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1. Structure of human MUTYH and functional profiling of cancer-associated variants reveal an allosteric network between its [4Fe-4S] cluster cofactor and active site required for DNA repair

   https://doi.org/10.1038/s41467-025-58361-w  

   Trasviña-Arenas, Carlos_H; Dissanayake, Upeksha_C; Tamayo, Nikole; Hashemian, Mohammad; Lin, W_Jonathan; Demir, Merve; Hoyos-Gonzalez, Nallely; Fisher, Andrew_J; Cisneros, G_Andrés; Horvath, Martin_P; et al (April 2025, Nature Communications)

   Abstract MUTYH is a clinically important DNA glycosylase that thwarts mutations by initiating base-excision repair at 8-oxoguanine (OG):A lesions. The roles for its [4Fe-4S] cofactor in DNA repair remain enigmatic. Functional profiling of cancer-associated variants near the [4Fe-4S] cofactor reveals that most variations abrogate both retention of the cofactor and enzyme activity. Surprisingly, R241Q and N238S retained the metal cluster and bound substrate DNA tightly, but were completely inactive. We determine the crystal structure of human MUTYH bound to a transition state mimic and this shows that Arg241 and Asn238 build an H-bond network connecting the [4Fe-4S] cluster to the catalytic Asp236 that mediates base excision. The structure of the bacterial MutY variant R149Q, along with molecular dynamics simulations of the human enzyme, support a model in which the cofactor functions to position and activate the catalytic Asp. These results suggest that allosteric cross-talk between the DNA binding [4Fe-4S] cofactor and the base excision site of MUTYH regulate its DNA repair function. 

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2. Crystal structure of MutYX: A novel clusterless adenine DNA glycosylase with a distinct C-terminal domain and 8-Oxoguanine recognition sphere

   https://doi.org/10.1101/2025.01.03.631205  

   Trasviña-Arenas, CH; Hashemian, Mohammad; Malek, Melody; Merrill, Steven; Fisher, Andrew J; David, Sheila S (January 2025, bioRxiv)

   Abstract The [4Fe-4S] cluster is an important cofactor of the base excision repair (BER) adenine DNA glycosylase MutY to prevent mutations associated with 8-oxoguanine (OG). Several MutYs lacking the [4Fe-4S] cofactor have been identified. Phylogenetic analysis shows that clusterless MutYs are distributed in two clades suggesting cofactor loss in two independent evolutionary events. Herein, we determined the first crystal structure of a clusterless MutY complexed with DNA. On the basis of the dramatic structural divergence from canonical MutYs, we refer to this as representative of a clusterless MutY subgroup “MutYX”. Interestingly, MutYX compensates for the missing [4Fe-4S] cofactor to maintain positioning of catalytic residues by expanding a pre-existing α-helix and acquisition of the new α-helix. Surprisingly, MutYX also acquired a new C-terminal domain that uniquely recognizes OG using residue Gln201 and Arg209. Adenine glycosylase assays and binding affinity measurements indicate that Arg209 is the primary residue responsible to specificity for OG:A lesions, while Gln201 bridges OG and Arg209. Surprisingly, replacement of Arg209 and Gln201 with Ala increases activity toward G:A mismatches. The MutYX structure serves as an example of devolution, capturing structural features required to retain function in the absence of a metal cofactor considered indispensable. 

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3. Crystal structure of MutYX: a novel clusterless adenine DNA glycosylase with a distinct C-terminal domain and 8-oxoguanine recognition sphere

   https://doi.org/10.1093/nar/gkaf1127  

   Trasviña-Arenas, Carlos H; Hashemian, Mohammad; Malek, Melody; Merrill, Steven; Fisher, Andrew J; David, Sheila S (November 2025, Nucleic Acids Research)

   The [4Fe–4S] cluster is an important cofactor of the base excision repair (BER) adenine DNA glycosylase MutY to prevent mutations associated with 8-oxoguanine (OG). Several MutYs lacking the [4Fe–4S] cofactor have been identified. Phylogenetic analysis shows that clusterless MutYs are distributed in two clades suggesting cofactor loss has occurred in multiple independent evolutionary events. Herein, we determined the first crystal structure of a clusterless MutY complexed with DNA. On the basis of the dramatic structural divergence from canonical MutYs, we refer to this as representative of a clusterless MutY subgroup “MutYX.” Interestingly, MutYX compensates for the missing [4Fe–4S] cofactor to maintain positioning of catalytic residues by expanding a pre-existing α-helix and acquisition of a new α-helix. Surprisingly, MutYX also acquired a new C-terminal domain that uniquely recognizes OG using residues Gln201 and Arg209. Adenine glycosylase assays and binding affinity measurements indicate that Arg209 is the primary residue responsible for OG:A lesion specificity, while Gln201 assists by bridging OG and Arg209. Surprisingly, replacement of Arg209 and Gln201 with Ala increased activity toward G:A mismatches. The MutYX structure serves as an example of devolution, capturing structural features required to retain function in the absence of a metal cofactor considered indispensable. 

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4. Active Site Characterization of a Campylobacter jejuni Nitrate Reductase Variant Provides Insight into the Enzyme Mechanism

   https://doi.org/10.1021/acs.inorgchem.4c01991  

   Yang, Jing; Mintmier, Breeanna; KC, Khadanand; Metzger, Mikayla C; Radhakrishnan, Manohar; McGarry, Jennifer; Wilcoxen, Jarett; Basu, Partha; Kirk, Martin L (July 2024, Inorganic Chemistry)

   Mo K-edge X-ray absorption spectroscopy (XAS) is used to probe the structure of wild-type Campylobacter jejuni nitrate reductase NapA and the C176A variant. The results of extended X-ray absorption fine structure (EXAFS) experiments on wt NapA support an oxidized Mo(VI) hexacoordinate active site coordinated by a single terminal oxo donor, four sulfur atoms from two separate pyranopterin dithiolene ligands, and an additional S atom from a conserved cysteine amino acid residue. We found no evidence of a terminal sulfido ligand in wt NapA. EXAFS analysis shows the C176A active site to be a 6-coordinate structure, and this is supported by EPR studies on C176A and small molecule analogs of Mo(V) enzyme forms. The SCys is replaced by a hydroxide or water ligand in C176A, and we find no evidence of a coordinated sulfhydryl (SH) ligand. Kinetic studies show that this variant has completely lost its catalytic activity toward nitrate. Taken together, the results support a critical role for the conserved C176 in catalysis and an oxygen atom transfer mechanism for the catalytic reduction of nitrate to nitrite that does not employ a terminal sulfido ligand in the catalytic cycle. 

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5. Trapping a cross-linked lysine–tryptophan radical in the catalytic cycle of the radical SAM enzyme SuiB

   https://doi.org/10.1073/pnas.2101571118  

   Balo, Aidin R.; Caruso, Alessio; Tao, Lizhi; Tantillo, Dean J.; Seyedsayamdost, Mohammad R.; Britt, R. David (May 2021, Proceedings of the National Academy of Sciences)

   The radicalS-adenosylmethionine (rSAM) enzyme SuiB catalyzes the formation of an unusual carbon–carbon bond between the sidechains of lysine (Lys) and tryptophan (Trp) in the biosynthesis of a ribosomal peptide natural product. Prior work on SuiB has suggested that the Lys–Trp cross-link is formed via radical electrophilic aromatic substitution (rEAS), in which an auxiliary [4Fe-4S] cluster (AuxI), bound in the SPASM domain of SuiB, carries out an essential oxidation reaction during turnover. Despite the prevalence of auxiliary clusters in over 165,000 rSAM enzymes, direct evidence for their catalytic role has not been reported. Here, we have used electron paramagnetic resonance (EPR) spectroscopy to dissect the SuiB mechanism. Our studies reveal substrate-dependent redox potential tuning of the AuxI cluster, constraining it to the oxidized [4Fe-4S]²⁺state, which is active in catalysis. We further report the trapping and characterization of an unprecedented cross-linked Lys–Trp radical (Lys–Trp•) in addition to the organometallic Ω intermediate, providing compelling support for the proposed rEAS mechanism. Finally, we observe oxidation of the Lys–Trp• intermediate by the redox-tuned [4Fe-4S]²⁺AuxI cluster by EPR spectroscopy. Our findings provide direct evidence for a role of a SPASM domain auxiliary cluster and consolidate rEAS as a mechanistic paradigm for rSAM enzyme-catalyzed carbon–carbon bond-forming reactions. 

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