Biosynthesis of Chuangxinmycin Featuring a Deubiquitinase‐like Sulfurtransferase

Abstract

The knowledge on sulfur incorporation mechanism involved in sulfur-containing molecule biosynthesis remains limited. Chuangxinmycin is a sulfur-containing antibiotic with a unique thiopyrano[4,3,2-cd]indole (TPI) skeleton and selective inhibitory activity against bacterial tryptophanyl-tRNA synthetase. Despite the previously reported biosynthetic gene clusters and the recent functional characterization of a P450 enzyme responsible for CÀS bond formation, the enzymatic mechanism for sulfur incorporation remains unknown. Here, we resolve this central biosynthetic problem by in vitro biochemical characterization of the key enzymes and reconstitute the TPI skeleton in a one-pot enzymatic reaction. We reveal that the JAMM/MPN + protein Cxm3 functions as a deubiquitinase-like sulfurtransferase to catalyze a non-classical sulfur-transfer reaction by interacting with the ubiquitinlike sulfur carrier protein Cxm4GG. This finding adds a new mechanism for sulfurtransferase in nature.

Biochemical and Structural Characterization of OvoATh2: A Mononuclear Nonheme Iron Enzyme from Hydrogenimonas thermophila for Ovothiol Biosynthesis

“…For sulfur carrier proteins generating thiocarboxylate intermediates, they terminate with a C -terminal Gly-Gly. Some exceptions to this general structural motif have been reported, in which the C -terminal Gly-Gly motif was released from the carrier protein after proteolysis through the removal of the C -terminal residues by a dedicated protease. , Recently, persulfides and thiocarboxylates have been implicated as key intermediates in the biosynthesis of thioamides, functional groups in sulfur-modified tRNA, and many small molecular natural products . Similar to thiazoline/thiazole in peptide-based natural products, thioamide generation is generally an ATP-dependent process involving either phosphorylation or adenosylation activation and l -cysteine, protein-bound thiocarboxylates, or l -cysteine persulfides have been suggested as sulfur sources, while in many cases, the sulfur sources are unknown …”

Cytochrome P450s in algae: Bioactive natural product biosynthesis and light-driven bioproduction

“…During the biosynthesis of natural products, generally, P450s implement their functions by catalyzing the common monooxygenation ( e.g ., hydroxylation and epoxidation) reactions to decorate the core structure, improve the aqueous solubility of products, and provide chemical handles for further modifications 15 . Intriguingly, in an increasing number of cases, P450s have been found capable of mediating diverse “uncommon” reactions for skeleton construction, including C–X (X = C/N/S) bond formation/scission, ring formation/expansion/contraction and even more vagarious reactions, which dramatically expand the chemical space of natural products 16 , 17 , 18 , 19 , 20 . As a result, the P450-mediated reactions greatly increase the diversity of both the chemical structures and biological activities of natural products.…”

Iterative Methylation Leads to 3-Methylchuangxinmycin Production in Actinoplanes tsinanensis CPCC 200056

“…It is converted to an α-thione derivative by the JAMM/ MPN + protein CxnF functioning as a deubiquitinase-like sulfurtransferase, in which sulfur carrier protein CxnE is also employed. 6 The α-thione derivative is then reduced by NAD(P)H-dependent reductase CxnC to generate (R)-3-(1H-indol-3-yl)-2-mercaptopropanoic acid. This thiol intermediate is cyclized to 3-demethylchuangxinmycin (DCM, Figure 1) bearing the dihydrothiopyrano[4,3,2-cd]indole scaffold by cytochrome P450 CxnD.…”

“…Recently, the biosynthetic gene cluster for CM was identified from A. tsinanensis and the biosynthetic pathway was proposed for CM (Figure S1). − In the pathway, tryptophan is transformed by aminotransferase CxnB to indole-3-pyruvic acid. It is converted to an α-thione derivative by the JAMM/MPN + protein CxnF functioning as a deubiquitinase-like sulfurtransferase, in which sulfur carrier protein CxnE is also employed .…”

“… − In the pathway, tryptophan is transformed by aminotransferase CxnB to indole-3-pyruvic acid. It is converted to an α-thione derivative by the JAMM/MPN + protein CxnF functioning as a deubiquitinase-like sulfurtransferase, in which sulfur carrier protein CxnE is also employed . The α-thione derivative is then reduced by NAD­(P)­H-dependent reductase CxnC to generate ( R )-3-(1 H -indol-3-yl)-2-mercaptopropanoic acid.…”