Logan A. Calderone

SAMHD1 is the lone human dNTP triphosphohydrolase and is linked to antiviral defense, nucleotide pool homeostasis, chemotherapy resistance, and the autoinflammatory Aicardi-Goutières syndrome. Although its substrate specificity and nucleotide-dependent oligomerization have been extensively studied, the identity and mechanistic roles of its metal cofactors remain poorly understood. Here, we integrate selective metal enrichment, spectroscopy, biochemical reconstitution, and enzyme kinetics to define the metal requirements underlying SAMHD1 activation and catalysis. We show that robust SAMHD1 activity is preferentially supported by transition metals and that the enzyme readily assembles multiple iron-containing dinuclear active sites in solution. Iron preferentially binds to one position of the bimetallic core and promotes recruitment of a second divalent metal required for catalysis. Although manganese can substitute for iron, it alters metal-binding equilibria and less efficiently supports dinuclear cofactor assembly, highlighting a specialized organizational role for iron. In contrast, the second site remains comparatively permissive and accommodates various divalent metal ions with distinct functional consequences. Mixed-metal active sites further retain catalytic activity across redox conditions that otherwise suppress activity in homodinuclear diiron configurations, suggesting that metal plasticity buffers SAMHD1 against oxidative inhibition. Transition metals additionally act as higher-affinity allosteric activators than Mg2+, revealing that metal identity contributes to both catalytic and regulatory layers of SAMHD1 function. Cumulatively, these findings redefine the metal requirements of SAMHD1 and establish a framework in which iron-dependent active site organization and mixed-metal flexibility cooperate to sustain dNTP hydrolysis under changing cellular environments and metal flux conditions.

SAMHD1 is the lone human dNTP triphosphohydrolase and is intimately linked to HIV viral restriction, dNTP pool maintenance, resistance to chemotherapy, and the autoinflammatory Aicardi-Goutières syndrome. While its substrate promiscuity and nucleotide basis of activity have been extensively studied, the identity and mechanistic roles of its metal cofactors remain poorly defined. Here, we integrate elemental analysis, spectroscopy, protein cross-linking, and enzyme kinetics to elucidate the molecular mechanisms underlying metal-dependent activation and catalysis in SAMHD1. Our findings establish that transition metals are essential components of SAMHD1 function, highlight their overlooked role in allosteric regulation, and reveal a central role for iron in organizing the dinuclear active site. We show that iron is preferentially incorporated in one position of the bimetallic core, where it promotes recruitment of a second divalent metal ion required for activity. While manganese can substitute for iron, it alters the metal binding equilibria, highlighting the unique functional properties of iron. Notably, SAMHD1 exhibits metal cofactor promiscuity at the second metal site, accommodating diverse divalent metals with distinct effects on activity. Cumulatively, our findings establish iron as a core structural and functional determinant of SAMHD1 catalysis and reveal how transition metal selectivity and flexibility enable enzymatic activity across diverse cellular environments and metal flux conditions.

The General Stress Response promotes survival of bacteria in adverse conditions, but how sensor proteins transduce species-specific signals to initiate the response is not known. The serine/threonine phosphatase RsbU initiates the General Stress Response in Bacillus subtilis upon binding a partner protein (RsbT) that is released from sequestration by environmental stresses. We report that RsbT activates RsbU by inducing otherwise flexible linkers of RsbU to form a short coiled-coil that dimerizes and activates the phosphatase domains. Importantly, we present evidence that related coiled-coil linkers and phosphatase dimers transduce signals from diverse sensor domains to control the General Stress Response and other signaling across bacterial phyla. This coiled-coil linker transduction mechanism additionally suggests a resolution to the mystery of how shared sensory domains control serine/threonine phosphatases, diguanylate cyclases and histidine kinases. We propose that this provides bacteria with a modularly exchangeable toolkit for the evolution of diverse signaling pathways.

The General Stress Response promotes survival of bacteria in adverse conditions, but how sensor proteins transduce species-specific signals to initiate the response is not known. The serine/threonine phosphatase RsbU initiates the General Stress Response in B. subtilis upon binding a partner protein (RsbT) that is released from sequestration by environmental stresses. We report that RsbT activates RsbU by inducing otherwise flexible linkers of RsbU to form a short coiled-coil that dimerizes and activates the phosphatase domains. Importantly, we present evidence that related coiled-coil linkers and phosphatase dimers transduce signals from diverse sensor domains to control the General Stress Response and other signaling across bacterial phyla. This coiled-coil linker transduction mechanism additionally suggests a resolution to the mystery of how shared sensory domains control serine/threonine phosphatases, diguanylate cyclases and histidine kinases. We propose that this provides bacteria with a modularly exchangeable toolkit for the evolution of diverse signaling pathways.

Skatole is a pungent heterocyclic compound derived from the essential amino acid l-tryptophan by bacteria in the mammalian digestive tract. The four-step anaerobic conversion of tryptophan to skatole is well-established; though, to date, no aerobic counterpart has been reported. Herein, we report the discovery of the oxygen-dependent skatole synthase SktA that single-handedly converts 5-bromo-l-tryptophan to 5-bromoskatole, obviating the need for a multienzyme process. SktA is part of a three-gene biosynthetic gene cluster (BGC) in the cyanobacterium Nostoc punctiforme NIES-2108 and functions as a nonheme diiron enzyme belonging to the heme oxygenase-like domain-containing oxidase (HDO) superfamily. Our detailed biochemical analyses revealed cyanide and bicarbonate as biosynthetic coproducts, while stopped-flow experiments showed the hallmark formation of a substrate-triggered peroxo Fe₂(III) intermediate. Overall, this work unravels an alternative pathway for converting tryptophan to skatole while also expanding the functional repertoire of HDO enzymes.

How can a single protein domain encode a conformational landscape with multiple stably folded states, and how do those states interconvert? Here, we use real-time and relaxation-dispersion NMR to characterize the conformational landscape of the circadian rhythm protein KaiB from Rhodobacter sphaeroides . Unique among known natural metamorphic proteins, this KaiB variant spontaneously interconverts between two monomeric states: the “Ground” and “Fold-switched” (FS) states. KaiB in its FS state interacts with multiple binding partners, including the central KaiC protein, to regulate circadian rhythms. We find that KaiB itself takes hours to interconvert between the Ground and FS state, underscoring the ability of a single-sequence to encode the slow process needed for function. We reveal the rate-limiting step between the Ground and FS state is the cis–trans isomerization of three prolines in the fold-switching region by demonstrating interconversion acceleration by the prolyl isomerase Cyclophilin A. The interconversion proceeds through a “partially disordered” (PD) state, where the C-terminal half becomes disordered while the N-terminal half remains stably folded. We found two additional properties of KaiB’s landscape. First, the Ground state experiences cold denaturation: At 4 °C, the PD state becomes the majorly populated state. Second, the Ground state exchanges with a fourth state, the “Enigma” state, on the millisecond-timescale. We combine AlphaFold2-based predictions and NMR chemical shift predictions to predict this Enigma state is a beta-strand register shift that relieves buried charged residues, and support this structure experimentally. These results provide mechanistic insight into how evolution can design a single-sequence that achieves specific timing needed for its function.

Nitriles are uncommon in nature and are typically constructed from oximes through the oxidative decarboxylation of amino acid substrates or from the derivatization of carboxylic acids. Here we report a third nitrile biosynthesis strategy featuring the cyanobacterial nitrile synthase AetD. During the biosynthesis of the eagle-killing neurotoxin, aetokthonotoxin, AetD transforms the 2-aminopropionate portion of 5,7-dibromo-l-tryptophan to a nitrile. Employing a combination of structural, biochemical and biophysical techniques, we characterized AetD as a non-haem diiron enzyme that belongs to the emerging haem-oxygenase-like dimetal oxidase superfamily. High-resolution crystal structures of AetD together with the identification of catalytically relevant products provide mechanistic insights into how AetD affords this unique transformation, which we propose proceeds via an aziridine intermediate. Our work presents a unique template for nitrile biogenesis and portrays a substrate binding and metallocofactor assembly mechanism that may be shared among other haem-oxygenase-like dimetal oxidase enzymes.

Heme oxygenase-like domain-containing oxidases (HDOs) are a rapidly expanding enzyme family that typically use dinuclear metal cofactors instead of heme. FlcD, an HDO from the opportunistic pathogen Pseudomonas aeruginosa, catalyzes the excision of an oxime carbon in the biosynthesis of the copper-containing antibiotic fluopsin C. We show that FlcD is a dioxygenase that catalyzes a four-electron oxidation. Crystal structures of FlcD reveal a mononuclear iron in the active site, which is coordinated by two histidines, one glutamate, and the oxime of the substrate. Enzyme activity, Mössbauer spectroscopy, and electron paramagnetic resonance spectroscopy analyses support the usage of a mononuclear iron cofactor. This cofactor resembles that of mononuclear non-heme iron-dependent enzymes and breaks the paradigm of dinuclear HDO cofactors. This study begins to illuminate the catalytic mechanism of methine excision and indicates convergent evolution of different lineages of mononuclear iron-dependent enzymes.

The General Stress Response promotes survival of bacteria in adverse conditions, but how sensor proteins transduce species-specific signals to initiate the response is not known. The serine/threonine phosphatase RsbU initiates the General Stress Response in B. subtilis upon binding a partner protein (RsbT) that is released from sequestration by environmental stresses. We report that RsbT activates RsbU by inducing otherwise flexible linkers of RsbU to form a short coiled-coil that dimerizes and activates the phosphatase domains. Importantly, we present evidence that related coiled-coil linkers and phosphatase dimers transduce signals from diverse sensor domains to control the General Stress Response and other signaling across bacterial phyla. This coiled-coil linker transduction mechanism additionally suggests a resolution to the mystery of how shared sensory domains control serine/threonine phosphatases, diguanylate cyclases and histidine kinases. We propose that this provides bacteria with a modularly exchangeable toolkit for the evolution of diverse signaling pathways.