is one of the few homopoly­(amino-acid)­s occurring in nature. ε-PL, which possesses multiple amino groups, is highly soluble in water, where it forms the antimicrobial polycationic chain (PL^<i>n</i>+). Although the high water-solubility is advantageous for the use of ε-PL as a food preservative, it has limited the applicability of ε-PL as a biopolymer plastic. Here, we report on the preparation and availability of a water-insoluble complex formed with PL^<i>n</i>+ and an anionic surfactant, bis­(2-ethylhexyl) sulfosuccinate (BEHS^–, is also commercialized as AOT) anion. The PLⁿ⁺/BEHS^–-complex, which is soluble in organic solvents, was successfully used as a coating material for a cellulose acetate membrane to create a water-resistant antimicrobial membrane. In addition, the thermoplastic PLⁿ⁺/BEHS^–-complex was able to be uniformly mixed with polypropylene by heating, resulting in materials exhibiting antimicrobial activities

Mechanism of ɛ-Poly-l-Lysine Production and Accumulation Revealed by Identification and Analysis of an ɛ-Poly-l-Lysine-Degrading Enzyme ▿

ɛ-Poly-l-lysine (ɛ-PL) is produced by Streptomyces albulus NBRC14147 as a secondary metabolite and can be detected only when the fermentation broth has an acidic pH during the stationary growth phase. Since strain NBRC14147 produces ɛ-PL-degrading enzymes, the original chain length of the ɛ-PL polymer product synthesized by ɛ-PL synthetase (Pls) is unclear. Here, we report on the identification of the gene encoding the ɛ-PL-degrading enzyme (PldII), which plays a central role in ɛ-PL degradation in this strain. A knockout mutant of the pldII gene was found to produce an ɛ-PL of unchanged polymer chain length, demonstrating that the length is not determined by ɛ-PL-degrading enzymes but rather by Pls itself and that the 25 to 35 l-lysine residues of ɛ-PL represent the original chain length of the polymer product synthesized by Pls in vivo. Transcriptional analysis of pls and a kinetic study of Pls further suggested that the Pls catalytic function is regulated by intracellular ATP, high levels of which are required for full enzymatic activity. Furthermore, it was found that acidic pH conditions during ɛ-PL fermentation, rather than the inhibition of the ɛ-PL-degrading enzyme, are necessary for the accumulation of intracellular ATP