Fumitaka Kudo

Cyclopropane-containing amino acids are found in diverse natural products and contribute to their structural complexity and biological activity. However, the distribution of bacterial biosynthetic pathways that produce methylated cyclopropane amino acids and the structural basis of methyl-SAM recognition remain poorly understood. Here, we show that pyridoxal 5′-phosphate (PLP)-dependent 1-amino-2-methylcyclopropane-1-carboxylic acid (2-methyl-ACC) synthases are widely distributed in bacteria. Although Orf30 preferentially converts (R)-4″-methyl-SAM into (1S,2S)-methyl-ACC, some homologues also accept the (S)-4″-methyl-SAM stereoisomer. Structural, computational, and mutational analyses of Orf30, the PLP-dependent enzyme responsible for formation of the 2-methyl-ACC moiety in the natural product Q6402A, suggest that the shape and hydrophobicity of the C4″-methyl-binding region contribute to productive substrate positioning and stereochemical preference. Molecular dynamics simulations, density functional theory calculations, and mutational analyses further support direct cyclopropane formation from a PLP-bound quinonoid intermediate without requiring a substrate intermediate covalently bound to the enzyme at C4″. Together, these findings show how active-site architecture couples substrate stereochemical recognition with quinonoid-driven cyclopropanation, expanding our understanding of ACC synthase-like PLP enzymes.

Nonribosomal peptide synthetases (NRPSs) assemble structurally diverse bioactive natural products through selective communication between adenylation (A) and carrier protein (CP) domains. Although rational rewiring of these protein–protein interactions could enable customized NRPS design, this remains challenging due to the dynamic nature of protein interfaces. Here we show that structure-guided interface reprogramming enables productive non-cognate A– CP pairings across enterobactin, vibriobactin, pyochelin, and vicenistatin biosynthetic systems. Engineered interactions between the A domains EntE, VibE, or PchD and the non-cognate CPs VinL or EntB were validated by biochemical, kinetic, and structural analyses. Reconstituted pathways containing engineered VibE, EntB, and EntF restored enterobactin production and increased yield to 2.2-fold that of the native EntE–EntB–EntF system. Interface-engineered PchD also enhanced production of a non-native salicylic acid–norspermidine conjugate. An X-ray structure and molecular dynamics simulations of an engineered non-cognate A–CP complex reveal recognition principles for programmable NRPS interface design.

Adenylation enzymes transfer acyl substrates selectively onto carrier proteins (CPs) in natural product biosynthesis. Despite the importance of adenylation enzyme–CP interactions, structural information on these transient complexes remains limited. Previously, we developed a pantetheine cross‐linking probe (named C2Br), which contains an ethylenediamine linker with a reactive bromoacetamide group, and determined the structure of the cross–linked complex of the adenylation enzyme HitB with the CP HitD. Here, we investigated the linker‐length effects of pantetheine probes in the cross‐linking reactions of two adenylation enzymes, HitB and EntE, with CPs using probes with different diamine linkers, such as C2Br and C4Br, the latter containing a longer butanediamine linker moiety. Both adenylation enzymes formed cross‐linked complexes with CPs irrespective of the probe used, but the reaction efficiencies depended on the linker length. Crystal structural analysis showed that the HitB–HitD interface interactions in the HitB–C4Br‐HitD complex are essentially identical to those in the HitB–C2Br‐HitD complex. In contrast, the diamine moieties of probes adopt different interaction modes, accounting for the observed variations in cross‐linking efficiencies. A repertoire of pantetheine probes with varying linker lengths will facilitate structural studies on adenylation enzyme–CP interactions by enabling optimization for each adenylation enzyme.

4-Methylazetidinecarboxylic acids (MeAZEs) are unusual nonproteinogenic amino acids found in nonribosomal peptides, such as bonnevillamide D and vioprolide A. The biosynthesis of MeAZEs is thought to proceed from S-adenosyl-l-methionine (SAM) via C-methylation and azetidine ring formation, but the order of these steps has remained unclear. Guided by our previous findings, we proposed that C4″-methylation of SAM by the cobalamin-dependent radical SAM enzyme BnvC, which is encoded in the biosynthetic gene cluster (BGC) of bonnevillamides, precedes azetidine ring formation. In this study, we identified the DUF364-containing enzyme Orf5, which is associated with the bnvC gene in the BGC of bonnevillamides, converts (4″R)-4″-methyl-SAM into cis-MeAZE. Further, we found that VioH, previously shown to catalyze azetidine ring formation from SAM in vioprolide A biosynthesis, transforms (4″S)-4″-methyl-SAM into trans-MeAZE. Structural modeling indicated that the C-methyl group was essential for efficient cyclization. These results elucidated the biosynthetic logic of MeAZEs and established C-methylation as a prerequisite for azetidine ring formation.

C -Methylation by cobalamin-dependent radical SAM methyltransferases functions as a molecular switch that alters substrate properties and drives natural product biosynthesis.

Hitachimycin is a bicyclic macrolactam antibiotic with (S)‐β‐phenylalanine (β‐Phe) at the starter position of the polyketide skeleton. While the enzymes that recognize β‐amino acids, modify the aminoacyl groups, and transfer the resultant dipeptide groups to the acyl carrier protein domains of polyketide synthases (PKSs) have been studied extensively, the post‐PKS modification mechanism responsible for constructing the unique bicyclic structure of hitachimycin remains elusive. In this study, we first inactivated six genes encoding putative post‐PKS modification enzymes, namely hitM1 to hitM6, in Streptomyces scabrisporus to determine their involvement in hitachimycin biosynthesis. The ΔhitM4 strain accumulated an all‐trans‐2,4,6,8,18‐pentaene macrolactam, which was confirmed as a true intermediate in hitachimycin biosynthesis by cellular feeding experiments, and appears to be the initial intermediate in the post‐PKS modification pathway. The ΔhitM1 strain accumulated 10‐O‐demethyl‐10‐oxohitachimycin (M1‐A). In enzymatic experiments, M1‐A was reduced by the NAD(P)H‐dependent reductase HitM1 in the presence of NADPH. The product of the reaction catalyzed by HitM1 was converted to hitachimycin by the methyltransferase HitM6. We thus propose a plausible post‐PKS modification mechanism for the biosynthesis of hitachimycin.

Adenylation enzymes catalyze the selective incorporation of aminoacyl building blocks in the biosynthesis of nonribosomal peptides and related natural products. Although β‐amino acid units are one of the important aminoacyl building blocks in natural product biosynthesis, very little is known about the engineering of β‐amino acid adenylation enzymes. In this study, we engineered the substrate specificity of the (S)‐β‐phenylalanine adenylation enzyme, HitB, involved in the biosynthesis of macrolactam polyketide hitachimycin. Based on the previously determined structure of HitB wild‐type, we mutated Phe328 and Ser293, which are located near the meta and ortho position of the (S)‐β‐phenylalanine moiety, respectively. As a result, the HitB F328V and F328L mutants efficiently activated meta‐substituted (S)‐β‐phenylalanine analogs, and the HitB T293G and T293S mutants efficiently activated ortho‐substituted (S)‐β‐phenylalanine analogs. Structural analysis of the HitB F328L and T293G mutants with the corresponding nonhydrolyzable intermediate analogs revealed an enlarged substrate binding pocket for (S)‐β‐phenylalanine analogs, providing detailed insights into the structural basis for creating enzyme substrate promiscuity. Our findings may be useful for production of various β‐amino acid‐containing natural product analogs.

Macrolactam antibiotics incorporating β-amino acid polyketide starter units, isolated primarily from Actinomycetes species, show significant biological activities. This review provides a detailed analysis into the biosynthetic studies of vicenistatin, a macrolactam antibiotic with a 3-aminoisobutyrate starter unit, as well as biosynthetic research on related macrolactam compounds. Firstly, the elucidation of a common mechanism for the incorporation of β-amino acid starter units into the polyketide synthase (PKS) is described. Secondly, the unique biosynthetic mechanisms of the β-amino acids that are used to supply the main macrolactam biosynthetic pathways with starter units are discussed. Thirdly, some distinctive post-PKS modification mechanisms that complete macrolactam antibiotic biosynthesis are summarized. Finally, future directions for creating new macrolactam compounds through engineered biosynthesis pathways are described.

Adenylation enzymes activate amino acid substrates to aminoacyl adenylates and generally transfer this moiety onto the thiol group of the phosphopantetheine arm of a carrier protein for the selective incorporation of aminoacyl building blocks in natural product biosynthesis. In contrast to the canonical thioester-forming adenylation enzymes, the amide-forming adenylation enzyme VinM transfers an l-alanyl group onto the amino group of the aminoacyl unit attached to the phosphopantetheine arm of the carrier protein VinL to generate dipeptidyl-VinL in vicenistatin biosynthesis. It is unclear how VinM distinguishes aminoacyl-VinL from VinL for amide bond formation. Herein we describe structural and biochemical analyses of VinM. We determined the crystal structure of VinM in complex with VinL using a designed pantetheine-type cross-linking probe. The VinM-VinL complex structure in combination with site-directed mutagenesis analysis revealed that the interactions with both the phosphopantetheine arm and VinL are critical for the amide-forming activity of VinM.

Ketosynthase-like decarboxylase (KS_Q) domains are widely distributed in the loading modules of modular type I polyketide synthases (PKSs) and catalyze the decarboxylation of the (alkyl-)malonyl unit bound to the acyl carrier protein (ACP) in the loading module for the construction of the PKS starter unit. Previously, we performed a structural and functional analysis of the GfsA KS_Q domain involved in the biosynthesis of macrolide antibiotic FD-891. We furthermore revealed the recognition mechanism for the malonic acid thioester moiety of the malonyl-GfsA loading module ACP (ACP_L) as a substrate. However, the exact recognition mechanism for the GfsA ACP_L moiety remains unclear. Here, we present a structural basis for the interactions between the GfsA KS_Q domain and GfsA ACP_L. We determined the crystal structure of the GfsA KS_Q-acyltransferase (AT) didomain in complex with ACP_L (ACP_L=KS_QAT complex) by using a pantetheine crosslinking probe. We identified the key amino acid residues involved in the KS_Q domain-ACP_L interactions and confirmed the importance of these residues by mutational analysis. The binding mode of ACP_L to the GfsA KS_Q domain is similar to that of ACP to the ketosynthase domain in modular type I PKSs. Furthermore, comparing the ACP_L=KS_QAT complex structure with other full-length PKS module structures provides important insights into the overall architectures and conformational dynamics of the type I PKS modules.