Regeneration of insulin-producing β-cells is an alternative avenue to manage diabetes, and it is crucial to unravel this process in vivo during physiological responses to the lack of β-cells. Here, we aimed to characterize how hepatocytes can contribute to β-cell regeneration, either directly or indirectly via secreted proteins or metabolites, in a zebrafish model of β-cell loss. Using lineage tracing, we show that hepatocytes do not directly convert into β-cells even under extreme β-cell ablation conditions. A transcriptomic analysis of isolated hepatocytes after β-cell ablation displayed altered lipid- and glucose-related processes. Based on the transcriptomics, we performed a genetic screen that uncovers a potential role of the molybdenum cofactor (Moco) biosynthetic pathway in β-cell regeneration and glucose metabolism in zebrafish. Consistently, molybdenum cofactor synthesis 2 (Mocs2) haploinsufficiency in mice indicated dysregulated glucose metabolism and liver function. Together, our study sheds light on the liver-pancreas crosstalk and suggests that the molybdenum cofactor biosynthesis pathway should be further studied in relation to glucose metabolism and diabetes.

Polarization and charge-transfer interactions play an important role in ligand-receptor complexes containing metals, and only quantum mechanics methods can adequately describe their contribution to the binding energy. In this work, we selected a set of benzenesulfonamide ligands of human Carbonic Anhydrase II (hCA II)-an important druggable target containing a Zn2+ ion in the active site-as a case study to predict the binding free energy in metalloprotein-ligand complexes and designed specialized computational methods that combine the ab initio fragment molecular orbital (FMO) method and GRID approach. To reproduce the experimental binding free energy in these systems, we adopted a machine-learning approach, here named formula generator (FG), considering different FMO energy terms, the hydrophobic interaction energy (computed by GRID) and logP. The main advantage of the FG approach is that it can find nonlinear relations between the energy terms used to predict the binding free energy, explicitly showing their mathematical relation. This work showed the effectiveness of the FG approach, and therefore, it might represent an important tool for the development of new scoring functions. Indeed, our scoring function showed a high correlation with the experimental binding free energy (R2 = 0.76-0.95, RMSE = 0.34-0.18), revealing a nonlinear relation between energy terms and highlighting the relevant role played by hydrophobic contacts. These results, along with the FMO characterization of ligand-receptor interactions, represent important information to support the design of new and potent hCA II inhibitors.

The 2011 discovery of the first rare earth-dependent enzyme in methylotrophic Methylobacterium extorquens AM1 prompted intensive research toward understanding the unique chemistry at play in these systems. This enzyme, an alcohol dehydrogenase (ADH), features a La3+ ion closely associated with redox-active coenzyme pyrroloquinoline quinone (PQQ) and is structurally homologous to the Ca2+-dependent ADH from the same organism. AM1 also produces a periplasmic PQQ-binding protein, PqqT, which we have now structurally characterized to 1.46-Å resolution by X-ray diffraction. This crystal structure reveals a Lys residue hydrogen-bonded to PQQ at the site analogously occupied by a Lewis acidic cation in ADH. Accordingly, we prepared K142A- and K142D-PqqT variants to assess the relevance of this site toward metal binding. Isothermal titration calorimetry experiments and titrations monitored by UV-Vis absorption and emission spectroscopies support that K142D-PqqT binds tightly (Kd = 0.6 ± 0.2 μM) to La3+ in the presence of bound PQQ and produces spectral signatures consistent with those of ADH enzymes. These spectral signatures are not observed for WT- or K142A-variants or upon addition of Ca2+ to PQQ ⸦ K142D-PqqT. Addition of benzyl alcohol to La3+-bound PQQ ⸦ K142D-PqqT (but not Ca2+-bound PQQ ⸦ K142D-PqqT, or La3+-bound PQQ ⸦ WT-PqqT) produces spectroscopic changes associated with PQQ reduction, and chemical trapping experiments reveal the production of benzaldehyde, supporting ADH activity. By creating a metal binding site that mimics native ADH enzymes, we present a rare earth-dependent artificial metalloenzyme primed for future mechanistic, biocatalytic, and biosensing applications.

Metal-dependent enzymes are abundant and vital catalytic agents in nature. The functional versatility of metalloenzymes has made them common targets for improvement by protein engineering as well as mimicry by de novo designed sequences. In both strategies, the incorporation of non-canonical cofactors and/or non-canonical side chains has proved a useful tool. Less explored-but similarly powerful-is the utilization of non-canonical covalent modifications to the polypeptide backbone itself. Such efforts can entail either introduction of limited artificial monomers in natural chains to produce heterogeneous backbones or construction of completely abiotic oligomers that adopt defined folds. Herein, we review recent research applying artificial protein-like backbones in the construction of metalloenzyme mimics, highlighting progress as well as open questions in this emerging field.

Embedding a catalytically competent transition metal into a protein scaffold affords an artificial metalloenzyme (ArM). Such hybrid catalysts display features that are reminiscent of both homogeneous and enzymatic catalysts. Pioneered by Whitesides and Kaiser in the late 1970s, this field of ArMs has expanded over the past two decades, marked by ever-increasing diversity in reaction types, cofactors, and protein scaffolds. Recent noteworthy developments include i) the use of earth-abundant metal cofactors, ii) concurrent cascade reactions, iii) synergistic catalysis, and iv) in vivo catalysis. Thanks to significant progress in computational protein design, ArMs based on de novo-designed proteins and tailored chimeric proteins promise a bright future for this exciting field.

Hydrogen-bonding (H-bonding) interactions in metalloprotein active sites can critically regulate enzyme function. Changes in the protein structure triggered by interplay with substrates, products, and partner proteins are often translated to the metallocofactor by way of specific changes in H-bond networks connected to the active site. However, the complexities of metalloprotein architecture and mechanism often preclude our ability to define the precise molecular interactions giving rise to these intricate regulatory pathways. To address this shortcoming, we have developed conformationally switchable artificial metalloproteins (swArMs) in which allosteric Gln-binding triggers protein conformational changes that impact the microenvironment surrounding an installed metallocofactor. Herein, we report a combined structural, spectroscopic, and computational approach to enhance the conformation-dependent changes in H-bond interactions surrounding the metallocofactor site of a swArM. Structure-informed molecular dynamics simulations were employed to predict point mutations that could enhance active site H-bond interactions preferentially in the Gln-bound holo-conformation of the swArM. Testing our predictions via the unique infrared spectral signals associated with the metallocofactor site, we have identified three key residues capable of imparting conformational control over the metallocofactor microenvironment. The resultant swArMs not only model biologically relevant structural regulation but also provide an enhanced Gln-responsive biological probe to be leveraged in future biosensing applications.

The continuously increasing level of atmospheric CO2 in the atmosphere has led to global warming. Converting CO2 into other carbon compounds could mitigate its atmospheric levels and produce valuable products, as CO2 also serves as a plentiful and inexpensive carbon feedstock. However, the inert nature of CO2 poses a major challenge for its reduction. To meet the challenge, nature has evolved metalloenzymes using transition metal ions like Fe, Ni, Mo, and W, as well as electron-transfer partners for their functions. Mimicking these enzymes, artificial metalloenzymes (ArMs) have been designed using alternative protein scaffolds and various metallocofactors like Ni, Co, Re, Rh, and FeS clusters. Both the catalytic efficiency and the scope of CO2-reduction product of these ArMs have been improved over the past decade. This review first focuses on the natural metalloenzymes that directly reduce CO2 by discussing their structures and active sites, as well as the proposed reaction mechanisms. It then introduces the common strategies for electrochemical, photochemical, or photoelectrochemical utilization of these native enzymes for CO2 reduction and highlights the most recent advancements from the past five years. We also summarize principles of protein design for bio-inspired ArMs, comparing them with native enzymatic systems and outlining challenges and opportunities in enzymatic CO2 reduction.

About 50 proteins expressed in plastids of photosynthetic eukaryotes ligate iron‑sulfur (Fe-S) clusters and ensure vital functions in photosynthesis, sulfur and nitrogen assimilation, but also in the synthesis of pigments, vitamins and hormones. The synthesis of these Fe-S clusters, which are co- or post-translationally incorporated into these proteins, relies on several proteins belonging to the so-called sulfur mobilization (SUF) machinery. An Fe-S cluster is first de novo synthesized on a scaffold protein complex before additional late-acting maturation factors act in the specific transfer, possible conversion and insertion of this cluster into target recipient proteins. In this review, we will summarize what is known about the molecular mechanisms responsible for both the synthesis and transfer steps, focusing in particular on the structural aspects that allow the formation of the required protein complexes.

Weak magnetic field (WMF) has been recognized to promote biological denitrification processes; however, the underlying mechanisms remain largely unexplored, hindering the optimization of its effectiveness. Here, we systematically investigated the effects of WMF on denitrification performance, enzyme activity, microbial community, and metaproteome in packed bed bioreactors treating high nitrate wastewater under different WMF intensities and C:N ratios. Results showed that WMFs significantly promoted denitrification by consistently stimulating the activities of denitrifying reductases and NAD+/NADH biosynthesis across decreasing C:N ratios. Reductases and electron transfer enzymes involved in denitrification were overproduced due to the significantly enriched overexpression of ferromagnetic ion-containing (FIC) metalloproteins. We also observed WMFs\' intensity-dependent selective pressure on microbial community structures despite the effects being limited compared to those caused by changing C:N ratios. By coupling genome-centric metaproteomics and structure prediction, we found the dominant denitrifier, Halomonas, was outcompeted by Pseudomonas and Azoarcus under WMFs, likely due to its structural deficiencies in iron uptake, suggesting that advantageous ferromagnetic ion acquisition capacity was necessary to satisfy the substrate demand for FIC metalloprotein overproduction. This study advances our understanding of the biomagnetic effects in the context of complex communities and highlights WMF\'s potential for manipulating FIC protein-associated metabolism and fine-tuning community structure.

Methanogens often inhabit sulfidic environments that favor the precipitation of transition metals such as iron (Fe) as metal sulfides, including mackinawite (FeS) and pyrite (FeS2). These metal sulfides have historically been considered biologically unavailable. Nonetheless, methanogens are commonly cultivated with sulfide (HS-) as a sulfur source, a condition that would be expected to favor metal precipitation and thus limit metal availability. Recent studies have shown that methanogens can access Fe and sulfur (S) from FeS and FeS2 to sustain growth. As such, medium supplied with FeS2 should lead to higher availability of transition metals when compared to medium supplied with HS-. Here, we examined how transition metal availability under sulfidic (i.e., cells provided with HS- as sole S source) versus non-sulfidic (cells provided with FeS2 as sole S source) conditions impact the metalloproteome of Methanosarcina barkeri Fusaro. To achieve this, we employed size exclusion chromatography coupled with inductively coupled plasma mass spectrometry and shotgun proteomics. Significant changes were observed in the composition and abundance of iron, cobalt, nickel, zinc, and molybdenum proteins. Among the differences were alterations in the stoichiometry and abundance of multisubunit protein complexes involved in methanogenesis and electron transport chains. Our data suggest that M. barkeri utilizes the minimal iron-sulfur cluster complex and canonical cysteine biosynthesis proteins when grown on FeS2 but uses the canonical Suf pathway in conjunction with the tRNA-Sep cysteine pathway for iron-sulfur cluster and cysteine biosynthesis under sulfidic growth conditions.IMPORTANCEProteins that catalyze biochemical reactions often require transition metals that can have a high affinity for sulfur, another required element for life. Thus, the availability of metals and sulfur are intertwined and can have large impacts on an organismismal biochemistry. Methanogens often occupy anoxic, sulfide-rich (euxinic) environments that favor the precipitation of transition metals as metal sulfides, thereby creating presumed metal limitation. Recently, several methanogens have been shown to acquire iron and sulfur from pyrite, an abundant iron-sulfide mineral that was traditionally considered to be unavailable to biology. The work presented here provides new insights into the distribution of metalloproteins, and metal uptake of Methanosarcina barkeri Fusaro grown under euxinic or pyritic growth conditions. Thorough characterizations of this methanogen under different metal and sulfur conditions increase our understanding of the influence of metal availability on methanogens, and presumably other anaerobes, that inhabit euxinic environments.