Prosthetic Group Research Articles

Hypochlorous acid inactivates myeloperoxidase inside phagocytosing neutrophils

When neutrophils phagocytose bacteria, they release myeloperoxidase (MPO) into phagosomes to catalyse the conversion of superoxide to the potent antimicrobial oxidant hypochlorous acid (HOCl). Here we show that within neutrophils, MPO is inactivated by HOCl. In this study, we aimed to identify the effects of HOCl on the structure and function of MPO, and determine the enzyme's susceptibility to oxidative inactivation during phagocytosis. When hydrogen peroxide was added to a neutrophil granule extract containing chloride, MPO activity was rapidly lost in a HOCl-dependent reaction. With high concentrations of hydrogen peroxide, western blotting demonstrated that MPO was both fragmented and converted to high molecular weight aggregates. Using the purified enzyme, we showed that HOCl generated by MPO inactivated the enzyme by destroying its prosthetic heme groups and releasing iron. MPO protein was additionally modified by forming high molecular weight aggregates. Before inactivation occurred, MPO chlorinated itself to convert most of its amine groups to dichloramines. When human neutrophils phagocytosed Staphylococcus aureus, they released MPO that was largely inactivated in a process that required production of superoxide. Enzyme inactivation occurred inside neutrophils because it was not blocked when extracellular HOCl was scavenged with methionine. The inactivated enzyme contained a chlorinated tyrosine residue, establishing that it had reacted with HOCl. Our results demonstrate that MPO will substantially inactivate itself during phagocytosis, which may limit oxidant production inside phagosomes. Other neutrophil proteins are also likely to be inactivated. The chloramines formed on neutrophil proteins may contribute to the bactericidal milieu of the phagosome.

Comparison of clinical effects of minimally invasive femoral head replacement and proximal femoral nail antirotation in the treatment of comminuted intertrochanteric fracture in the elderly

To analyze the clinical effect of minimally invasive femoral head replacement and proximal femoral nail antirotation(PFNA) internal fixation at the same time in the treatment of elderly patients with comminuted intertrochanteric fracture. From April 2020 to October 2020, 76 elderly patients with comminuted intertrochanteric fracture treated by minimally invasive femoral head replacement and PFNA were analyzed retrospectively. There were 35 patients in the prosthetic group, including 24 females and 11 males with an average age of (86.2±6.1) years old. There were 41 patients in PFNA group including 28 females and 13 males with an average age of (84.6±5.3) years old. The operation time, intraoperative blood loss, postoperative ambulation time, hospitalization time and complications were observed and compared between two groups. Harris hip score was performed at 1, 6 and 12 months after operation. All patients were followed up for 13 to 17 months with an average of (14.3±1.4) months. The operation time of the prosthesis group was longer than that of the PFNA group (P<0.05);the amount of bleeding in PFNA group was less than that in prosthesis group (P>0.05);the time of ambulation in prosthetic group was earlier than that in PFNA group(P<0.05);the number of complications in the prosthesis group was less than that in the PFNA group(P<0.05); the Harris score of prosthesis group was significantly higher than that of PFNA group at 1 and 6 months after operation (P<0.05), but there was no significant difference in Harris score between two groups at 12 months after operation(P>0.05);the number of complications in the prosthesis group was less than that in the PFNA group (P<0.05). Minimally invasive femoral head replacement is a good choice for the elderly patients with commuited intertrochanteric fracture. It can improve the quality of life and reduce the burden of family members and society.

AlphaFold-predicted protein structures and small-angle X-ray scattering: insights from an extended examination of selected data in the Small-Angle Scattering Biological Data Bank.

By providing predicted protein structures from nearly all known protein sequences, the artificial intelligence program AlphaFold (AF) is having a major impact on structural biology. While a stunning accuracy has been achieved for many folding units, predicted unstructured regions and the arrangement of potentially flexible linkers connecting structured domains present challenges. Focusing on single-chain structures without prosthetic groups, an earlier comparison of features derived from small-angle X-ray scattering (SAXS) data taken from the Small-Angle Scattering Biological Data Bank (SASBDB) is extended to those calculated using the corresponding AF-predicted structures. Selected SASBDB entries were carefully examined to ensure that they represented data from monodisperse protein solutions and had sufficient statistical precision and q resolution for reliable structural evaluation. Three examples were identified where there is clear evidence that the single AF-predicted structure cannot account for the experimental SAXS data. Instead, excellent agreement is found with ensemble models generated by allowing for flexible linkers between high-confidence predicted structured domains. A pool of representative structures was generated using a Monte Carlo method that adjusts backbone dihedral allowed angles along potentially flexible regions. A fast ensemble modelling method was employed that optimizes the fit of pair distance distribution functions [P(r) versus r] and intensity profiles [I(q) versus q] computed from the pool to their experimental counterparts. These results highlight the complementarity between AF prediction, solution SAXS and molecular dynamics/conformational sampling for structural modelling of proteins having both structured and flexible regions.

Radiofluorination of an Anionic, Azide-Functionalized Teroligomer by Copper-Catalyzed Azide-Alkyne Cycloaddition.

This study describes the synthesis, radiofluorination and purification of an anionic amphiphilic teroligomer developed as a stabilizer for siRNA-loaded calcium phosphate nanoparticles (CaP-NPs). As the stabilizing amphiphile accumulates on nanoparticle surfaces, the fluorine-18-labeled polymer should enable to track the distribution of the CaP-NPs in brain tumors by positron emission tomography after application by convection-enhanced delivery. At first, an unmodified teroligomer was synthesized with a number average molecular weight of 4550 ± 20 Da by free radical polymerization of a defined composition of methoxy-PEG-monomethacrylate, tetradecyl acrylate and maleic anhydride. Subsequent derivatization of anhydrides with azido-TEG-amine provided an azido-functionalized polymer precursor (o14PEGMA-N3) for radiofluorination. The 18F-labeling was accomplished through the copper-catalyzed cycloaddition of o14PEGMA-N3 with diethylene glycol-alkyne-substituted heteroaromatic prosthetic group [18F]2, which was synthesized with a radiochemical yield (RCY) of about 38% within 60 min using a radiosynthesis module. The 18F-labeled polymer [18F]fluoro-o14PEGMA was obtained after a short reaction time of 2-3 min by using CuSO4/sodium ascorbate at 90 °C. Purification was performed by solid-phase extraction on an anion-exchange cartridge followed by size-exclusion chromatography to obtain [18F]fluoro-o14PEGMA with a high radiochemical purity and an RCY of about 15%.

Heme induced cell death: You can run from TLR4 but you cannot hide from ROS

Heme is the prosthetic group of several enzymes and proteins, including cytochrome c oxidase and hemoglobin. During hemolytic events, high amounts of heme derived from hemoglobin can be released from red blood cells, contributing to the pathogenesis of different hemolytic diseases. Heme-induced cell death is an important player in this situation, and different studies report that heme stimulus can lead to distinct types of regulated cell death. Here, I hypothesize that, when incubated in the presence of high heme concentration, macrophages display concomitant and distinct types of cell death, including necroptosis and a yet to be identified type of cell death. This hypothesis is based on recently published results discussed throughout this manuscript.

Myoglobin content and oxidative status to understand meat products’ color: Phenomenological based model

Meat color is attributed to myoglobin concentration and redox state. Myoglobin is a water-soluble hemoprotein with iron in its prosthetic group, which defines myoglobin’s redox state via its valence state and ligand linked to the free coordination site. In fresh meat, myoglobin can be in one of three states in dynamic equilibrium: deoxymyoglobin, oxymyoglobin, and metmyoglobin. Myoglobin can chemically interact with numerous components in meat products, affecting its redox state. These structural alterations in myoglobin caused by processing conditions will affect its thermal stability and consequently meat product color. Presently, cured meat products are made with sodium nitrite, a curing agent that reacts with myoglobin in meat, forming nitrosomyoglobin, a stable compound that gives the final meat product a distinctive pink color. This work proposes a mathematical model for understanding myoglobin structural changes during the production of a cured meat product. Because of the phenomenological structure of the established model, valuable knowledge is obtained, allowing new possibilities to be explored in the color of meat products while considering the chemical state of myoglobin. The proposed phenomenological model is validated with an error of roughly 10%.

Structural and Biochemical Investigation of Selected Pathogenic Mutants of the Human Dihydrolipoamide Dehydrogenase.

Clinically relevant disease-causing variants of the human dihydrolipoamide dehydrogenase (hLADH, hE3), a common component of the mitochondrial α-keto acid dehydrogenase complexes, were characterized using a multipronged approach to unravel the molecular pathomechanisms that underlie hLADH deficiency. The G101del and M326V substitutions both reduced the protein stability and triggered the disassembly of the functional/obligate hLADH homodimer and significant FAD losses, which altogether eventually manifested in a virtually undetectable catalytic activity in both cases. The I12T-hLADH variant proved also to be quite unstable, but managed to retain the dimeric enzyme form; the LADH activity, both in the forward and reverse catalytic directions and the affinity for the prosthetic group FAD were both significantly compromised. None of the above three variants lent themselves to an in-depth structural analysis via X-ray crystallography due to inherent protein instability. Crystal structures at 2.89 and 2.44 Å resolutions were determined for the I318T- and I358T-hLADH variants, respectively; structure analysis revealed minor conformational perturbations, which correlated well with the residual LADH activities, in both cases. For the dimer interface variants G426E-, I445M-, and R447G-hLADH, enzyme activities and FAD loss were determined and compared against the previously published structural data.

Effect of long-term influx of tertiary treated wastewater on native bacterial communities in a dry valley topsoil: 16S rRNA gene-based metagenomic analysis of composition and functional profile.

Although dumping treated wastewater into soil might provide nutrients and organic matter, it can also expose the ecosystem to biological and chemical risks. A vital indication of soil health and quality is the soil microbial community. The current work used next-generation 16S rRNA gene amplicon sequencing to evaluate the effects of the long-term influx of tertiary treated wastewater (TWW) into Wadi Uranah, a dry valley in Makkah city, Saudi Arabia, on native topsoil bacterial community composition and predicted functions. The findings demonstrated that neither the compositions of microbial communities nor their predicted functions using PICRUSt2 differed significantly (p>0.05) between polluted valley soil (PolVS) and unpolluted valley soil (UPVS). Alpha and beta diversity, however, showed that the PolVS samples had a considerably higher level of diversity and variability. Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidetes were the most prevalent phyla in both groups. Noticeable relative variations existed in some metabolic pathways such as cofactor, prosthetic group, electron carrier degradation, aldehyde degradation, and Entner-Doudoroff (ED) pathways. Overall, our findings suggest that because both groups have very similar core microbiomes and functions, the long-term disposal of tertiary TWW into Wadi Uranah may have little to no influence on the composition and function of soil bacterial communities. In addition, the long-term discharge of tertiary TWW after partially treated wastewater's initial disposal may have helped the native soil microbial community recover.

Shapes and Patterns of Heme-Binding Motifs in Mammalian Heme-Binding Proteins.

Heme is a double-edged sword. On the one hand, it has a pivotal role as a prosthetic group of hemoproteins in many biological processes ranging from oxygen transport and storage to miRNA processing. On the other hand, heme can transiently associate with proteins, thereby regulating biochemical pathways. During hemolysis, excess heme, which is released into the plasma, can bind to proteins and regulate their activity and function. The role of heme in these processes is under-investigated, with one problem being the lack of knowledge concerning recognition mechanisms for the initial association of heme with the target protein and the formation of the resulting complex. A specific heme-binding sequence motif is a prerequisite for such complex formation. Although numerous short signature sequences indicating a particular protein function are known, a comprehensive analysis of the heme-binding motifs (HBMs) which have been identified in proteins, concerning specific patterns and structural peculiarities, is missing. In this report, we focus on the evaluation of known mammalian heme-regulated proteins concerning specific recognition and structural patterns in their HBMs. The Cys-Pro dipeptide motifs are particularly emphasized because of their more frequent occurrence. This analysis presents a comparative insight into the sequence and structural anomalies observed during transient heme binding, and consequently, in the regulation of the relevant protein.

Proteomic analysis of the effect of hemin in breast cancer

Heme, an iron-containing prosthetic group found in many proteins, carries out diverse biological functions such as electron transfer, oxygen storage and enzymatic reactions. Hemin, the oxidised form of heme, is used to treat porphyria and also to activate heme-oxygenase (HO) which catalyses the rate-limiting step in heme degradation. Our group has previously demonstrated that hemin displays antitumor activity in breast cancer (BC). The aim of this work has been to study the effect of hemin on protein expression modifications in a BC cell line to gain insight into the molecular mechanisms of hemin antitumor activity. For this purpose, we carried out proteome analysis by Mass Spectrometry (MS) which showed that 1309 proteins were significantly increased in hemin-treated cells, including HO-1 and the proteases that regulate HO-1 function, and 921 proteins were significantly decreased. Furthermore, the MS-data analysis showed that hemin regulates the expression of heme- and iron-related proteins, adhesion and cytoskeletal proteins, cancer signal transduction proteins and enzymes involved in lipid metabolism. By biochemical and cellular studies, we further corroborated the most relevant in-silico results. Altogether, these results show the multiple physiological effects that hemin treatment displays in BC and demonstrate its potential as anticancer agent.

Molecular Taphonomy of Heme: Chemical Degradation of Hemin under Presumed Fossilization Conditions.

The metalloporphyrin heme acts as the oxygen-complexing prosthetic group of hemoglobin in blood. Heme has been noted to survive for many millions of years in fossils. Here, we investigate its stability and degradation under various conditions expected to occur during fossilization. Oxidative, reductive, aerobic, and anaerobic conditions were studied at neutral and alkaline pH values. Elevated temperatures were applied to accelerate degradation. High-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) identified four main degradation products. The vinyl residues are oxidized to formyl and further to carboxylate groups. In the presence of air or H2O2, cleavage of the tetrapyrrole ring occurs, and hematinic acid is formed. The highest stability of heme was observed under anaerobic reductive conditions (half-life 9.5 days), while the lowest stability was found in the presence of H2O2 (half-life 1 min). We confirmed that the iron cation plays a crucial role in degradation, since protoporphyrin IX, lacking iron, remained significantly more stable. Under anaerobic, reductive conditions, the above-mentioned degradation products were not observed, suggesting a different degradation pathway. To our knowledge, this is the first molecular taphonomy study on heme, which will be useful for understanding its fate during fossilization.

Heme Interactions as Regulators of the Alternative Pathway Complement Responses and Implications for Heme-Associated Pathologies.

Heme (Fe2+-protoporphyrin IX) is a pigment of life, and as a prosthetic group in several hemoproteins, it contributes to diverse critical cellular processes. While its intracellular levels are tightly regulated by networks of heme-binding proteins (HeBPs), labile heme can be hazardous through oxidative processes. In blood plasma, heme is scavenged by hemopexin (HPX), albumin and several other proteins, while it also interacts directly with complement components C1q, C3 and factor I. These direct interactions block the classical pathway (CP) and distort the alternative pathway (AP). Errors or flaws in heme metabolism, causing uncontrolled intracellular oxidative stress, can lead to several severe hematological disorders. Direct interactions of extracellular heme with alternative pathway complement components (APCCs) may be implicated molecularly in diverse conditions at sites of abnormal cell damage and vascular injury. In such disorders, a deregulated AP could be associated with the heme-mediated disruption of the physiological heparan sulphate-CFH coat of stressed cells and the induction of local hemostatic responses. Within this conceptual frame, a computational evaluation of HBMs (heme-binding motifs) aimed to determine how heme interacts with APCCs and whether these interactions are affected by genetic variation within putative HBMs. Combined computational analysis and database mining identified putative HBMs in all of the 16 APCCs examined, with 10 exhibiting disease-associated genetic (SNPs) and/or epigenetic variation (PTMs). Overall, this article indicates that among the pleiotropic roles of heme reviewed, the interactions of heme with APCCs could induce differential AP-mediated hemostasis-driven pathologies in certain individuals.

Influence of N-arylsulfonamido d-valine N-substituents on the selectivity and potency of matrix metalloproteinase inhibitors

To develop matrix metalloproteinase inhibitors (MMPIs) for both therapy and medicinal imaging by fluorescence-based techniques or positron-emission tomography (PET), a small library of eighteen N-substituted N-arylsulfonamido d-valines were synthesized and their potency to inhibit two gelatinases (MMP-2, and MMP-9), two collagenases (MMP-8, and MMP-13) and macrophage elastase (MMP-12) was determined in a Structure-Activity-Relation study with ({4-[3-(5-methylthiophen-2-yl)-1,2,4-oxadiazol-5-yl]phenyl}sulfonyl)-d-valine (1) as a lead. All compounds were shown to be more potent MMP-2/-9 inhibitors (nanomolar range) compared to other tested MMPs. This is a remarkable result considering that a carboxylic acid group is the zinc binding moiety. The compound with a terminal fluoropropyltriazole group at the furan ring (P1′ substituent) was only four times less potent in inhibiting MMP-2 activity than the lead compound 1, making this compound a promising probe for PET application (after using a prosthetic group approach to introduce fluorine-18). Compounds with a TEG spacer and a terminal azide or even a fluorescein moiety at the sulfonylamide N atom (P2′ substituent) were almost as active as the lead structure 1, making the latter derivative a suitable fluorescence imaging tool.

Evaluation of Substituted N-Aryl Maleimide and Acrylamides for Bioconjugation

Novel SF5-bearing maleimide and acrylamide derivatives were synthesised as potential [18F]radio-prosthetic groups for radiolabelling peptides and proteins. The efficacy of selected prosthetic groups was first assessed through bioconjugation with protected model amino acid derivatives. These reactions were investigated on an analytical scale via LC-MS across a pH range to quantitatively evaluate this prosthetic group’s reactivity and stability. Model bioconjugate reactions were then replicated using analogous para-substituted derivatives to determine the influence of the electronic effects of -SF5. Finally, the SF5-bearing prosthetic groups were utilised for bioconjugation with cancer-targeting c-RGD peptides. N-aryl maleimides reacted extremely efficiently with the model amino acid N-acetyl-L-cysteine. The subsequent conjugates were obtained as regio-isomeric mixtures of the corresponding thio-succinamic acids in yields of 80–96%. Monitoring the bioconjugate reaction by LC-MS revealed that ring hydrolysis of the intermediate SF5–thio-succinimide conjugate occurred instantaneously, an advantageous quality in minimising undesirable thiol exchange reactions with non-targeted cysteine residues. In contrast, N-aryl acrylamides demonstrated poor solubility in semi-aqueous media (&lt;1 mM). In turn, synthetic-scale model bioconjugations with Nα-acetyl-L-lysine were performed in methanol, affording the corresponding acrylamide conjugates in modest to high yield (58–89%). Including electron-deficient, fluorinated prosthetic groups for bioconjugation will broaden their applicability within the fields of 19F-MRI and PET imaging.

Heme: The Lord of the Iron Ring.

Heme is an iron-protoporphyrin complex with an essential physiologic function for all cells, especially for those in which heme is a key prosthetic group of proteins such as hemoglobin, myoglobin, and cytochromes of the mitochondria. However, it is also known that heme can participate in pro-oxidant and pro-inflammatory responses, leading to cytotoxicity in various tissues and organs such as the kidney, brain, heart, liver, and in immune cells. Indeed, heme, released as a result of tissue damage, can stimulate local and remote inflammatory reactions. These can initiate innate immune responses that, if left uncontrolled, can compound primary injuries and promote organ failure. In contrast, a cadre of heme receptors are arrayed on the plasma membrane that is designed either for heme import into the cell, or for the purpose of activating specific signaling pathways. Thus, free heme can serve either as a deleterious molecule, or one that can traffic and initiate highly specific cellular responses that are teleologically important for survival. Herein, we review heme metabolism and signaling pathways, including heme synthesis, degradation, and scavenging. We will focus on trauma and inflammatory diseases, including traumatic brain injury, trauma-related sepsis, cancer, and cardiovascular diseases where current work suggests that heme may be most important.

Development of brain-penetrable antibody radioligands for in vivo PET imaging of amyloid-β and tau.

Alzheimer's disease (AD) is characterized by the misfolding and aggregation of two major proteins: amyloid-beta (Aβ) and tau. Antibody-based PET radioligands are desirable due to their high specificity and affinity; however, antibody uptake in the brain is limited by the blood-brain barrier (BBB). Previously, we demonstrated that antibody transport across the BBB can be facilitated through interaction with the transferrin receptor (TfR), and the bispecific antibody-based PET ligands were capable of detecting Aβ aggregates via ex vivo imaging. Since tau accumulation in the brain is more closely correlated with neuronal death and cognition, we report here our strategies to prepare four F-18-labeled specifically engineered bispecific antibody probes for the selective detection of tau and Aβ aggregates to evaluate their feasibility and specificity, particularly for in vivo PET imaging. We first created and evaluated (via both in vitro and ex vivo studies) four specifically engineered bispecific antibodies, by fusion of single-chain variable fragments (scFv) of a TfR antibody with either a full-size IgG antibody of Aβ or tau or with their respective scFv. Using [18F]SFB as the prosthetic group, all four 18F-labeled bispecific antibody probes were then prepared by conjugation of antibody and [18F]SFB in acetonitrile/0.1 M borate buffer solution (final pH ~ 8.5) with an incubation of 20 min at room temperature, followed by purification on a PD MiniTrap G-25 size exclusion gravity column. Based on both in vitro and ex vivo evaluation, the bispecific antibodies displayed much higher brain concentrations than the unmodified antibody, supporting our subsequent F18-radiolabeling. [18F]SFB was produced in high yields in 60 min (decay-corrected radiochemical yield (RCY) 46.7 ± 5.4) with radiochemical purities of >95%, confirmed by analytical high performance liquid chromatography (HPLC) and radio-TLC. Conjugation of [18F]SFB and bispecific antibodies showed a 65%-83% conversion efficiency with radiochemical purities of 95%-99% by radio-TLC. We successfully labeled four novel and specifically engineered bispecific antibodies with [18F]SFB under mild conditions with a high RCY and purities. This study provides strategies to create brain-penetrable F-18 radiolabeled antibody probes for the selective detection of tau and Aβ aggregates in the brain of transgenic AD mice via in vivo PET imaging.

In silico Study on the Binding Interactions of SSTA and 18F-SSTA Towards Somatostatin Receptor Subtype 2.

Somatostatin analogs (SSTAs) are versatile drugs that target a group of proteins known as somatostatin receptors. SSTAs are used for the treatment and PET-molecular imaging of Neuro Endocrine Tumors (NET), for they are labeled with the radionuclide 18F, a positron emitter radionuclide. The aim of this work was to theoretically study the binding interactions of SSTA labeled with 18F (half-life of 109.7 min) and somatostatin receptor subtype 2. As the labeling of SSTA with 18F required the use of a prosthetic group, a hydrophilicity enhancer, and a linker, the influence of these traits on the interactions of 18F-SSTA with the SSTR-2 binding site was studied. The binding modes of 18F-labeled analogues with SSTR-2 were studied by using protein homology modelling, non-equilibrium molecular dynamics, and molecular docking calculations, by means of three docking software: MVD, MOE, and VINA. The results showed the main role of Asp122, Asn276, Phe272 and Phe294 from the SSTR-2 binding site, which form interactions with residues Lys, Trp, Tyr, and Thr from 18F-labeled somatostatin analogues. The interaction between Lys (from 18F-SSTA) and Asp122 (from SSTR-2) was identified as the most energetic and considered the one that drives the binding between 18F-SSTA and SSTR-2 (the anchor interaction). Despite the presence of prosthetic groups, linkers, and hydrophilicity enhancers, all the studied 18F-SSTA formed the anchor interaction. The trend in the results agreed with the experimental reports, identifying the main role of Asp122 in the binding of somatostatin-14 to SSTR-2.

Crystal structure of FAD-independent methylene-tetrahydrofolate reductase from Mycobacterium hassiacum.

FAD-independent methylene-tetrahydrofolate (methylene-H4 F) reductase (Mfr), recently identified in mycobacteria, catalyzes the reduction of methylene-H4 F to methyl-H4 F with NADH as hydride donor by a ternary complex mechanism. This biochemical reaction corresponds to that of the ubiquitous FAD-dependent methylene-H4 F reductase (MTHFR), although the latter uses a ping-pong mechanism with the prosthetic group as intermediate hydride carrier. Comparative genomics and genetic analyses indicated that Mfr is indispensable for the growth of Mycobacterium tuberculosis, which lacks the MTHFR encoding gene. Therefore, Mfr appears to be an excellent target for the design of antimycobacterial drugs. Here, we report the heterologous production, enzymological characterization, and the crystal structure of Mfr from the thermophilic mycobacterium Mycobacterium hassiacum (hMfr), which shows 78% sequence identity to Mfr from M. tuberculosis. Although hMfr and MTHFR have minor sequence identity and different catalytic mechanisms, their structures are highly similar, thus suggesting a divergent evolution of Mfr and MTHFR from a common ancestor. Most of the important active site residues of MTHFR are conserved and equivalently positioned in the tertiary structure of hMfr. The Glu9Gln variant of hMfr exhibits a drastic reduction of the catalytic activity, which supports the predicted function of the glutamate residue as proton donor in both hMfr and MTHFR. Thus, highly similar binding modes for the C1 -carriers and the reducing agents in hMfr and MTHFR are assumed.

Structural and Biochemical Characterization of Mycobacterium tuberculosis Zinc SufU-SufS Complex.

Iron-sulfur (Fe-S) clusters are inorganic prosthetic groups in proteins composed exclusively of iron and inorganic sulfide. These cofactors are required in a wide range of critical cellular pathways. Iron-sulfur clusters do not form spontaneously in vivo; several proteins are required to mobilize sulfur and iron, assemble and traffic-nascent clusters. Bacteria have developed several Fe-S assembly systems, such as the ISC, NIF, and SUF systems. Interestingly, in Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), the SUF machinery is the primary Fe-S biogenesis system. This operon is essential for the viability of Mtb under normal growth conditions, and the genes it contains are known to be vulnerable, revealing the Mtb SUF system as an interesting target in the fight against tuberculosis. In the present study, two proteins of the Mtb SUF system were characterized for the first time: Rv1464(sufS) and Rv1465(sufU). The results presented reveal how these two proteins work together and thus provide insights into Fe-S biogenesis/metabolism by this pathogen. Combining biochemistry and structural approaches, we showed that Rv1464 is a type II cysteine-desulfurase enzyme and that Rv1465 is a zinc-dependent protein interacting with Rv1464. Endowed with a sulfurtransferase activity, Rv1465 significantly enhances the cysteine-desulfurase activity of Rv1464 by transferring the sulfur atom from persulfide on Rv1464 to its conserved Cys40 residue. The zinc ion is important for the sulfur transfer reaction between SufS and SufU, and His354 in SufS plays an essential role in this reaction. Finally, we showed that Mtb SufS-SufU is more resistant to oxidative stress than E. coli SufS-SufE and that the presence of zinc in SufU is likely responsible for this improved resistance. This study on Rv1464 and Rv1465 will help guide the design of future anti-tuberculosis agents.

Microbial Synthesis of Heme b: Biosynthetic Pathways, Current Strategies, Detection, and Future Prospects.

Heme b, which is characterized by a ferrous ion and a porphyrin macrocycle, acts as a prosthetic group for many enzymes and contributes to various physiological processes. Consequently, it has wide applications in medicine, food, chemical production, and other burgeoning fields. Due to the shortcomings of chemical syntheses and bio-extraction techniques, alternative biotechnological methods have drawn increasing attention. In this review, we provide the first systematic summary of the progress in the microbial synthesis of heme b. Three different pathways are described in detail, and the metabolic engineering strategies for the biosynthesis of heme b via the protoporphyrin-dependent and coproporphyrin-dependent pathways are highlighted. The UV spectrophotometric detection of heme b is gradually being replaced by newly developed detection methods, such as HPLC and biosensors, and for the first time, this review summarizes the methods used in recent years. Finally, we discuss the future prospects, with an emphasis on the potential strategies for improving the biosynthesis of heme b and understanding the regulatory mechanisms for building efficient microbial cell factories.