The nickel-pincer nucleotide (NPN) is an organometallic cofactor that was first discovered in lactate racemase from Lactiplantibacillus plantarum. In this review, we provide an overview on the structure-function relationships of enzymes that utilize or are involved in the biosynthesis of the NPN cofactor. Recent structural advances have greatly extended our understanding of the biological role of the NPN cofactor in a diverse family of 2-hydroxyacid racemases and epimerases. Moreover, structural studies of the accessory proteins LarB (a combined carboxylase/hydrolase), two distinct forms of LarE (an ATP-dependent sulfur transferase), and LarC (a CTP-dependent nickel insertase) have elucidated key features in the biosynthetic pathway for the NPN cofactor. Finally, we discuss the potential of future structural investigations to uncover additional enzymes that synthesize and use the NPN cofactor to catalyze new reactions.

In 1926, James B. Sumner crystallized jack bean urease-the first enzyme to be obtained in crystalline form-thus demonstrating that enzymes are proteinaceous. To honor the 100-year anniversary of that momentous event, this review highlights critical findings leading up to Sumner's efforts, explains the significance of his results, and describes subsequent experimental findings related to urease. For example, nearly five decades after crystals became available Burt Zerner and colleagues identified urease as the first known nickel-containing enzyme. The surprising discovery of nickel in urease raised questions about the structure of the metal-containing active site, the enzyme mechanism, and pathway by which the catalytic center is synthesized - each of which is addressed here. Finally, I reflect on remaining open questions related to this remarkable enzyme and potential experimental directions that could be employed to provide corresponding insights.

The nickel pincer nucleotide (NPN) cofactor catalyzes the racemization/epimerization of α-hydroxy acids in enzymes of the LarA family. The established proton-coupled hydride transfer mechanism requires two catalytic histidine residues that alternately act as general acids and general bases. Notably, however, a fraction of LarA homologs (LarAHs) lack one of the active site histidine residues, replacing it with an asparaginyl side chain that cannot participate in acid/base catalysis. Here, we investigated two such LarAHs and solved their cryo-electron microscopic structures with and without loaded NPN cofactor, respectively. The structures revealed a consistent octameric assembly that is unprecedented in the LarA family and unveiled a new set of active site residues that likely recognize and process substrates differently from those of the well-studied LarAHs. Genomic context analysis suggested their potential involvement in carbohydrate metabolism. Together, these findings lay the groundwork for expanding the breadth of reactions and the range of mechanisms of LarA enzymes.

The ethylene-forming enzyme (EFE) from the fungus Penicillium digitatum strain Pd1 was heterologously produced in Escherichia coli and its properties were compared to the extensively characterized bacterial enzyme from Pseudomonas savastanoi strain PK2. Both enzymes catalyze four reactions: the conversion of 2-oxoglutarate (2OG) to ethylene and CO2, oxidative decarboxylation of 2OG coupled to l-arginine (l-Arg) hydroxylation, uncoupled oxidative decarboxylation of 2OG, and the production of 3-hydroxypropionate (3-HP) from 2OG. The strain Pd1 enzyme exhibited a greater ratio of ethylene production over l-Arg hydroxylation than the PK2 strain EFE. The uncoupled decarboxylation of 2OG and 3-HP production are minor reactions in both cases, but they occur to a greater extent using the fungal enzyme. Additional distinctions of the fungal versus bacterial enzyme are noted in the absorbance maxima and l-Arg dependence of their anaerobic electronic spectra. The structures of the Pd1 EFE apoprotein and the EFE·Mn(II)·2OG complex resembled the corresponding structures of the PK2 enzyme, but notable structural differences were observed in the computationally predicted Pd1 EFE·Fe(II)·2OG·l-Arg complex versus the PK2 EFE·Mn(II)·2OG·l-Arg crystal structure. These studies extend our biochemical understanding and represent the first structural and conformational characterization of a eukaryotic EFE.

The ethylene-forming enzyme (EFE) catalyzes two main reactions: the conversion of 2-oxoglutarate (2OG) to ethylene plus CO2 and the oxidative decarboxylation of 2OG coupled to the C5 hydroxylation of l-arginine (l-Arg). EFE also facilitates two minor reactions: the uncoupled oxidative decarboxylation of 2OG and the generation of 3-hydroxypropionate (3HP) from 2OG. To better understand the evolution of this enzyme's diverse activities, we demonstrated that two distantly related extant enzymes produce trace levels of ethylene and 3HP, and we examined the reactivities of 11 reconstructed ancestors. The structure of one ancestral protein was resolved by X-ray crystallography, while the others were modeled with AlphaFold2. These studies highlight the importance of residues located at the 2OG and l-Arg binding pockets for the varied activities. For example, effective formation of ethylene requires that the 2OG binding pocket be hydrophobic except for interactions with the substrate carboxylates. Newly identified changes near the l-Arg binding site exhibit significant effects on the reactivities of the enzyme's reactions. Analysis of the reconstructed ancestors suggests that the primordial enzyme exhibited both ethylene-forming and l-Arg hydroxylation activities with partition ratios like the extant examples; i.e., an enzyme capable of catalyzing predominantly one of these reactions did not subsequently develop the ability to affect the secondary reaction.

The LarA family consists of diverse racemases/epimerases that interconvert the diastereomers of alpha-hydroxyacids by using a nickel-pincer nucleotide (NPN) cofactor. The hidden redox reaction catalyzed by the NPN cofactor makes LarA enzymes attractive engineering targets for various applications. However, how a LarA enzyme binds its natural substrate and recognizes different alpha-hydroxyacids has not been elucidated. Here, we report three high-resolution structures of the enzyme-substrate complexes of a broad-spectrum LarA enzyme from Isosphaera pallida (LarA Ip ). The substrate binding mode reveals a near-optimal orientation and distance between the hydride donor and acceptor, consistent with an updated proton-coupled hydride transfer mechanism. The experimentally solved structures, together with the structural models of other LarA enzymes, lead to the identification of the residues/structural elements that are critically involved in the interactions with different alpha-hydroxyacids. Collectively, this work provides a structural basis for the catalysis and substrate specificity of the LarA enzymes.

Bile acids (BAs) are steroid detergents in bile that contribute to the absorption of fats and fat-soluble vitamins while shaping the gut microbiome because of their antimicrobial properties1-4. Here we identify the enzyme responsible for a mechanism of BA metabolism by the gut microbiota involving amino acid conjugation to the acyl-site of BAs, thus producing a diverse suite of microbially conjugated bile acids (MCBAs). We show that this transformation is mediated by acyltransferase activity of bile salt hydrolase (bile salt hydrolase/transferase, BSH/T). Clostridium perfringens BSH/T rapidly performed acyl transfer when provided various amino acids and taurocholate, glycocholate or cholate, with an optimum at pH 5.3. Amino acid conjugation by C. perfringens BSH/T was diverse, including all proteinaceous amino acids except proline and aspartate. MCBA production was widespread among gut bacteria, with strain-specific amino acid use. Species with similar BSH/T amino acid sequences had similar conjugation profiles and several bsh/t alleles correlated with increased conjugation diversity. Tertiary structure mapping of BSH/T followed by mutagenesis experiments showed that active site structure affects amino acid selectivity. These MCBA products had antimicrobial properties, where greater amino acid hydrophobicity showed greater antimicrobial activity. Inhibitory concentrations of MCBAs reached those measured natively in the mammalian gut. MCBAs fed to mice entered enterohepatic circulation, in which liver and gallbladder concentrations varied depending on the conjugated amino acid. Quantifying MCBAs in human faecal samples showed that they reach concentrations equal to or greater than secondary and primary BAs and were reduced after bariatric surgery, thus supporting MCBAs as a significant component of the BA pool that can be altered by changes in gastrointestinal physiology. In conclusion, the inherent acyltransferase activity of BSH/T greatly diversifies BA chemistry, creating a set of previously underappreciated metabolites with the potential to affect the microbiome and human health.

The nickel-pincer nucleotide (NPN) cofactor is a modified pyridinium mononucleotide that tri-coordinates nickel and is crucial for the activity of certain racemases and epimerases. LarB, LarC, and LarE are responsible for NPN synthesis, with the cofactor subsequently installed into LarA homologs. Hurdles for investigating the functional properties of such proteins arise from the difficulty of obtaining the active, NPN cofactor-loaded enzymes and in assaying their diverse reactivities. Here, we show that when the Lactiplantibacillus plantarum lar genes are cloned into the Duet expression system and cultured in Escherichia coli, they confer lactate racemase activity to the cells. By replacing L. plantarum larA with related genes from other microorganisms, this system allows for the generation of active LarA homologs. Furthermore, the Duet system enables the functional testing of LarB, LarC, and LarE homologs from other microorganisms. In addition to applying the Duet expression system for synthesis of active, NPN cofactor-containing enzymes in E. coli, we demonstrate that circular dichroism spectroscopy provides a broadly applicable means of assaying these enzymes. By selecting a wavelength of high molar ellipticity and low absorbance for a given 2-hydroxy acid substrate enantiomer, the conversion of one enantiomer/epimer into the other can be monitored for LarA homologs without the need for any coupling enzymes or reagents. The methods discussed here further our abilities to investigate the unique activities of Lar proteins. IMPORTANCE:Enzymes containing the nickel-pincer nucleotide (NPN) cofactor are prevalent in a wide range of microorganisms and catalyze various critical biochemical reactions, yet they remain underexplored due, in part, to limitations in current research methodologies. The two significant advancements described here, the heterologous production of active NPN-cofactor containing enzymes in Escherichia coli and the use of a circular dichroism-based assay to monitor enzyme activities, expand our capacity to analyze these enzymes. Such additional detailed characterization will deepen our understanding of the diverse chemistry catalyzed by the NPN cofactor and potentially uncover novel roles for this organometallic species in microbial metabolism.

Biometals webinar presentation and International Biometals Society This short presentation will be an introduction to the Biometals webinars series organized by the International Biometals society and the Biometals Journal. Biometal webinars will be monthly in 2024 jointly organized by the Cassiny platform, Springer and the International Biometals society. It will be with two speakers, one senior scientist one younger. One of them at least author of a recent Biometals article. confirmed senior speakers: January 16th, 2024:Bob Hausinger (Ni) ; February 13th, 2024: David Frazer (Fe) ; March 12th , 2024: Wolfgang Maret (Zn) ; April 9th 2024: Alison Butler (Fe) ; May 7th 2024: John Helmann (Fe) ; Free of access, each webinar will be live and accessible live on the Cassyni Platform followed by questions and answers time. It will also be recorded and be accessible (if accepted by the speaker) on the platform. A DOI number will be assigned to the recorded presentation. This will not replace the face-to-face Biometals symposium that should be take place in summer 2025. Biosynthesis and Functions of the Nickel-Pincer Nucleotide (NPN) Cofactor The nickel-pincer nucleotide (NPN) cofactor catalyzes the proton-coupled hydride-transfer reactions of selected racemases and epimerases (1-2). This novel organometallic molecule has a square-planar nickel atom that is tri-coordinated by a modified pyridinium mononucleotide, forming C-Ni and two S-Ni bonds (3). Within the active site of enzyme, the nickel is additionally bound by a histidyl residue and in some cases the NPN cofactor is covalently tethered to a lysyl group. Biosynthesis of NPN is a three-step process. LarB uses nicotinic acid adenine dinucleotide (NaAD) as its substrate and catalyzes C5-carboxylation and phosphoanhydride hydrolysis to produce the dicarboxylated pyridine mononucleotide (P2CMN) (4). LarE catalyzes an ATP-dependent sulfur insertion reaction that converts P2CMN into a species with two thiocarboxylic acids (P2TMN) by sacrificial loss of a cysteinyl sulfur atom or by sulfur donation from a [4Fe-5S] cluster (5-6). LarC is a CTP-dependent nickel insertase or cyclometallase that transforms P2TMN into NPN (7-8). Recent developments in understanding the biosynthesis and utilization of the NPN cofactor will be described (9).

The ethylene-forming enzyme (EFE) is an Fe(II), 2-oxoglutarate (2OG), and l-arginine (l-Arg)-dependent oxygenase that either forms ethylene and three CO2/bicarbonate from 2OG or couples the decarboxylation of 2OG to C5 hydroxylation of l-Arg. l-Arg binds with C5 toward the metal center, causing 2OG to change from monodentate to chelate metal interaction and OD1 to OD2 switch of D191 metal coordination. We applied anaerobic UV-visible spectroscopy, X-ray crystallography, and computational approaches to three EFE systems with high-resolution structures. The ineffective l-Arg analogue l-canavanine binds to the EFE with O5 pointing away from the metal center while promoting chelate formation by 2OG but fails to switch the D191 metal coordination from OD1 to OD2. Substituting alanine for R171 that interacts with 2OG and l-Arg inactivates the protein, prevents metal chelation by 2OG, and weakens l-Arg binding. The R171A EFE had electron density at the 2OG binding site that was identified by mass spectrometry as benzoic acid. The substitution by alanine of Y306 in the EFE, a residue 12 & Aring; away from the catalytic metal center, generates an interior cavity that leads to multiple local and distal structural changes that reduce l-Arg binding and significantly reduce the enzyme activity. Flexibility analyses revealed correlated and anticorrelated motions in each system, with important distinctions from the wild-type enzyme. In combination, the results are congruent with the currently proposed enzyme mechanism, reinforce the importance of metal coordination by OD2 of D191, and highlight the importance of the second coordination sphere and longer range interactions in promoting EFE activity.

The cover shows the oxygen diffusion channel in class 7 histone demethylase (PHF8) and ethylene-forming enzyme (EFE). PHF8 catalyzes the hydroxylation of its H3 K9me2 histone substrate, and EFE catalyzes two competing reactions of ethylene generation and substrate l-Arg hydroxylation. Although both enzymes initially have an off-line 2-oxoglutarate (2OG) conformation, upon oxygen binding, PHF8 forms an in-line superoxo complex, whereas EFE uses the off-line superoxo complex. More information can be found in the Research Article by C. Z. Christov and co-workers. (DOI: 10.1002/chem.202300138).

LarB catalyzes the first step of biosynthesis for the nickel-pincer nucleotide cofactor by converting nicotinic acid adenine dinucleotide (NaAD) to AMP and pyridinium-3,5-biscarboxylic acid mononucleotide (P2CMN). Prior studies had shown that LarB uses CO2 for substrate carboxylation and reported the structure of a Lactiplantibacillus plantarum LarB·NAD+ complex, revealing a covalent linkage between Cys221 and C4 of the pyridine ring. This interaction was proposed to promote C5 carboxylation, with C5-carboxylated-NaAD suggested to activate magnesium-bound water, leading to phosphoanhydride hydrolysis. Here, we extended the analysis of wild-type LarB by using ultraviolet-visible spectroscopy to obtain additional evidence for cysteinyl side chain attachment to the ring of NAD+, thus demonstrating that this linkage is not a crystallization artifact. Using the S127A variant of L. plantarum LarB, a form of the enzyme with a reduced rate of NaAD hydrolysis, we examined its interaction with the authentic substrate. The intermediate arising from C5 carboxylation of NaAD, dinicotinic acid adenine dinucleotide (DaAD), was identified by using mass spectrometry. S127A LarB exhibited spectroscopic evidence of a Cys221-NAD+ adduct, but a covalent enzyme-NaAD linkage was not detectable. We determined the S127A LarB·NaAD structure, providing new insights into the enzyme mechanism, and tentatively identified the position and mode of CO2 binding. The crystal structure revealed the location of the side chain for Glu180, which was previously disordered, but showed that it is not well positioned to abstract the C5 proton in the adduct species to restore aromaticity as Cys221 is expelled. Based on these combined results, we propose a revised catalytic mechanism of LarB..

Cofactors are essential components of numerous enzymes, therefore their characterization by structural, biophysical, and biochemical approaches is crucial for understanding the resulting catalytic and regulatory mechanisms. In this chapter, we present a case study of a recently discovered cofactor, the nickel-pincer nucleotide (NPN), by demonstrating how we identified and thoroughly characterized this unprecedented nickel-containing coenzyme that is tethered to lactase racemase from Lactiplantibacillus plantarum. In addition, we describe how the NPN cofactor is biosynthesized by a panel of proteins encoded in the lar operon and describe the properties of these novel enzymes. Comprehensive protocols for conducting functional and mechanistic studies of NPN-containing lactate racemase (LarA) and the carboxylase/hydrolase (LarB), sulfur transferase (LarE), and metal insertase (LarC) used for NPN biosynthesis are provided for potential applications towards characterizing enzymes in the same or homologous families.

Metalloenzymes have been detailed in The Enzymes since its inception over half a century ago. Here, I review selected metal-containing enzyme highlights from early chapters in this series and I describe advances made since those contributions. Three topics are emphasized: nickel-containing enzymes, Fe(II)/2-oxoglutarate-dependent oxygenases, and enzymes containing non-canonical iron-sulfur clusters.

This review summarizes the structures, biochemical properties, and mechanisms of two major biological sources of ethylene, the ethylene-forming enzyme (EFE) and 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACCO). EFE is found in selected bacteria and fungi where it catalyzes two reactions: (1) the oxygen-dependent conversion of 2-oxoglutarate (2OG) to ethylene plus three molecules of CO2/bicarbonate and (2) the oxidative decarboxylation of 2OG while transforming l-arginine to guanidine and l-Δ1-pyrroline-5-carboxylic acid. ACCO is present in plants where it makes the plant hormone by transforming ACC, O2, and an external reductant to ethylene, HCN, CO2, and water. Despite catalyzing distinct chemical reactions, EFE and ACCO are related in sequence and structure, and both enzymes require Fe(ii) for their activity. Advances in our understanding of EFE, derived from both experimental and computational approaches, have clarified how this enzyme catalyzes its dual reactions. Drawing on the published mechanistic studies of ACCO and noting the parallels between this enzyme and EFE, we propose a novel reaction mechanism for ACCO.

The non-heme Fe(II) and 2-oxoglutarate (2OG) dependent ethylene-forming enzyme (EFE) catalyzes both ethylene generation and L-Arg hydroxylation. Despite experimental and computational progress in understanding the mechanism of EFE, no EFE variant has been optimized for ethylene production while simultaneously reducing the L-Arg hydroxylation activity. In this study, we show that the two L-Arg binding conformations, associated with different reactivity preferences in EFE, lead to differences in the intrinsic electric field (IntEF) of EFE. Importantly, we suggest that applying an external electric field (ExtEF) along the Fe-O bond in the EFE·Fe(III)·OO-˙·2OG·L-Arg complex can switch the EFE reactivity between L-Arg hydroxylation and ethylene generation. Furthermore, we explored how applying an ExtEF alters the geometry, electronic structure of the key reaction intermediates, and the individual energy contributions of second coordination sphere (SCS) residues through combined quantum mechanics/molecular mechanics (QM/MM) calculations. Experimentally generated variant forms of EFE with alanine substituted for SCS residues responsible for stabilizing the key intermediates in the two reactions of EFE led to changes in enzyme activity, thus demonstrating the key role of these residues. Overall, the results of applying an ExtEF indicate that making the IntEF of EFE less negative and stabilizing the off-line binding of 2OG is predicted to increase ethylene generation while reducing L-Arg hydroxylation.

The nickel-pincer nucleotide (NPN) cofactor discovered in lactate racemase from Lactiplantibacillus plantarum (LarALp) is essential for the activities of racemases/epimerases in the highly diverse LarA superfamily. Prior mechanistic studies have established a proton-coupled hydride-transfer mechanism for LarALp, but direct evidence showing that hydride attacks the C4 atom in the pyridinium ring of NPN has been lacking. Here, we show that sodium borohydride (NaBH4) irreversibly inactivates LarALp accompanied by a rapid color change of the enzyme. The altered ultraviolet-visible spectra during NaBH4 titration supported hydride transfer to C4 of NPN, and the concomitant Ni loss unraveled by mass spectrometry experiments accounted for the irreversible inactivation. High-resolution structures of LarALp revealed a substantially weakened C- Ni bond in the metastable sulfite-NPN adduct where the NPN cofactor is in the reduced state. These findings allowed us to propose a mechanism of LarALp inactivation by NaBH4 that provides key insights into the enzyme-catalyzed reaction and sheds light on the reactivity of small molecule NPN mimetics.

This study investigates dioxygen binding and 2-oxoglutarate (2OG) coordination by two model non-heme Fe(II)/2OG enzymes: a class 7 histone demethylase (PHF8) that catalyzes the hydroxylation of its H3K9me2 histone substrate leading to demethylation reactivity and the ethylene-forming enzyme (EFE) that catalyzes two competing reactions of ethylene generation and substrate L-Arg hydroxylation. Although both enzymes initially bind 2OG using an off-line 2OG coordination mode, in PHF8, the substrate oxidation requires a transition to an in-line mode, whereas EFE is catalytically productive for ethylene production from 2OG via the off-line mode. We utilized classical molecular dynamics, quantum mechanics/molecular mechanics (QM/MM) MD and QM/MM metadynamics simulations to reveal that it is the dioxygen binding process and, ultimately, the protein environment that control the formation of the in-line Fe(III)-OO-. intermediate in PHF8 and the off-line Fe(III)-OO-. intermediate in EFE.

LarC catalyzes the CTP-dependent insertion of nickel ion into pyridinium-3,5-bisthiocarboxylic acid mononucleotide (P2TMN), the final biosynthetic step for generating the nickel-pincer nucleotide (NPN) enzyme cofactor. In this study, we characterized a LarC homolog from Moorella thermoacetica (LarC(Mt)) and characterized selected properties of the protein. We ruled out the hypothesis that enzyme inhibition by its product pyrophosphate accounts for its apparent single-turnover activity. Most notably, we identified a cytidinylylated-substrate intermediate that is formed during the reaction of LarC(Mt). Selected LarC(Mt) variants with substitutions at the predicted CTP-binding site retained substantial amounts of activity, but exhibited greatly reduced levels of the CMP-P2TMN intermediate. In contrast, enhanced amounts of the CMP-P2TMN intermediate were generated when using LarC(Mt) from cells grown on medium without supplemental nickel. On the basis of these results, we propose a functional role for CTP in the unprecedented nickel-insertase reaction during NPN biosynthesis.

Lactate racemase interconverts the L- and D-isomers of lactic acid, a central metabolic intermediate in cells. In 2015, the Hausinger laboratory discovered that the enzyme responsible for this activity in Lactobacillus plantarum, LarA, possesses a covalently-tethered coenzyme they named the nickel-pincer nucleotide (NPN) cofactor. This novel organometallic molecule contains a square-planar nickel atom bound by one histidine residue and tri-coordinated by a modified pyridinium mononucleotide forming C-Ni and two S-Ni bonds. In this seminar, Dr. Hausinger will describe an array of structure/function studies that characterize the three enzymes used for biosynthesis of the NPN cofactor. LarB is a carboxylase/hydrolase of nicotinic acid adenine dinucleotide that forms a dicarboxylated pyridine mononucleotide (P2CMN). LarE is an ATP-dependent sacrificial sulfur insertase that converts P2CMN into a species with two thiocarboxylic acids (P2TMN). Finally, LarC is a CTP-dependent nickel insertase or cyclometallase that transforms P2TMN into NPN. The NPN cofactor is incorporated into a variety of 2-hydroxyacid racemases and selected sugar epimerases where it catalyzes proton-coupled hydride transfer reactions.