Organic Cofactor Research Articles

Presence of vitamin B12 metabolism in the last common ancestor of land plants.

Vitamin B12, also known as cobalamin, is an essential organic cofactor for methionine synthase (METH), and is only synthesized by a subset of bacteria. Plants and fungi have an alternative methionine synthase (METE) that does not need B12 and are typically considered not to utilize it. Some algae facultatively utilize B12 because they encode both METE and METH, while other algae are dependent on B12 as they encode METH only. We performed phylogenomic analyses of METE, METH and 11 further proteins involved in B12 metabolism across more than 1600 plant and algal genomes and transcriptomes (e.g. from OneKp), demonstrating the presence of B12-associated metabolism deep into the streptophytes. METH and five further accessory proteins (MTRR, CblB, CblC, CblD and CblJ) were detected in the hornworts (Anthocerotophyta), and two (CblB and CblJ) were identified in liverworts (Marchantiophyta) in the bryophytes, suggesting a retention of B12-metabolism in the last common land plant ancestor. Our data further show more limited distributions for other B12-related proteins (MCM and RNR-II) and B12 dependency in several algal orders. Finally, considering the collection sites of algae that have lost B12 metabolism, we propose freshwater-to-land transitions and symbiotic associations to have been constraining factors for B12 availability in early plant evolution. This article is part of the theme issue 'The evolution of plant metabolism'.

Investigation of numerical transport models in protein-based molecular junctions with cofactors of diverse chemical natures

The cofactors of proteins dictate the charge transport mechanism across molecular junctions when self-assembled protein monolayers are sandwiched between two metal electrodes. Here, we summarized how the chemical coordination nature of cofactors in various proteins modulates electrical conductance by investigating electronic transport studies across different protein-based molecular junctions under various forces applied under the AFM tip. We have utilized several numerical techniques of electronic transport to analyse the experimentally obtained current–voltage measurements across various protein-based molecular junctions and depicted the origin of electronic modulation in the electrical conductance under different external stimuli. We could also find the origin of electronic conductance modulation under external stimuli at various applied forces by obtaining several analytical transport parameters such as energy barrier, coupling strength, and electrical conductance values. Utilizing density-functional-theory calculations, we further validate that the electronic density of states present in the cofactors within the proteins dominates the electronic transport behaviours across protein-based molecular junctions. Our findings reveal the limiting factor for applying various external stimuli on different proteins, which could be further valuable in bioelectronic applications. We have also found that the organic cofactor containing protein follows all the tunneling mechanism-related numerical transport models and the electronic transport across proteins with pure inorganic cofactors follows Landauer transport formalism.

Peptides En Route from Prebiotic to Biotic Catalysis.

ConspectusIn the quest to understand prebiotic catalysis, different molecular entities, mainly minerals, metal ions, organic cofactors, and ribozymes, have been implied as key players. Of these, inorganic and organic cofactors have gained attention for their ability to catalyze a wide array of reactions central to modern metabolism and frequently participate in these reactions within modern enzymes. Nevertheless, bridging the gap between prebiotic and modern metabolism remains a fundamental question in the origins of life.In this Account, peptides are investigated as a potential bridge linking prebiotic catalysis by minerals/cofactors to enzymes that dominate modern life's chemical reactions. Before ribosomal synthesis emerged, peptides of random sequences were plausible on early Earth. This was made possible by different sources of amino acid delivery and synthesis, as well as their condensation under a variety of conditions. Early peptides and proteins probably exhibited distinct compositions, enriched in small aliphatic and acidic residues. An increase in abundance of amino acids with larger side chains and canonical basic groups was most likely dependent on the emergence of their more challenging (bio)synthesis. Pressing questions thus arise: how did this composition influence the early peptide properties, and to what extent could they contribute to early metabolism?Recent research from our group and colleagues shows that highly acidic peptides/proteins comprising only the presumably "early" amino acids are in fact competent at secondary structure formation and even possess adaptive folding characteristics such as spontaneous refoldability and chaperone independence to achieve soluble structures. Moreover, we showed that highly acidic proteins of presumably "early" composition can still bind RNA by utilizing metal ions as cofactors to bridge carboxylate and phosphoester functional groups. And finally, ancient organic cofactors were shown to be capable of binding to sequences from amino acids considered prebiotically plausible, supporting their folding properties and providing functional groups, which would nominate them as catalytic hubs of great prebiotic relevance.These findings underscore the biochemical plausibility of an early peptide/protein world devoid of more complex amino acids yet collaborating with other catalytic species. Drawing from the mechanistic properties of protein-cofactor catalysis, it is speculated here that the early peptide/protein-cofactor ensemble could facilitate a similar range of chemical reactions, albeit with lower catalytic rates. This hypothesis invites a systematic experimental test.Nonetheless, this Account does not exclude other scenarios of prebiotic-to-biotic catalysis or prioritize any specific pathways of prebiotic syntheses. The objective is to examine peptide availability, composition, and functional potential among the various factors involved in the emergence of early life.

Recent Advances of Oxalate Decarboxylase: Biochemical Characteristics, Catalysis Mechanisms, and Gene Expression and Regulation.

Oxalate decarboxylase (OXDC) is a typical Mn2+/Mn3+ dependent metal enzyme and splits oxalate to formate and CO2 without any organic cofactors. Fungi and bacteria are the main organisms expressing the OXDC gene, but with a significantly different mechanism of gene expression and regulation. Many articles reported its potential applications in the clinical treatment of hyperoxaluria, low-oxalate food processing, degradation of oxalate salt deposits, oxalate acid diagnostics, biocontrol, biodemulsifier, and electrochemical oxidation. However, some questions still remain to be clarified about the role of substrate binding and/or protein environment in modulating the redox properties of enzyme-bound Mn(II)/Mn(III), the nature of dioxygen involved in the catalytic mechanism, and how OXDC acquires Mn(II) /Mn(III). This review mainly summarizes its biochemical and structure characteristics, gene expression and regulation, and catalysis mechanism. We also deep-mined oxalate decarboxylase gene data from National Center for Biotechnology Information to give some insights to explore new OXDC with diverse biochemical properties.

History of Maturation of Prokaryotic Molybdoenzymes-A Personal View.

In prokaryotes, the role of Mo/W enzymes in physiology and bioenergetics is widely recognized. It is worth noting that the most diverse family of Mo/W enzymes is exclusive to prokaryotes, with the probable existence of several of them from the earliest forms of life on Earth. The structural organization of these enzymes, which often include additional redox centers, is as diverse as ever, as is their cellular localization. The most notable observation is the involvement of dedicated chaperones assisting with the assembly and acquisition of the metal centers, including Mo/W-bisPGD, one of the largest organic cofactors in nature. This review seeks to provide a new understanding and a unified model of Mo/W enzyme maturation.

Anchoring a Structurally Editable Proximal Cofactor‐like Module to Construct an Artificial Dual‐center Peroxygenase

AbstractA recent novel strategy for constructing artificial metalloenzymes (ArMs) that target new‐to‐nature functions uses dual‐functional small molecules (DFSMs) with catalytic and anchoring groups for converting P450BM3 monooxygenase into a peroxygenase. However, this process requires excess DFSMs (1000 equivalent of P450) owing to their low binding affinity for P450, thus severely limiting its practical application. Herein, structural optimization of the DFSM‐anchoring group considerably enhanced their binding affinity by three orders of magnitude (Kd≈10−8 M), thus approximating native cofactors, such as FMN or FAD in flavoenzymes. An artificial cofactor‐driven peroxygenase was thus constructed. The co‐crystal structure of P450BM3 bound to a DFSM clearly revealed a precatalytic state in which the DFSM participates in H2O2 activation, thus facilitating peroxygenase activity. Moreover, the increased binding affinity substantially decreases the DFSM load to as low as 2 equivalents of P450, while maintaining increased activity. Furthermore, replacement of catalytic groups showed disparate selectivity and activity for various substrates. This study provides an unprecedented approach for assembling ArMs by binding editable organic cofactors as a co‐catalytic center, thereby increasing the catalytic promiscuity of P450 enzymes.

Anchoring a Structurally Editable Proximal Cofactor-like Module to Construct an Artificial Dual-center Peroxygenase.

A recent novel strategy for constructing artificial metalloenzymes (ArMs) that target new-to-nature functions uses dual-functional small molecules (DFSMs) with catalytic and anchoring groups for converting P450BM3 monooxygenase into a peroxygenase. However, this process requires excess DFSMs (1000 equivalent of P450) owing to their low binding affinity for P450, thus severely limiting its practical application. Herein, structural optimization of the DFSM-anchoring group considerably enhanced their binding affinity by three orders of magnitude (Kd ≈10-8 M), thus approximating native cofactors, such as FMN or FAD in flavoenzymes. An artificial cofactor-driven peroxygenase was thus constructed. The co-crystal structure of P450BM3 bound to a DFSM clearly revealed a precatalytic state in which the DFSM participates in H2 O2 activation, thus facilitating peroxygenase activity. Moreover, the increased binding affinity substantially decreases the DFSM load to as low as 2 equivalents of P450, while maintaining increased activity. Furthermore, replacement of catalytic groups showed disparate selectivity and activity for various substrates. This study provides an unprecedented approach for assembling ArMs by binding editable organic cofactors as a co-catalytic center, thereby increasing the catalytic promiscuity of P450 enzymes.

Electron transport chains as a window into the earliest stages of evolution

The origin and early evolution of life is generally studied under two different paradigms: bottom up and top down. Prebiotic chemistry and early Earth geochemistry allow researchers to explore possible origin of life scenarios. But for these “bottom–up” approaches, even successful experiments only amount to a proof of principle. On the other hand, “top–down” research on early evolutionary history is able to provide a historical account about ancient organisms, but is unable to investigate stages that occurred during and just after the origin of life. Here, we consider ancient electron transport chains (ETCs) as a potential bridge between early evolutionary history and a protocellular stage that preceded it. Current phylogenetic evidence suggests that ancestors of several extant ETC components were present at least as late as the last universal common ancestor of life. In addition, recent experiments have shown that some aspects of modern ETCs can be replicated by minerals, protocells, or organic cofactors in the absence of biological proteins. Here, we discuss the diversity of ETCs and other forms of chemiosmotic energy conservation, describe current work on the early evolution of membrane bioenergetics, and advocate for several lines of research to enhance this understanding by pairing top–down and bottom–up approaches.

Interaction of Organisms in Abandoned Tin Mining Pits: Perspective of Life in Acid Mine Drainage Environment

Acid Mine Drainage (AMD) occured after mining activity exposes metal sulphides to oxidizing conditions that impact acidic condition with low pH value in the waters and heavy metals contamination. These conditions are also occurred in abandoned tin mining pits, one of placed in Bangka Belitung Archipelago Province. This mini review aimed to elaborate information of micro- and macro-organism’ life in acid mine drainage to be associated with the possibility of life in abandoned tin mining pits. Acidophilic and acidotolerant organisms such as bacterial, phyto- and zooplankton, and some macroorganism included invertebrate or vertebrate like fishes and also water plants were found in these waters. Their presence developed a symbiosis interaction in aquatic environment. Phytoplankton is an autotroph organism, despite being considered autotroph organism, many phytoplankton require exogenous organic cofactors and nutrients for their life. These cofactors were often served by heterotroph bacterial to sustain the growth of phytoplankton. Instead, bacterial obtained dissolved organic matter derived from phytoplankton to survive in the aquatic environment. Furthermore, phytoplankton was consumed by zooplankton; zooplankton was consumed by small fish to big fish in the waters. In addition, water plants also support the interaction of organisms in the water by supplying dissolved oxygen, also anorganic and organic material for their life. The symbiosis and quorum sensing plays an important role in structuring the aquatic food web and creating a life in the acidic water-polluted heavy metal.

Biotin Binding Hardly Affects Electron Transport Efficiency across Streptavidin Solid-State Junctions

The electron transport (ETp) efficiency of solid-state protein-mediated junctions is highly influenced by the presence of electron-rich organic cofactors or transition metal ions. Hence, we chose to investigate an interesting cofactor-free non-redox protein, streptavidin (STV), which has unmatched strong binding affinity for an organic small-molecule ligand, biotin, which lacks any electron-rich features. We describe for the first time meso-scale ETp via electrical junctions of STV monolayers and focus on the question of whether the rate of ETp across both native and thiolated STV monolayers is influenced by ligand binding, a process that we show to cause some structural conformation changes in the STV monolayers. Au nanowire-electrode-protein monolayer-microelectrode junctions, fabricated by modifying an earlier procedure to improve the yields of usable junctions, were employed for ETp measurements. Our results on compactly integrated, dense, uniform, ∼3 nm thick STV monolayers indicate that, notwithstanding the slight structural changes in the STV monolayers upon biotin binding, there is no statistically significant conductance change between the free STV and that bound to biotin. The ETp temperature (T) dependence over the 80-300 K range is very small but with an unusual, slightly negative (metallic-like) dependence toward room temperature. Such dependence can be accounted for by the reversible structural shrinkage of the STV at temperatures below 160 K.

Conformational change of organic cofactor PLP is essential for catalysis in PLP-dependent enzymes

Pyridoxal 5’-phosphate (PLP)-dependent enzymes are ubiquitous, catalyzing various biochemical reactions of approximately 4% of all classified enzymatic activities. They transform amines and amino acids into important metabolites or signaling molecules and are important drug targets in many diseases. In the crystal structures of PLP-dependent enzymes, organic cofactor PLP showed diverse conformations depending on the catalytic step. The conformational change of PLP is essential in the catalytic mechanism. In the study, we review the sophisticated catalytic mechanism of PLP, especially in transaldimination reactions. Most drugs targeting PLP-dependent enzymes make a covalent bond to PLP with the transaldimination reaction. A detailed understanding of organic cofactor PLP will help develop a new drug against PLP-dependent enzymes.

The pathway for coenzyme M biosynthesis in bacteria

Mercaptoethane sulfonate or coenzyme M (CoM) is the smallest known organic cofactor and is most commonly associated with the methane-forming step in all methanogenic archaea but is also associated with the anaerobic oxidation of methane to CO2 in anaerobic methanotrophic archaea and the oxidation of short-chain alkanes in Syntrophoarchaeum species. It has also been found in a small number of bacteria capable of the metabolism of small organics. Although many of the steps for CoM biosynthesis in methanogenic archaea have been elucidated, a complete pathway for the biosynthesis of CoM in archaea or bacteria has not been reported. Here, we present the complete CoM biosynthesis pathway in bacteria, revealing distinct chemical steps relative to CoM biosynthesis in methanogenic archaea. The existence of different pathways represents a profound instance of convergent evolution. The five-step pathway involves the addition of sulfite, the elimination of phosphate, decarboxylation, thiolation, and the reduction to affect the sequential conversion of phosphoenolpyruvate to CoM. The salient features of the pathway demonstrate reactivities for members of large aspartase/fumarase and pyridoxal 5'-phosphate-dependent enzyme families.

A consensus view of the proteome of the last universal common ancestor.

The availability of genomic and proteomic data from across the tree of life has made it possible to infer features of the genome and proteome of the last universal common ancestor (LUCA). A number of studies have done so, all using a unique set of methods and bioinformatics databases. Here, we compare predictions across eight such studies and measure both their agreement with one another and with the consensus predictions among them. We find that some LUCA genome studies show a strong agreement with the consensus predictions of the others, but that no individual study shares a high or even moderate degree of similarity with any other individual study. From these observations, we conclude that the consensus among studies provides a more accurate depiction of the core proteome of the LUCA and its functional repertoire. The set of consensus LUCA protein family predictions between all of these studies portrays a LUCA genome that, at minimum, encoded functions related to protein synthesis, amino acid metabolism, nucleotide metabolism, and the use of common, nucleotide‐derived organic cofactors.

Small-molecule autocatalytic networks are universal metabolic fossils.

Life and the genetic code are self-referential and so are autocatalytic networks made of simpler, small molecules. Several origins of life theories postulate autocatalytic chemical networks preceding the primordial genetic code, yet demonstration with biochemical systems is lacking. Here, small-molecule reflexively autocatalytic food-generated networks (RAFs) ranging in size from 3 to 619 reactions were found in all of 6683 prokaryotic metabolic networks searched. The average maximum RAF size is 275 reactions for a rich organic medium and 93 for a medium with a single organic cofactor, NAD. In the rich medium, all universally essential metabolites are produced with the exception of glycerol-1-p (archaeal lipid precursor), phenylalanine, histidine and arginine. The 300 most common reactions, present in at least 2732 RAFs, are mostly involved in amino acid biosynthesis and the metabolism of carbon, 2-oxocarboxylic acid and purines. ATP and NAD are central in generating network complexity, and because ATP is also one of the monomers of RNA, autocatalytic networks producing redox and energy currencies are a strong candidate niche of the origin of a primordial information-processing system. The wide distribution of small-molecule autocatalytic networks indicates that molecular reproduction may be much more prevalent in the Universe than hitherto predicted. This article is part of the theme issue 'Emergent phenomena in complex physical and socio-technical systems: from cells to societies'.

Kinetic resolution of racemic benzofused alcohols catalysed by HMFO variants in presence of natural deep eutectic solvents

5-Hydroxymethylfurfural oxidase (HMFO) has demonstrated to be a useful biocatalyst for the selective oxidation of alcohols employing oxygen as mild oxidant with no requirement of expensive organic cofactors. This wild-type HMFO biocatalyst and an engineered thermostable variant have been tested in the kinetic resolution of different benzofused alcohols. The use of natural deep eutectic solvents was also explored in HMFO-catalysed oxidation of alcohols. The oxidation of racemic 1-indanol showed a higher conversion and selectivity in presence of 60% v/v of different NADES, especially for those containing carbohydrates. By choosing properly the biocatalyst and the NADES, good enantioselectivity values can be obtained, demonstrating the advantages of employing these neoteric solvents in biocatalysed processes.

D‐Band EPR and ENDOR Spectroscopy of 15N‐Labeled Photosystem I

For billions of years, nature has optimized the photosynthetic machinery that converts light energy into chemical energy. Key primary reactions of photosynthesis occur in large membrane protein–cofactor complexes. The light-induced sequential electron transfer reactions occur through a chain of donor/acceptor cofactors embedded in the protein matrix resulting in a long-lived transmembrane charge-separated state. EPR is the method of choice to study electron transfer and the interaction of protein environment with redox-active cofactors. However, the spectra of organic cofactor radicals typically are not fully resolved and severely overlap at conventional X-band EPR. Even at Q-band EPR, this overlap is present and often a serious problem. As a result, there is a large variation of the reported EPR data and limited understanding of electronic structures of several redox-active cofactors. These serious problems can often be overcome by the excellent spectral resolution provided by high-frequency EPR (HF EPR). Here, we study the electronic structure of the primary electron donor P700 and the secondary electron acceptor A1 of Photosystem I (PSI) using 130 GHz (D-band) EPR and Electron–Nuclear-Double-Resonance (ENDOR) spectroscopy. PSI was isotopically labeled with 15N (I = ½) to avoid quadrupolar interactions in the most abundant nitrogen isotope 14N (I = 1) and simplify the ENDOR spectra. ENDOR spectroscopy is central for determining the hyperfine coupling of nitrogen atoms of the two chlorophyll molecules comprising oxidized P700 and the involvement of protein nitrogen atoms with reduced A1. While HF ENDOR of A1− allows identification of two nitrogen atoms, HF ENDOR of P700+ still does not permit unique assignment of the recorded hyperfine couplings.

A shared mechanistic pathway for pyridoxal phosphate–dependent arginine oxidases

The mechanism by which molecular oxygen is activated by the organic cofactor pyridoxal phosphate (PLP) for oxidation reactions remains poorly understood. Recent work has identified arginine oxidases that catalyze desaturation or hydroxylation reactions. Here, we investigate a desaturase from the Pseudoalteromonas luteoviolacea indolmycin pathway. Our work, combining X-ray crystallographic, biochemical, spectroscopic, and computational studies, supports a shared mechanism with arginine hydroxylases, involving two rounds of single-electron transfer to oxygen and superoxide rebound at the 4' carbon of the PLP cofactor. The precise positioning of a water molecule in the active site is proposed to control the final reaction outcome. This proposed mechanism provides a unified framework to understand how oxygen can be activated by PLP-dependent enzymes for oxidation of arginine and elucidates a shared mechanistic pathway and intertwined evolutionary history for arginine desaturases and hydroxylases.

Calcium in Signaling: Its Specificity and Vulnerabilities toward Biogenic and Abiogenic Metal Ions.

Divalent calcium ion (Ca2+) plays an indispensable role as a second messenger in a myriad of signal transduction processes. Of utmost importance for the faultless functioning of calcium-modulated signaling proteins is their binding selectivity of the native metal cation over rival biogenic/abiogenic metal ion contenders in the intra/extracellular fluids. In this Perspective, we summarize recent findings on the competition between the cognate Ca2+ and other biogenic or abiogenic divalent cations for binding to Ca2+-signaling proteins or organic cofactors. We describe the competition between the two most abundant intracellular biogenic metal ions (Mg2+ and Ca2+) for Ca2+-binding sites in signaling proteins, followed by the rivalry between native Ca2+ and "therapeutic" Li+ as well as "toxic" Pb2+. We delineate the key factors governing the rivalry between the native and non-native cations in proteins and highlight key implications for the biological performance of the respective proteins/organic cofactors.

Mechanisms of O2 Activation by Mononuclear Non-Heme Iron Enzymes.

Two major subclasses of mononuclear non-heme ferrous enzymes use two electron-donating organic cofactors (α-ketoglutarate or pterin) to activate O2 to form FeIV═O intermediates that further react with their substrates through hydrogen atom abstraction or electrophilic aromatic substitution. New spectroscopic methodologies have been developed, enabling the study of the active sites in these enzymes and their oxygen intermediates. Coupled to electronic structure calculations, the results of these spectroscopies provide fundamental insight into mechanism. This Perspective summarizes the results of these studies in elucidating the mechanism of dioxygen activation to form the FeIV═O intermediate and the geometric and electronic structure of this intermediate that enables its high reactivity and selectivity in product formation.

Peroxisomal Cofactor Transport.

Peroxisomes are eukaryotic organelles that are essential for growth and development. They are highly metabolically active and house many biochemical reactions, including lipid metabolism and synthesis of signaling molecules. Most of these metabolic pathways are shared with other compartments, such as Endoplasmic reticulum (ER), mitochondria, and plastids. Peroxisomes, in common with all other cellular organelles are dependent on a wide range of cofactors, such as adenosine 5′-triphosphate (ATP), Coenzyme A (CoA), and nicotinamide adenine dinucleotide (NAD). The availability of the peroxisomal cofactor pool controls peroxisome function. The levels of these cofactors available for peroxisomal metabolism is determined by the balance between synthesis, import, export, binding, and degradation. Since the final steps of cofactor synthesis are thought to be located in the cytosol, cofactors must be imported into peroxisomes. This review gives an overview about our current knowledge of the permeability of the peroxisomal membrane with the focus on ATP, CoA, and NAD. Several members of the mitochondrial carrier family are located in peroxisomes, catalyzing the transfer of these organic cofactors across the peroxisomal membrane. Most of the functions of these peroxisomal cofactor transporters are known from studies in yeast, humans, and plants. Parallels and differences between the transporters in the different organisms are discussed here.