Cloning and Characterization of the Tetrocarcin A Gene Cluster from Micromonospora chalcea NRRL 11289 Reveals a Highly Conserved Strategy for Tetronate Biosynthesis in Spirotetronate Antibiotics

Abstract

Tetrocarcin A (TCA), produced by Micromonospora chalcea NRRL 11289, is a spirotetronate antibiotic with potent antitumor activity and versatile modes of action. In this study, the biosynthetic gene cluster of TCA was cloned and localized to a 108-kb contiguous DNA region. In silico sequence analysis revealed 36 putative genes that constitute this cluster (including 11 for unusual sugar biosynthesis, 13 for aglycone formation, and 4 for glycosylations) and allowed us to propose the biosynthetic pathway of TCA. The formation of D-tetronitrose, L-amicetose, and L-digitoxose may begin with D-glucose-1-phosphate, share early enzymatic steps, and branch into different pathways by competitive actions of specific enzymes. Tetronolide biosynthesis involves the incorporation of a 3-C unit with a polyketide intermediate to form the characteristic spirotetronate moiety and trans-decalin system. Further substitution of tetronolide with five deoxysugars (one being a deoxynitrosugar) was likely due to the activities of four glycosyltransferases. In vitro characterization of the first enzymatic step by utilization of 1,3-biphosphoglycerate as the substrate and in vivo cross-complementation of the bifunctional fused gene tcaD3 (with the functions of chlD3 and chlD4) to ⌬chlD3 and ⌬chlD4 in chlorothricin biosynthesis supported the highly conserved tetronate biosynthetic strategy in the spirotetronate family. Deletion of a large DNA fragment encoding polyketide synthases resulted in a non-TCA-producing strain, providing a clear background for the identification of novel analogs. These findings provide insights into spirotetronate biosynthesis and demonstrate that combinatorial-biosynthesis methods can be applied to the TCA biosynthetic machinery to generate structural diversity.

Probing the catalytic mechanism of a C‐3′‐methyltransferase involved in the biosynthesis of D‐tetronitrose

“…7 The mechanism was proposed under the assumption that the dTDP-sugar substrate binds in the active site with its C-3 0 amino group in the equatorial position as previously suggested. 4,6,9 The structures of the abortive complexes determined in this investigation, however, and the previously described structure of the TcaB9/SAH/dTDP-product complex, show that the stereochemistry around the C-3 0 position is the same for the substrate, the substrate analog, and the product sugar. As a consequence, His 181 and Glu 224 are not in the proper position to abstract the C-3 0 proton.…”

“…2 As shown in Scheme 1, it is composed of three structurally distinct moieties: a polycyclic tetronolide core, a monosaccharide referred to as D-tetronitrose (highlighted in purple), and a tetrasaccharide composed of alternating L-digitoxose and L-amicetose residues. 3,4 Interestingly, the antibacterial activities of the tetrocarcins are directly proportional to the number of deoxysugars decorating their aglycone rings. 5 The biosynthesis of D-tetronitrose requires 10 enzymes, many of which catalyze very intriguing reactions.…”

“…5 The biosynthesis of D-tetronitrose requires 10 enzymes, many of which catalyze very intriguing reactions. 4,6 One example is a C-3 0 -methyltransferase, which is referred to as TcaB9 and is the focus of this investigation. It catalyzes the fifth step in the biosynthetic pathway, namely the transfer of a methyl group from S-adenosyl-L-methionine (SAM) to the C-3 0 position of dTDP-3-amino-2,3,6-trideoxy-4-keto-D-glucose as illustrated in Scheme 2.…”

Antitumor Compounds from Actinomycetes in Deep-sea Water of Toyama Bay

“…This multi-catalytic enzyme system is feasible for combinatorial biosynthesis by genetic engineering of the biosynthetic pathway to produce novel analogues [11]. Biosynthetic genes for tetrocarcin A was characterized and its biosynthetic pathway has been proposed ( [12]; Fig. 17.5), which will open up future prospects of combinatorial biosynthesis of new leads based on the spirotetronate antibiotics.…”

The Diels–Alderase Never Ending Story

“…In this section, we thus focus on the unique spirotetronate scaffold that is proposed to be biosynthesized from the IMDA between a diene moiety and a γ -methylene-acyltetronate (19 to 20), a three-carbon unit derived from glycerol is necessary to attach the linear polyketide chain as shown in the Scheme 21.10. Comparative gene analysis between kijanimicin (kij) [45], chlorothricin (chl) [46], and tetrocarcin A (tca) [47] biosynthetic gene clusters indicates that the tetronate moiety might be biosynthesized by specifically conserved enzymes. A recent study on the tetronate biosynthetic pathway [48] allowed speculation that the tetronate formation is catalyzed by the FkbH homolog KijC, the stand-alone acyl carrier protein (ACP) KijD, the acyltransferase KijE, and the ketoacyl ACP synthase III homolog KijB.…”