Jinping Yang

All Publications

• Electrostatic loading and photoredox-accelerated release of antibiotics from oligoviologen-crosslinked hydrogels using red light MATERIALS TODAY CHEMISTRY Dorsainvil, J. M., Palmquist, M. S., Yang, J., Delawder, A. O., Danielson, M. K., Zhang, Y., Saak, T. M., Gruschka, M. C., Kong, X., Wencewicz, T. A., Barnes, J. C. 2024; 35

  View details for DOI 10.1016/j.mtchem.2023.101847

  View details for Web of Science ID 001142464100001

• Siderophore Synthetase DesD Catalyzes N-to-C Condensation in Desferrioxamine Biosynthesis. ACS chemical biology Yang, J., Banas, V. S., Rivera, G. S., Wencewicz, T. A. 2023; 18 (6): 1266-1270

  Abstract

  Desferrioxamine siderophores are assembled by the nonribosomal-peptide-synthetase-independent siderophore (NIS) synthetase enzyme DesD via ATP-dependent iterative condensation of three N1-hydroxy-N1-succinyl-cadaverine (HSC) units. Current knowledge of NIS enzymology and the desferrioxamine biosynthetic pathway does not account for the existence of most known members of this natural product family, which differ in substitution patterns of the N- and C-termini. The directionality of desferrioxamine biosynthetic assembly, N-to-C versus C-to-N, is a longstanding knowledge gap that is limiting further progress in understanding the origins of natural products in this structural family. Here, we establish the directionality of desferrioxamine biosynthesis using a chemoenzymatic approach with stable isotope incorporation and dimeric substrates. We propose a mechanism where DesD catalyzes the N-to-C condensation of HSC units to establish a unifying biosynthetic paradigm for desferrioxamine natural products in Streptomyces.

  View details for DOI 10.1021/acschembio.3c00167

  View details for PubMedID 37207292

• In Vitro Reconstitution of Fimsbactin Biosynthesis from Acinetobacter baumannii. ACS chemical biology Yang, J., Wencewicz, T. A. 2022; 17 (10): 2923-2935

  Abstract

  Siderophores produced via nonribosomal peptide synthetase (NRPS) pathways serve as critical virulence factors for many pathogenic bacteria. Improved knowledge of siderophore biosynthesis guides the development of inhibitors, vaccines, and other therapeutic strategies. Fimsbactin A is a mixed ligand siderophore derived from human pathogenic Acinetobacter baumannii that contains phenolate-oxazoline, catechol, and hydroxamate metal chelating groups branching from a central l-Ser tetrahedral unit via amide and ester linkages. Fimsbactin A is derived from two molecules of l-Ser, two molecules of 2,3-dihydroxybenzoic acid (DHB), and one molecule of l-Orn and is a product of the fbs biosynthetic operon. Here, we report the complete in vitro reconstitution of fimsbactin A biosynthesis in a cell-free system using purified enzymes. We demonstrate the conversion of l-Orn to N1-acetyl-N1-hydroxy-putrescine (ahPutr) via ordered action of FbsJ (decarboxylase), FbsI (flavin N-monooxygenase), and FbsK (N-acetyltransferase). We achieve conversion of l-Ser, DHB, and l-Orn to fimsbactin A using FbsIJK in combination with the NRPS modules FbsEFGH. We also demonstrate chemoenzymatic conversion of synthetic ahPutr to fimsbactin A using FbsEFGH and establish the substrate selectivity for the NRPS adenylation domains in FbsH (DHB) and FbsF (l-Ser). We assign a role for the type II thioesterase FbsM in producing the shunt metabolite 2-(2,3-dihydroxyphenyl)-4,5-dihydrooxazole-4-carboxylic acid (DHB-oxa) via cleavage of the corresponding thioester intermediate that is tethered to NRPS peptidyl carrier domains during biosynthetic assembly. We propose a mechanism for branching NRPS-derived peptides via amide and ester linkages via the dynamic equilibration of N-DHB-Ser and O-DHB-Ser thioester intermediates via hydrolysis of DHB-oxa thioester intermediates. We also propose a genetic signature for NRPS "branching" in the presence of a terminating C-T-C motif (FbsG).

  View details for DOI 10.1021/acschembio.2c00573

  View details for PubMedID 36122366

• An acyl-adenylate mimic reveals the structural basis for substrate recognition by the iterative siderophore synthetase DesD. The Journal of biological chemistry Yang, J., Banas, V. S., Patel, K. D., Rivera, G. S., Mydy, L. S., Gulick, A. M., Wencewicz, T. A. 2022; 298 (8): 102166

  Abstract

  Siderophores are conditionally essential metabolites used by microbes for environmental iron sequestration. Most Streptomyces strains produce hydroxamate-based desferrioxamine (DFO) siderophores composed of repeating units of N1-hydroxy-cadaverine (or N1-hydroxy-putrescine) and succinate. The DFO biosynthetic operon, desABCD, is highly conserved in Streptomyces; however, expression of desABCD alone does not account for the vast structural diversity within this natural product class. Here, we report the in vitro reconstitution and biochemical characterization of four DesD orthologs from Streptomyces strains that produce unique DFO siderophores. Under in vitro conditions, all four DesD orthologs displayed similar saturation steady-state kinetics (Vmax = 0.9-2.5 μM⋅min-1) and produced the macrocyclic trimer DFOE as the favored product, suggesting a conserved role for DesD in the biosynthesis of DFO siderophores. We further synthesized a structural mimic of N1-hydroxy-N1-succinyl-cadaverine (HSC)-acyl-adenylate, the HSC-acyl sulfamoyl adenosine analog (HSC-AMS), and obtained crystal structures of DesD in the ATP-bound, AMP/PPi-bound, and HSC-AMS/Pi-bound forms. We found HSC-AMS inhibited DesD orthologs (IC50 values = 48-53 μM) leading to accumulation of linear trimeric DFOG and di-HSC at the expense of macrocyclic DFOE. Addition of exogenous PPi enhanced DesD inhibition by HSC-AMS, presumably via stabilization of the DesD-HSC-AMS complex, similar to the proposed mode of adenylate stabilization where PPi remains buried in the active site. In conclusion, our data suggest that acyl-AMS derivatives may have utility as chemical probes and bisubstrate inhibitors to reveal valuable mechanistic and structural insight for this unique family of adenylating enzymes.

  View details for DOI 10.1016/j.jbc.2022.102166

  View details for PubMedID 35750210

  View details for PubMedCentralID PMC9356276

• Multifunctional P450 Monooxygenase CftA Diversifies the Clifednamide Pool through Tandem C-H Bond Activations. Journal of natural products Yang, J., Qi, Y., Blodgett, J. A., Wencewicz, T. A. 2022; 85 (1): 47-55

  Abstract

  Polycyclic tetramate macrolactams (PTMs) are a class of structurally complex hybrid polyketide-nonribosomal peptide (PK-NRP) natural products produced by diverse bacteria. Several PTMs display pharmaceutically interesting bioactivities, and the early stages of PTM biosynthesis involving polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) enzymology are well studied. However, the timing and mechanisms of post PKS-NRPS oxidations by P450 monooxygenases encoded in PTM biosynthetic gene clusters (BGCs) remain poorly characterized. Here we demonstrate that CftA, encoded in clifednamide-type PTM BGCs, is a multifunctional P450 monooxygenase capable of converting the C29-C30 ethyl side chain of ikarugamycin to either a C29-C30 methyl ketone or a C29-C30 hydroxymethyl ketone through C-H bond activation, resulting in the formation of clifednamide A or clifednamide C, respectively. We also report the complete structure of clifednamide C solved via multidimensional NMR (COSY, HSQC, HMBC, NOESY, and TOCSY) using material purified from an engineered Streptomyces strain optimized for production. Finally, the in vitro reconstitution of recombinant CftA catalytic activity revealed the oxidation cascade for sequential conversion of ikarugamycin to clifednamide A and clifednamide C. Our findings confirm prior genetics-based predictions on the origins of clifednamide complexity via P450s encoded in PTM BGCs and place CftA into a growing group of multifunctional P450s that tailor PTM natural products through late-stage regioselective C-H bond activation.

  View details for DOI 10.1021/acs.jnatprod.1c00606

  View details for PubMedID 35086337

• Emergence of Ferrichelatase Activity in a Siderophore-Binding Protein Supports an Iron Shuttle in Bacteria. ACS central science Endicott, N. P., Rivera, G. S., Yang, J., Wencewicz, T. A. 2020; 6 (4): 493-506

  Abstract

  Siderophores are small-molecule high-affinity multidentate chelators selective for ferric iron that are produced by pathogenic microbes to aid in nutrient sequestration and enhance virulence. In Gram-positive bacteria, the currently accepted paradigm in siderophore-mediated iron acquisition is that effluxed metal-free siderophores extract ferric iron from biological sources and the resulting ferric siderophore complex undergoes diffusion-controlled association with a surface-displayed siderophore-binding protein (SBP) followed by ABC permease-mediated translocation across the cell envelope powered by ATP hydrolysis. Here we show that a more efficient paradigm is possible in Gram-positive bacteria where extracellular metal-free siderophores associate directly with apo-SBPs on the cell surface and serve as non-covalent cofactors that enable the holo-SBPs to non-reductively extract ferric iron directly from host metalloproteins with so-called "ferrichelatase" activity. The resulting SBP-bound ferric siderophore complex is ready for import through an associated membrane permease and enzymatic turnover is achieved through cofactor replacement from the readily available pool of extracellular siderophores. This new "iron shuttle" model closes a major knowledge gap in microbial iron acquisition and defines new roles of the siderophore and SBP as cofactor and enzyme, respectively, in addition to the classically accepted roles as a transport substrate and receptor pair. We propose the formal name "siderophore-dependent ferrichelatases" for this new class of catalytic SBPs.

  View details for DOI 10.1021/acscentsci.9b01257

  View details for PubMedID 32341999

  View details for PubMedCentralID PMC7181320