X-ray free-electron laser studies reveal correlated motion during isopenicillin N synthase catalysis

Abstract

Isopenicillin N synthase (IPNS) catalyzes the unique reaction of l--(-aminoadipoyl)-l-cysteinyl-d-valine (ACV) with dioxygen giving isopenicillin N (IPN), the precursor of all natural penicillins and cephalosporins. X-ray free-electron laser studies including time-resolved crystallography and emission spectroscopy reveal how reaction of IPNS:Fe(II):ACV with dioxygen to yield an Fe(III) superoxide causes differences in active site volume and unexpected conformational changes that propagate to structurally remote regions. Combined with solution studies, the results reveal the importance of protein dynamics in regulating intermediate conformations during conversion of ACV to IPN. The results have implications for catalysis by multiple IPNS-related oxygenases, including those involved in the human hypoxic response, and highlight the power of serial femtosecond crystallography to provide insight into long-range enzyme dynamics during reactions presently impossible for nonprotein catalysts. RESULTSTo focus tr-SFX analyses on events associated with O 2 reaction rather than ACV binding and/or IPN release, we co-crystallized the anaerobic IPNS:Fe(II):ACV complex under anaerobic conditions to produce a dense microcrystal slurry (~3 m × 3 m × 60 m, needle

Can Second Coordination Sphere and Long-Range Interactions Modulate Hydrogen Atom Transfer in a Non-Heme Fe(II)-Dependent Histone Demethylase?

“… 22 , 23 Importantly, the ability of SCS residues to induce fast motions has been demonstrated by experimental work, including on a penicillin-forming oxidase, which is structurally and mechanistically related to the 2OG oxygenases. 24 − 26 Further, an SCS substitution of lysine (K241) to alanine in the 2OG-dependent JmjC KDM4A has been shown to ablate demethylation activity. 27 A computational study explored the effects of K241A, Y177A, and N290A variations in KDM4A and demonstrated that in these variants, the histone substrate (H3K4me3) adopts a nonproductive conformation; 28 these results are in agreement with experimental studies for K241A.…”

“… 39 , 40 However, identifying residues at LR from the active site relevant for catalysis remains challenging, and experimental approaches, e.g., employing X-ray free electron laser-based analyses to study correlated motions during specific steps in catalysis by oxygenases, are demanding and expensive. 26 , 41 Recent studies demonstrated that electrostatic perturbations could be a promising tool to modulate the rate and specificity of chemical reactions. 42 − 44 Computational methods thus have an important role in studying factors away from the active site that regulates the kinetics of individual steps in oxygenase catalysis.…”

“…Non-heme Fe­(II)/2-oxoglutarate (2OG) oxygenases, including histone demethylases (JmjC KDMs), DNA demethylases, TET enzymes, and ethylene-forming enzymes (EFE) perform HAT as a part of their reactions . Earlier studies have demonstrated that substitutions in both the SCS and LR regions can significantly affect their catalytic activity; − such substitutions are also linked to disease. , Importantly, the ability of SCS residues to induce fast motions has been demonstrated by experimental work, including on a penicillin-forming oxidase, which is structurally and mechanistically related to the 2OG oxygenases. − Further, an SCS substitution of lysine (K241) to alanine in the 2OG-dependent JmjC KDM4A has been shown to ablate demethylation activity . A computational study explored the effects of K241A, Y177A, and N290A variations in KDM4A and demonstrated that in these variants, the histone substrate (H3K4me3) adopts a nonproductive conformation; these results are in agreement with experimental studies for K241A .…”

XFEL serial crystallography reveals the room temperature structure of methyl-coenzyme M reductase

“…5). These data provide a real-time handle to monitor both oxidation state and electronic structure during Serial Femtosecond Crystallography [46,47]. The oxidized MCR data was compared against those of two water-soluble nickel(II) standards, [Ni (H2O)6] 2+ and [Ni(bpy)3] 2+ (bpy = 2,2 ′ -bipyridine), which were also collected in solution at room temperature at the XFEL using the DOT sample delivery system.…”

Integrative proteomic and physiological analyses of the molecular response to dessication-stress in Auricularia fibrillifera

“…FH facilitates the synthesis of cysteine, which is the precursor for penicillin and cephalosporin synthesis. IPNS can catalyze the specific reaction of L-δ-(α-aminoadipoyl)-Lcysteinyl-D-valine with dioxygen giving isopenicillin-N, a precursor of cephalosporins and penicillins (Rabe et al, 2021). The significant abundance increase of these DAPs under dessication-stress is beneficial to the accumulation of penicillin and cephalosporin in A. fibrillifera.…”