Scholars@Duke
Kenichi Yokoyama

Kenichi Yokoyama

Associate Professor of Biochemistry
Biochemistry
ken.yoko@duke.edu
307 Research Drive, 228B Nanaline H Duke., Durham, NC 27710
307 Research Drive, Rm 228B Nanaline H Duke Bldg, Durham, NC 27710

Selected Publications

Mechanism of controlled radical initiation in radical SAM GTP 3',8-cyclase.

Journal Article Proc Natl Acad Sci U S A · November 11, 2025 Metalloenzymes couple substrate binding and formation of oxidative intermediates to minimize unwanted side reactions. However, the molecular details of such coupling frequently remain ambiguous. Radical S-adenosyl-L-methionine (SAM) enzymes constitute one ... Full text Link to item Cite

Azetidine amino acid biosynthesis by non-haem iron-dependent enzymes.

Journal Article Nat Chem · October 21, 2025 Azetidine, a four-membered aza-cycle, is a crucial structure in many bioactive compounds and drugs. However, their biosynthesis is frequently enigmatic. Here we report the mechanism of azetidine amino acid (polyoximic acid) biosynthesis in the polyoxin ant ... Full text Link to item Cite

Unusual O-H Activation-Initiated C-C Bond Cleavage Reaction by a Nonheme Fe Enzyme in Antifungal Nucleoside Biosynthesis.

Journal Article J Am Chem Soc · August 20, 2025 Fe(II)- and α-ketoglutarate (α-KG)-dependent enzymes catalyze diverse reactions, generally initiated by FeIV=O mediated cleavage of C-H bonds with bond dissociation energies (BDE) of up to ∼100 kcal/mol. Here, we report the discovery of a novel reaction in ... Full text Link to item Cite

Radical-Mediated Nucleophilic Peptide Cross-Linking in Dynobactin Biosynthesis.

Journal Article J Am Chem Soc · November 20, 2024 Dynobactins are recently discovered ribosomally synthesized and post-translationally modified peptide (RiPP) antibiotics that selectively kill Gram-negative pathogens by inhibiting the β-barrel assembly machinery (Bam) located on their outer membranes. Suc ... Full text Link to item Cite

Biosynthesis and Genome Mining Potentials of Nucleoside Natural Products.

Journal Article Chembiochem · September 1, 2023 Nucleoside natural products show diverse biological activities and serve as leads for various application purposes, including human and veterinary medicine and agriculture. Studies in the past decade revealed that these nucleosides are biosynthesized throu ... Full text Link to item Cite

Genome- and metabolome-guided discovery of marine BamA inhibitors revealed a dedicated darobactin halogenase.

Journal Article Cell Chem Biol · August 17, 2023 Darobactins represent a class of ribosomally synthesized and post-translationally modified peptide (RiPP) antibiotics featuring a rare bicyclic structure. They target the Bam-complex of Gram-negative bacteria and exhibit in vivo activity against drug-resis ... Full text Link to item Cite

Characterization of a Radical SAM Oxygenase for the Ether Crosslinking in Darobactin Biosynthesis.

Journal Article J Am Chem Soc · October 19, 2022 Darobactin A is a ribosomally synthesized, post-translationally modified peptide (RiPP) with potent and broad-spectrum anti-Gram-negative antibiotic activity. The structure of darobactin A is characterized by an ether and C-C crosslinking. However, the spe ... Full text Link to item Cite

Structural basis for inhibition and regulation of a chitin synthase from Candida albicans.

Journal Article Nat Struct Mol Biol · July 2022 Chitin is an essential component of the fungal cell wall. Chitin synthases (Chss) catalyze chitin formation and translocation across the membrane and are targets of antifungal agents, including nikkomycin Z and polyoxin D. Lack of structural insights into ... Full text Link to item Cite

Resolving the Multidecade-Long Mystery in MoaA Radical SAM Enzyme Reveals New Opportunities to Tackle Human Health Problems.

Journal Article ACS Bio Med Chem Au · April 20, 2022 MoaA is one of the most conserved radical S-adenosyl-l-methionine (SAM) enzymes, and is found in most organisms in all three kingdoms of life. MoaA contributes to the biosynthesis of molybdenum cofactor (Moco), a redox enzyme cofactor used in various enzym ... Full text Link to item Cite

Cryptic Phosphorylation-Mediated Divergent Biosynthesis of High-Carbon Sugar Nucleoside Antifungals.

Journal Article ACS Chem Biol · April 15, 2022 Establishing a general biosynthetic scheme for natural products is critical for a broader understanding of natural product biosynthesis and the structural prediction of metabolites based on genome sequence information. High-carbon sugar nucleoside antimicr ... Full text Link to item Cite

Selenocysteine substitutions in thiyl radical enzymes.

Chapter · 2022 Cysteine thiyl radicals are implicated as cofactors in a variety of enzymatic transformations, as well as transient byproducts of oxidative stress, yet their reactivity has undermined their detailed study. Selenocysteine exhibits a lower corresponding sele ... Full text Link to item Cite

Addendum: Cryptic phosphorylation in nucleoside natural product biosynthesis.

Journal Article Nat Chem Biol · October 2021 A Correction to this paper has been published: https://doi.org/10.1038/s41589-021-00867-7. ... Full text Link to item Cite

Mechanism of Reduction of an Aminyl Radical Intermediate in the Radical SAM GTP 3',8-Cyclase MoaA.

Journal Article J Am Chem Soc · September 1, 2021 The diversity of the reactions catalyzed by radical S-adenosyl-l-methionine (SAM) enzymes is achieved at least in part through the variety of mechanisms to quench their radical intermediates. In the SPASM-twitch family, the largest family of radical SAM en ... Full text Link to item Cite

Conserved Mechanism of 2'-Phosphorylation-Aided Amide Ligation in Peptidyl Nucleoside Biosynthesis.

Journal Article Biochemistry · July 20, 2021 Peptidyl nucleoside antifungals, represented by nikkomycins and polyoxins, consist of an unusual six-carbon nucleoside [aminohexuronic acid (AHA)] ligated to a nonproteinogenic amino acid via an amide bond. A recent study suggested that AHA is biosynthesiz ... Full text Link to item Cite

Quantitative Characterization of the Amount and Length of (1,3)-β-d-glucan for Functional and Mechanistic Analysis of Fungal (1,3)-β-d-glucan Synthase.

Journal Article Bio Protoc · April 20, 2021 (1,3)-β-d-Glucan synthase (GS) is an essential enzyme for fungal cell wall biosynthesis that catalyzes the synthesis of (1,3)-β-d-glucan, a major and vital component of the cell wall. GS is a proven target of antifungal antibiotics including FDA-approved e ... Full text Link to item Cite

Cryptic phosphorylation in nucleoside natural product biosynthesis.

Journal Article Nat Chem Biol · February 2021 Kinases are annotated in many nucleoside biosynthetic gene clusters but generally are considered responsible only for self-resistance. Here, we report an unexpected 2'-phosphorylation of nucleoside biosynthetic intermediates in the nikkomycin and polyoxin ... Full text Link to item Cite

Mechanism of Rate Acceleration of Radical C-C Bond Formation Reaction by a Radical SAM GTP 3',8-Cyclase.

Journal Article J Am Chem Soc · May 20, 2020 While the number of characterized radical S-adenosyl-l-methionine (SAM) enzymes is increasing, the roles of these enzymes in radical catalysis remain largely ambiguous. In radical SAM enzymes, the slow radical initiation step kinetically masks the subseque ... Full text Link to item Cite

Length Specificity and Polymerization Mechanism of (1,3)-β-d-Glucan Synthase in Fungal Cell Wall Biosynthesis.

Journal Article Biochemistry · February 11, 2020 (1,3)-β-d-Glucan synthase (GS) catalyzes formation of the linear (1,3)-β-d-glucan in the fungal cell wall and is a target of clinically approved antifungal antibiotics. The catalytic subunit of GS, FKS protein, does not exhibit significant sequence homolog ... Full text Link to item Cite

Radical-mediated C-C bond formation in natural product and cofactor biosynthesis

Conference ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY · August 25, 2019 Link to item Cite

The Elusive 5'-Deoxyadenosyl Radical: Captured and Characterized by Electron Paramagnetic Resonance and Electron Nuclear Double Resonance Spectroscopies.

Journal Article J Am Chem Soc · July 31, 2019 The 5'-deoxyadenosyl radical (5'-dAdo·) abstracts a substrate H atom as the first step in radical-based transformations catalyzed by adenosylcobalamin-dependent and radical S-adenosyl-l-methionine (RS) enzymes. Notwithstanding its central biological role, ... Full text Link to item Cite

Characterization of Acyl Carrier Protein-Dependent Glycosyltransferase in Mitomycin C Biosynthesis.

Journal Article Biochemistry · June 25, 2019 Mitomycins make up a group of antitumor natural products that are biosynthesized from aminohydroxybenzoic acid (AHBA) and N-acetylglucosamine (GlcNAc). While the biosynthetic gene cluster was reported two decades ago, the mechanism by which the two buildin ... Full text Link to item Cite

The Elusive 5′-Deoxyadenosyl Radical: Captured and Characterized by EPR and ENDOR Spectroscopies

Journal Article · June 10, 2019 The 5'-deoxyadenosyl radical (5'-dAdo•) abstracts a substrate H-atom as the first step in radical-based transformations catalyzed by adenosylcobalamin-dependent and radical S-adenosyl-l-methionine (RS) enzymes. Notwithstanding its central biological ... Full text Cite

Insights into the universal radical SAM organometallic intermediate, Omega

Conference ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY · March 31, 2019 Link to item Cite

Enfumafungin synthase represents a novel lineage of fungal triterpene cyclases.

Journal Article Environ Microbiol · September 2018 Enfumafungin is a glycosylated fernene-type triterpenoid produced by the fungus Hormonema carpetanum. Its potent antifungal activity, mediated by its interaction with β-1,3-glucan synthase and the fungal cell wall, has led to its development into the semi- ... Full text Link to item Cite

C-C bond forming radical SAM enzymes involved in the construction of carbon skeletons of cofactors and natural products.

Journal Article Nat Prod Rep · July 18, 2018 Covering: up to the end of 2017 C-C bond formations are frequently the key steps in cofactor and natural product biosynthesis. Historically, C-C bond formations were thought to proceed by two electron mechanisms, represented by Claisen condensation in fatt ... Full text Link to item Cite

Paradigm Shift for Radical S-Adenosyl-l-methionine Reactions: The Organometallic Intermediate Ω Is Central to Catalysis.

Journal Article J Am Chem Soc · July 18, 2018 Radical S-adenosyl-l-methionine (SAM) enzymes comprise a vast superfamily catalyzing diverse reactions essential to all life through homolytic SAM cleavage to liberate the highly reactive 5'-deoxyadenosyl radical (5'-dAdo·). Our recent observation of a cat ... Full text Link to item Cite

Radical Breakthroughs in Natural Product and Cofactor Biosynthesis.

Journal Article Biochemistry · January 30, 2018 The radical SAM (S-adenosyl-l-methionine) superfamily is one of the largest group of enzymes with >113000 annotated sequences [Landgraf, B. J., et al. (2016) Annu. Rev. Biochem. 85, 485-514]. Members of this superfamily catalyze the reductive cleavage of S ... Full text Link to item Cite

Lessons From the Studies of a CC Bond Forming Radical SAM Enzyme in Molybdenum Cofactor Biosynthesis.

Journal Article Methods Enzymol · 2018 MoaA is one of the founding members of the radical S-adenosyl-L-methionine (SAM) superfamily, and together with the second enzyme, MoaC, catalyzes the construction of the pyranopterin backbone structure of the molybdenum cofactor (Moco). However, the exact ... Full text Link to item Cite

Glutamate 350 Plays an Essential Role in Conformational Gating of Long-Range Radical Transport in Escherichia coli Class Ia Ribonucleotide Reductase.

Journal Article Biochemistry · February 14, 2017 Escherichia coli class Ia ribonucleotide reductase (RNR) is composed of two subunits that form an active α2β2 complex. The nucleoside diphosphate substrates (NDP) are reduced in α2, 35 Å from the essential diferric-tyrosyl radical (Y122•) cofactor in β2. T ... Full text Link to item Cite

Carbon extension in peptidylnucleoside biosynthesis by radical SAM enzymes.

Journal Article Nat Chem Biol · November 2016 Nikkomycins and polyoxins are antifungal peptidylnucleoside antibiotics active against human and plant pathogens. Here we report that during peptidylnucleoside biosynthesis in Streptomyces cacaoi and S. tendae, the C5' extension of the nucleoside essential ... Full text Link to item Cite

Engineering of New Pneumocandin Side-Chain Analogues from Glarea lozoyensis by Mutasynthesis and Evaluation of Their Antifungal Activity.

Journal Article ACS Chem Biol · October 21, 2016 Pneumocandins are lipohexapeptides of the echinocandin family that inhibit fungal 1,3-β-glucan synthase. Most of the pathway steps have been identified previously. However, the lipoinitiation reaction has not yet been experimentally verified. Herein, we in ... Full text Link to item Cite

Mechanistic Investigation of cPMP Synthase in Molybdenum Cofactor Biosynthesis Using an Uncleavable Substrate Analogue.

Journal Article Biochemistry · December 15, 2015 Molybdenum cofactor (Moco) is essential for all kingdoms of life, plays central roles in various biological processes, and must be biosynthesized de novo. During its biosynthesis, the characteristic pyranopterin ring is constructed by a complex rearrangeme ... Full text Link to item Cite

The role of FeS clusters for molybdenum cofactor biosynthesis and molybdoenzymes in bacteria.

Journal Article Biochim Biophys Acta · June 2015 The biosynthesis of the molybdenum cofactor (Moco) has been intensively studied, in addition to its insertion into molybdoenzymes. In particular, a link between the assembly of molybdoenzymes and the biosynthesis of FeS clusters has been identified in the ... Full text Link to item Cite

Mechanism of pyranopterin ring formation in molybdenum cofactor biosynthesis.

Journal Article Proc Natl Acad Sci U S A · May 19, 2015 The molybdenum cofactor (Moco) is essential for all kingdoms of life, plays central roles in various biological processes, and must be biosynthesized de novo. During Moco biosynthesis, the characteristic pyranopterin ring is constructed by a complex rearra ... Full text Link to item Cite

C-Terminal glycine-gated radical initiation by GTP 3',8-cyclase in the molybdenum cofactor biosynthesis.

Journal Article J Am Chem Soc · March 11, 2015 The molybdenum cofactor (Moco) is an essential redox cofactor found in all kingdoms of life. Genetic mutations in the human Moco biosynthetic enzymes lead to a fatal metabolic disorder, Moco deficiency (MoCD). Greater than 50% of all human MoCD patients ha ... Full text Link to item Cite

A chemically competent thiosulfuranyl radical on the Escherichia coli class III ribonucleotide reductase.

Journal Article J Am Chem Soc · June 25, 2014 The class III ribonucleotide reductases (RNRs) are glycyl radical (G•) enzymes that provide the balanced pool of deoxynucleotides required for DNA synthesis and repair in many facultative and obligate anaerobic bacteria and archaea. Unlike the class I and ... Full text Link to item Cite

Function of the diiron cluster of Escherichia coli class Ia ribonucleotide reductase in proton-coupled electron transfer.

Journal Article J Am Chem Soc · June 12, 2013 The class Ia ribonucleotide reductase (RNR) from Escherichia coli employs a free-radical mechanism, which involves bidirectional translocation of a radical equivalent or "hole" over a distance of ~35 Å from the stable diferric/tyrosyl-radical (Y122(•)) cof ... Full text Link to item Cite

Identification of a cyclic nucleotide as a cryptic intermediate in molybdenum cofactor biosynthesis.

Journal Article J Am Chem Soc · May 8, 2013 The molybdenum cofactor (Moco) is a redox cofactor found in all kingdoms of life, and its biosynthesis is essential for survival of many organisms, including humans. The first step of Moco biosynthesis is a unique transformation of guanosine 5'-triphosphat ... Full text Link to item Cite

Structural interconversions modulate activity of Escherichia coli ribonucleotide reductase.

Journal Article Proc Natl Acad Sci U S A · December 27, 2011 Essential for DNA biosynthesis and repair, ribonucleotide reductases (RNRs) convert ribonucleotides to deoxyribonucleotides via radical-based chemistry. Although long known that allosteric regulation of RNR activity is vital for cell health, the molecular ... Full text Link to item Cite

Equilibration of tyrosyl radicals (Y356•, Y731•, Y730•) in the radical propagation pathway of the Escherichia coli class Ia ribonucleotide reductase.

Journal Article J Am Chem Soc · November 16, 2011 Escherichia coli ribonucleotide reductase is an α2β2 complex that catalyzes the conversion of nucleotides to deoxynucleotides using a diferric tyrosyl radical (Y(122)(•)) cofactor in β2 to initiate catalysis in α2. Each turnover requires reversible long-ra ... Full text Link to item Cite

A hot oxidant, 3-NO2Y122 radical, unmasks conformational gating in ribonucleotide reductase.

Journal Article J Am Chem Soc · November 3, 2010 Escherichia coli ribonucleotide reductase is an α2β2 complex that catalyzes the conversion of nucleotides to deoxynucleotides and requires a diferric-tyrosyl radical (Y(•)) cofactor to initiate catalysis. The initiation process requires long-range proton-c ... Full text Link to item Cite

Site-specific incorporation of 3-nitrotyrosine as a probe of pKa perturbation of redox-active tyrosines in ribonucleotide reductase.

Journal Article J Am Chem Soc · June 23, 2010 E. coli ribonucleotide reductase catalyzes the reduction of nucleoside 5'-diphosphates into 2'-deoxynucleotides and is composed of two subunits: alpha2 and beta2. During turnover, a stable tyrosyl radical (Y*) at Y(122)-beta2 reversibly oxidizes C(439) in ... Full text Link to item Cite

Insight into the mechanism of inactivation of ribonucleotide reductase by gemcitabine 5'-diphosphate in the presence or absence of reductant.

Journal Article Biochemistry · December 15, 2009 Gemcitabine 5'-diphosphate (F(2)CDP) is a potent inhibitor of ribonucleotide reductases (RNRs), enzymes that convert nucleotides (NDPs) to deoxynucleotides and are essential for DNA replication and repair. The Escherichia coli RNR, an alpha2beta2 complex, ... Full text Link to item Cite

Unnatural amino acids: better than the real things?

Journal Article F1000 Biology Reports · November 26, 2009 Full text Cite

Enzymatic preparation of neomycin C from ribostamycin.

Journal Article J Antibiot (Tokyo) · November 2009 Full text Link to item Cite

Mechanistic study on the reaction of a radical SAM dehydrogenase BtrN by electron paramagnetic resonance spectroscopy.

Journal Article Biochemistry · August 26, 2008 BtrN is a radical SAM ( S-adenosyl- l-methionine) enzyme that catalyzes the oxidation of 2-deoxy- scyllo-inosamine (DOIA) into 3-amino-2,3-dideoxy- scyllo-inosose (amino-DOI) during the biosynthesis of 2-deoxystreptamine (DOS) in the butirosin producer Bac ... Full text Link to item Cite

Characterization and mechanistic study of a radical SAM dehydrogenase in the biosynthesis of butirosin.

Journal Article J Am Chem Soc · December 12, 2007 BtrN encoded in the butirosin biosynthetic gene cluster possesses a CXXXCXXC motif conserved within the radical S-adenosyl methionine (SAM) superfamily. Its gene disruption in the butirosin producer Bacillus circulans caused the interruption of the biosynt ... Full text Link to item Cite

Biosynthesis of 2-deoxystreptamine by three crucial enzymes in Streptomyces fradiae NBRC 12773.

Journal Article J Antibiot (Tokyo) · December 2005 NeoA, B, and C encoded in the neomycin biosynthetic gene cluster have been enzymatically confirmed to be responsible to the formation of 2-deoxystreptamine (DOS) in Streptomyces fradiae. NeoC was functionally characterized as 2-deoxy-scyllo-inosose synthas ... Full text Link to item Cite

Stereochemical recognition of doubly functional aminotransferase in 2-deoxystreptamine biosynthesis.

Journal Article J Am Chem Soc · April 27, 2005 The doubly functional aminotransferase BtrS in the 2-deoxystreptamine (DOS) biosynthesis, in which two transaminations are involved, was characterized by a genetic as well as a chemical approach with the heterologously expressed enzyme. The gene disruption ... Full text Link to item Cite