Cloning and characterization of the biosynthetic gene cluster for kutznerides

Abstract

Kutznerides, actinomycete-derived cyclic depsipetides, consist of six nonproteinogenic residues, including a highly oxygenated tricyclic hexahydropyrroloindole, a chlorinated piperazic acid, 2-(1-methylcyclopropyl)-glycine, a ␤-branched-hydroxy acid, and 3-hydroxy glutamic acid, for which biosynthetic logic has not been elucidated. Herein we describe the biosynthetic gene cluster for the kutzneride family, identified by degenerate primer PCR for halogenating enzymes postulated to be involved in biosyntheses of these unusual monomers. The 56-kb gene cluster encodes a series of six nonribosomal peptide synthetase (NRPS) modules distributed over three proteins and a variety of tailoring enzymes, including both mononuclear nonheme iron and two flavindependent halogenases, and an array of oxygen transfer catalysts. The sequence and organization of NRPS genes support incorporation of the unusual monomer units into the densely functionalized scaffold of kutznerides. Our work provides insight into the formation of this intriguing class of compounds and provides a foundation for elucidating the timing and mechanisms of their biosynthesis.chlorination ͉ halogenases ͉ nonribosomal peptide biosynthesis K utznerides are antifungal and antimicrobial cyclic hexadepsipeptides isolated from the soil actinomycete Kutzneria sp. 744 (1). Structural elucidation of these metabolites revealed nine related compounds composed of five unusual nonproteinogenic amino acids and one hydroxy acid ( Fig. 1) but differing in the extent of substitution and stereochemistry of constituent residues (2). All kutznerides contain 2-(1-methylcyclopropyl)-D-glycine (MecPGly) connected to the ␣-hydroxyl moiety of either (S)-2-hydroxy-3-methylbutyric or (S)-2-hydroxy-3,3-dimethylbutyric acid. The hydroxy acid residue is followed by a piperazic acid moiety, found in four distinct forms: as piperazic acid in kutznerides 1, 3, 5, and 7; as dehydropiperazate in kutznerides 4 and 9; as ␥-chloro-piperazate in kutznerides 2 and 8; and as ␥-hydroxy-dehydropiperazate in kutzneride 6. Furthermore, kutznerides contain O-methyl-L-serine and either the threo or erythro isomer of 3-hydroxy-D-glutamate. Finally, an unusual tricyclic dihalogenated (2S,3aR,8aS)-6,7-dichloro-3a-hydroxy-hexahydropyrrolo[2,3-b]indole-2-carboxylic acid (PIC) is conserved in all structurally characterized kutznerides. The structural subunits of kutznerides suggest unusual enzymatic mechanisms involved in their biosynthesis.Recently, our laboratory has elucidated the enzymatic logic of carbon-chlorine bond formation during the biosynthesis of several chlorinated secondary metabolites. Among these, dichlorination of the pyrrole moiety in the biosynthesis of pyoluteorin (3) and chlorination of tryptophan in rebeccamycin biosynthesis (4) are carried out by the flavin-dependent halogenases PltA and RebH, respectively. The work of van Pee and coworkers (5) on the pyrrolnitrin halogenase PrnA demonstrated the necessity of FADH 2 , chloride, and oxygen for catalytic activity of flavoprotein halogenase...

A free-standing condensation enzyme catalyzing ester bond formation in C-1027 biosynthesis

“…For example, variations to the standard C-A-PCP module were found in the kutzneride (9) and bleomycin NRPSs (10), and iteratively acting domains have been observed for capreomycin (11), saframycin (12), and enterobactin (13) biosynthesis. Moreover, freestanding C, A, or PCP proteins may function in trans to form an NRPS module (8), and such freestanding proteins or didomain proteins have also been identified in biosynthetic machinery for natural products that do not even contain a peptide moiety.…”

Fungal cyclooligomerdepsipeptides: From classical biochemistry to combinatorial biosynthesis

“…90 Interestingly, no similarity is detected between the hydroxycarboxylic acid-activating A 1 domain signatures of the fungal CODSs and the 2-ketocarboxylic acid-activating A domain signatures derived from bacterial depsipeptide NRPSs. 22,57,108,109 4.2.2 Amino acid activating domains (A 2 ). The 10-amino acid specificity codes of the A 2 domains of CODSs show a higher degree of variation (Table 2).…”

“…98,108,109,133 Instead of utilizing preformed 2-hydroxycarboxylic acids as substrates, the respective bacterial NRPS A domains activate and load the corresponding 2-ketocarboxylic acids onto the multienzymes. Here, the ketocarboxylic acyl-thioesters undergo stereospecific reduction in cis by a ketoreductase (KR) domain, yielding the D-or L-hydroxycarboxylic acyl-thioesters ready for condensation.…”

At the Intersection of Chemistry, Biology, and Medicine

“…These included the siderophore assembly lines for yersiniabactin, pyochelin, vibriobactin, www.annualreviews.org • Chemistry, Biology, and Medicineand pseudomonine (44) and the antibiotic assembly lines for vancomycin and teicoplanin (32), the aminocoumarin class of antibiotics (45), the aclacinomycin class of aromatic polyketides and the peptidyl nucleoside antibiotics exemplified by pacidamycins (46,47), the indolecarbazoles rebeccamycin and staurosporine, the antitumor agents epothilones and bleomycins, the immunosuppressant agent FK506, and the antifungal kutznerides (47).…”