About

Craig A. Townsend is the Alsoph H. Corwin Professor of Chemistry at Johns Hopkins University, with joint appointments in the Departments of Biology and Biophysics, and collaborations with the Johns Hopkins School of Medicine. His research interests encompass the chemistry of natural products and their interface with biology and medicine, focusing on stereochemical and mechanistic studies of biosynthetic enzymes, enzyme engineering, chemoenzymatic synthesis, enzymology, and molecular biology related to polyketides and beta-lactam antibiotics. His work includes drug design and clinical applications targeting antibiotic-resistant bacteria, tuberculosis, and malaria, as well as structural studies of biosynthetic enzymes.

Research topics

• Biochemistry
• Biology
• Chemistry
• Stereochemistry
• Combinatorial chemistry
• Computational biology
• Philosophy
• Genetics
• Organic chemistry

Selected publications

• Sequential Reconstruction of Calicheamicin γ ₁ ^I Iodo‐Thiobenzoate by Selective Carrier Protein Trapping Reveals a Flavin‐Dependent Iodinase

  Angewandte Chemie · 2025-06-20

  articleSenior author

  Abstract Flavin‐dependent halogenases (FDHs) play important roles in natural product biosynthesis, particularly chlorinases and brominases. Apart from ubiquitous mammalian iodination in thyroxine biosynthesis, iodinated natural products are extremely rare, and enzymes responsible for iodination are even less well described. A notable exception is calicheamicin γ 1 I , a potent antitumor compound containing an aromatic iodide, for which a halogenase has been proposed to mediate iodine incorporation. Despite predictions regarding the enzymes involved in iodinated aryl ring biosynthesis, experimental evidence remains limited due to challenges in substrate identification and reaction monitoring. In this study, we successfully reconstituted the enzymatic activities required for iodination and all embellishments of the highly substituted benzene ring in calicheamicin γ 1 I . Using intact‐protein mass spectrometry combined with protease cleavage, we demonstrated formation of the orsellinate thioester followed by sequential C‐2 O ‐methylation, C‐5 iodination, C‐3 oxidation, and C‐3 O ‐methylation. This research characterizes the first flavin‐dependent iodinase that acts on a carrier protein‐dependent substrate, identifies the natural substrates of a cytochrome P450 oxygenase and two O ‐methyltransferases, and provides valuable insights for biocatalysis. Additionally, these findings could facilitate the engineering of other polyketide biosynthetic pathways and contribute to optimizing fermentation conditions to generate new calicheamicin derivatives.

  PublisherDOI

• Sequential Reconstruction of Calicheamicin γ ₁ ^I Iodo‐Thiobenzoate by Selective Carrier Protein Trapping Reveals a Flavin‐Dependent Iodinase

  Angewandte Chemie International Edition · 2025-06-20 · 3 citations

  articleOpen accessSenior authorCorresponding

  Abstract Flavin‐dependent halogenases (FDHs) play important roles in natural product biosynthesis, particularly chlorinases and brominases. Apart from ubiquitous mammalian iodination in thyroxine biosynthesis, iodinated natural products are extremely rare, and enzymes responsible for iodination are even less well described. A notable exception is calicheamicin γ 1 I , a potent antitumor compound containing an aromatic iodide, for which a halogenase has been proposed to mediate iodine incorporation. Despite predictions regarding the enzymes involved in iodinated aryl ring biosynthesis, experimental evidence remains limited due to challenges in substrate identification and reaction monitoring. In this study, we successfully reconstituted the enzymatic activities required for iodination and all embellishments of the highly substituted benzene ring in calicheamicin γ 1 I . Using intact‐protein mass spectrometry combined with protease cleavage, we demonstrated formation of the orsellinate thioester followed by sequential C‐2 O ‐methylation, C‐5 iodination, C‐3 oxidation, and C‐3 O ‐methylation. This research characterizes the first flavin‐dependent iodinase that acts on a carrier protein‐dependent substrate, identifies the natural substrates of a cytochrome P450 oxygenase and two O ‐methyltransferases, and provides valuable insights for biocatalysis. Additionally, these findings could facilitate the engineering of other polyketide biosynthetic pathways and contribute to optimizing fermentation conditions to generate new calicheamicin derivatives.

  PublisherOA PDFDOI

• Structural Basis for 3-Amino-3-carboxypropyl Transfer in Nocardicin Biosynthesis

  Journal of the American Chemical Society · 2025-09-08 · 2 citations

  articleOpen access

  S-Adenosyl-l-methionine (SAM) is well-known as a methyl donor for methyltransferases but also functions as a 3-amino-3-carboxypropyl (3-ACP) donor for 3-ACP transferases. NAT is a 3-ACP transferase which accepts β-lactam antibiotic nocardicin G (1) and SAM to produce isonocardicin C. Due to the lack of structural information about this enzyme, its reaction mechanism has not been fully identified. In this study, we report two X-ray crystal structures of NAT, including its apo and complex structure with 1 and SAH. Examination of them identified the structural basis for substrate recognition. Comprehensive approach integrating site-directed mutagenesis, thermal shift assay, MD simulation, and QM/MM calculation revealed that the Cα-amino group of SAM functions as a Brønsted base to enhance the nucleophilicity of the C6′-OH of 1, with the assistance of E143, thereby facilitating SN2 attack on the Cγ of SAM. This study presents structural and computational analysis leading to more precise understanding of 3-ACP transfer.

  PublisherOA PDFDOI

• <scp>l</scp>-2,3-Diaminopropionate Binding Mode of the SulM Adenylation Domain Limits Engineering Monobactam Analogue Biosynthesis with Larger Substrates

  JACS Au · 2025-04-16 · 1 citations

  articleOpen accessSenior author

  )-methyl-Dap adenylate bound. The ligand-bound structures rationalize the inability of SulA3 to incorporate larger substrates. Comparisons with the structures of other diamino acid-activating adenylation domains identify alternate binding modes that may be more suitable for the production of sulfazecin analogues. The impact of these structures on the further engineering of the SulA3 domain and its relation to monobactam synthesis in the recently structurally characterized SulTE are discussed.

  PublisherDOI

• The dabABC operon is a marker of C4-alkylated monobactam biosynthesis and responsible for (2S,3R)-diaminobutyrate production

  iScience · 2024-02-12 · 4 citations

  articleOpen accessSenior author

  )-Dab synthesis depends on an l-threonine kinase (DabA), a β-replacement reaction with l-aspartate (DabB) and an argininosuccinate lyase-like protein (DabC). The growing clinical importance of monobactams to both withstand Ambler Class B metallo-β-lactamases and retain their antibiotic activity make reprogrammed precursor and NRPS synthesis of modified monobactams a feasible and attractive goal.

  PublisherDOI

• The structure of the monobactam-producing thioesterase domain of SulM forms a unique complex with the upstream carrier protein domain

  Journal of Biological Chemistry · 2024-06-20 · 6 citations

  articleOpen access

  Nonribosomal peptide synthetases (NRPSs) are responsible for the production of important biologically active peptides. The large, multidomain NRPSs operate through an assembly line strategy in which the growing peptide is tethered to carrier domains that deliver the intermediates to neighboring catalytic domains. While most NRPS domains catalyze standard chemistry of amino acid activation, peptide bond formation, and product release, some canonical NRPS catalytic domains promote unexpected chemistry. The paradigm monobactam antibiotic sulfazecin is produced through the activity of a terminal thioesterase domain of SulM, which catalyzes an unusual β-lactam-forming reaction in which the nitrogen of the C-terminal N-sulfo-2,3-diaminopropionate residue attacks its thioester tether to release the monobactam product. We have determined the structure of the thioesterase domain as both a free-standing domain and a didomain complex with the upstream holo peptidyl-carrier domain. The position of variant lid helices results in an active site pocket that is quite constrained, a feature that is likely necessary to orient the substrate properly for β-lactam formation. Modeling of a sulfazecin tripeptide into the active site identifies a plausible binding mode identifying potential interactions for the sulfamate and the peptide backbone with Arg2849 and Asn2819, respectively. The overall structure is similar to the β-lactone-forming thioesterase domain that is responsible for similar ring closure in the production of obafluorin. We further use these insights to enable bioinformatic analysis to identify additional, uncharacterized β-lactam-forming biosynthetic gene clusters by genome mining.

  PublisherDOI

• The structure of the monobactam-producing thioesterase domain of SulM forms a unique complex with the upstream carrier protein domain

  bioRxiv (Cold Spring Harbor Laboratory) · 2024-04-06 · 1 citations

  preprintOpen access

  ABSTRACT Nonribosomal peptide synthetases (NRPSs) are responsible for the production of important biologically active peptides. The large, multidomain NRPSs operate through an assembly line strategy in which the growing peptide is tethered to carrier domains that deliver the intermediates to neighboring catalytic domains. While most NRPS domains catalyze standard chemistry of amino acid activation, peptide bond formation and product release, some canonical NRPS catalytic domains promote unexpected chemistry. The paradigm monobactam antibiotic sulfazecin is produced through the activity of a terminal thioesterase domain that catalyzes an unusual β-lactam forming reaction in which the nitrogen of the C-terminal N -sulfo-2,3-diaminopropionate residue attacks its thioester tether to release the β-lactam product. We have determined the structure of the thioesterase domain as both a free-standing domain and a didomain complex with the upstream holo peptidyl-carrier domain. The structure illustrates a constrained active site that orients the substrate properly for β-lactam formation. In this regard, the structure is similar to the β-lactone forming thioesterase domain responsible for the production of obafluorin. Analysis of the structure identifies features that are responsible for this four-membered ring closure and enable bioinformatic analysis to identify additional, uncharacterized β-lactam-forming biosynthetic gene clusters by genome mining.

  PublisherOA PDFDOI

• The dabABC operon is a marker of C4-alkylated monobactam biosynthesis and responsible for (2S,3R)-diaminobutyrate production

  iScience · 2024-04-01

  erratumOpen accessSenior author

  Publisher of over 50 scientific journals across the life, physical, earth, and health sciences, both independently and in partnership with scientific societies including Cell, Neuron, Immunity, Current Biology, AJHG, and the Trends Journals.

  PublisherOA PDFDOI

• CCDC 2207290: Experimental Crystal Structure Determination

  The Cambridge Structural Database · 2023-03-25

  datasetOpen accessSenior author

  An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

  PublisherDOI

• Normal and Aberrant Methyltransferase Activities Give Insights into the Final Steps of Dynemicin A Biosynthesis

  Journal of the American Chemical Society · 2023-06-05 · 3 citations

  articleOpen accessSenior authorCorresponding

  The naturally occurring enediynes are notable for their complex structures, potent DNA cleaving ability, and emerging usefulness in cancer chemotherapy. They can be classified into three distinct structural families, but all are thought to originate from a common linear C15-heptaene. Dynemicin A (DYN) is the paradigm member of anthraquinone-fused enediynes, one of the three main classes and exceptional among them for derivation of both its enediyne and anthraquinone portions from this same early biosynthetic building block. Evidence is growing about how two structurally dissimilar, but biosynthetically related, intermediates combine in two heterodimerization reactions to create a nitrogen-containing C30-coupled product. We report here deletions of two genes that encode biosynthetic proteins that are annotated as S-adenosylmethionine (SAM)-dependent methyltransferases. While one, DynO6, is indeed the required O-methyltransferase implicated long ago in the first studies of DYN biosynthesis, the other, DynA5, functions in an unanticipated manner in the post-heterodimerization events that complete the biosynthesis of DYN. Despite its removal from the genome of Micromonospora chersina, the ΔdynA5 strain retains the ability to synthesize DYN, albeit in reduced titers, accompanied by two unusual co-metabolites. We link the appearance of these unexpected structures to a substantial and contradictory body of other recent experimental data to advance a biogenetic rationale for the downstream steps that lead to the final formation of DYN. A sequence of product-forming transformations that is in line with new and existing experimental results is proposed and supported by a model reaction that also encompasses the formation of the crucial epoxide essential for the activation of DYN for DNA cleavage.

  PublisherOA PDFDOI

Recent grants

• NIH Grant S10RR013823

  NIH · $198k · 2001

• NIH Grant R01AI014937

  NIH · $5.4M · 2004

• NIH Grant R37AI014937

  NIH · $5.2M · 2014

• NIH Grant R56AI014937

  NIH · $383k · 2016

• Iterative Polyketide Synthase Function, Structure and Pathways

  NIH · $12.2M · 1978–2025

Frequent coauthors

• Rongfeng Li

  Nanjing Medical University

  24 shared

• Anna L. Vagstad

  ETH Zurich

  21 shared

• Jason M. Crawford

  Yale University

  17 shared

• Robert Busby

  Incorporated Research Institutions For Seismology

  17 shared

• Gyanu Lamichhane

  Johns Hopkins Medicine

  17 shared

• Michele Gunsior

  15 shared

• Brian O. Bachmann

  Vanderbilt University

  15 shared

• Francis P. Kuhajda

  Johns Hopkins Medicine

  14 shared

Labs

• Townsend LabPI

Awards & honors

• Research Fellow of the A. P. Sloan Foundation
• Camille and Henry Dreyfus Teacher-Scholar
• Maryland Chemist of the Year award
• Arthur C. Cope Scholar Award
• A. I. Scott Medal from the American Chemical Society