Magnesium-dependent Enzyme Research Articles

Sequential magnesium binding facilitates lysyl-tRNA synthetase to recognize ATP

Aminoacyl-tRNA synthetases (aaRSs) catalyze the ligation of amino acids to cognate tRNAs by consuming one molecule of ATP. Magnesium is essential for the enzymes’ activity. Certain class II aaRSs, such as lysyl-tRNA synthetase (LysRS) and seryl-tRNA synthetase (SerRS), recognize ATP together with three magnesium ions in the active site. The detailed role of how these magnesium ions facilitate the ATP recognition by the enzyme is unclear. Here, we report analyses of a crystal structure of human LysRS, in which the two enzymatic pockets of the LysRS dimer are in different states. One pocket is vacant of ATP, and the other is in an intermediate state of ATP recognition. Interestingly, only one magnesium ion instead of three is bound in both states. Compared with our previously solved LysRS structures, we proposed the order of binding for the three magnesium ions. These structures also reveal multiple intermediate ATP-bound states during the amino acid activation reaction, providing critical insights into the mechanisms of the magnesium-dependent enzyme activity of class II aaRSs.

Evidence for the Chemical Mechanism of RibB (3,4-Dihydroxy-2-butanone 4-phosphate Synthase) of Riboflavin Biosynthesis.

RibB (3,4-dihydroxy-2-butanone 4-phosphate synthase) is a magnesium-dependent enzyme that excises the C4 of d-ribulose-5-phosphate (d-Ru5P) as formate. RibB generates the four-carbon substrate for lumazine synthase that is incorporated into the xylene moiety of lumazine and ultimately the riboflavin isoalloxazine. The reaction was first identified by Bacher and co-workers in the 1990s, and their chemical mechanism hypothesis became canonical despite minimal direct evidence. X-ray crystal structures of RibB typically show two metal ions when solved in the presence of non-native metals and/or liganding non-substrate analogues, and the consensus hypothetical mechanism has incorporated this cofactor set. We have used a variety of biochemical approaches to further characterize the chemistry catalyzed by RibB from Vibrio cholera (VcRibB). We show that full activity is achieved at metal ion concentrations equal to the enzyme concentration. This was confirmed by electron paramagnetic resonance of the enzyme reconstituted with manganese and crystal structures liganded with Mn2+ and a variety of sugar phosphates. Two transient species prior to the formation of products were identified using acid quench of single turnover reactions in combination with NMR for singly and fully 13C-labeled d-Ru5P. These data indicate that dehydration of C1 forms the first transient species, which undergoes rearrangement by a 1,2 migration, fusing C5 to C3 and generating a hydrated C4 that is poised for elimination as formate. Structures determined from time-dependent Mn2+ soaks of VcRibB-d-Ru5P crystals show accumulation in crystallo of the same intermediates. Collectively, these data reveal for the first time crucial transient chemical states in the mechanism of RibB.

The ‘bald’ phenotype (androgenetic alopecia) is caused by the high glycaemic, high cholesterol and low mineral ‘western diet’

BackgroundThe success of 5α-reductase inhibitors in the 1990s vindicated the role of androgens and cast doubt on the role of diet in androgenetic alopecia (AGA). However, poor glucose control and high cholesterol are now recognised as comorbidities, which are salient characters of the ‘western diet’. Scope and approachIn glucose potentiated hair loss, continuous monosaccharide flux to the liver promotes the polyol pathway, causing fatty liver and attenuating synthesis of sex hormone binding globulin, accommodating the increased ratio of dihydrotestosterone (DHT) to testosterone. The scalp of the balding phenotype is characterised by overactive PPAR-γ receptors, increased fatty acid synthesis, enlarged sebaceous glands and sebum secretions. Sebum feeds lipophilic bacteria, such as Propionibacterium acnes, which augment the expression of prostaglandin-type (PGD2 & 15d-PGJ2) ligands of PPAR-γ and increase local insulin sensitivity via Akt/mTOR pathways. In hyperglycaemic events the androgen dependent polyol pathway depletes glucose and generates purine by-products that antagonise adenosine receptors. Mitochondrial reactive oxygen species accumulate, and ATP levels reduce, slowing gluconeogenesis in the outer root sheath keratinocytes of the hair follicle. Furthermore, the current commentary suggests that an important mineral in hair health is magnesium, which is relevant to both glucose and cholesterol potentiated hair loss. Magnesium deficiency not only reinforces insulin resistance, but in cholesterol potentiated hair loss, local magnesium dependent monooxygenase enzymes that metabolise cholesterol and vitamin D are impaired. Furthermore, magnesium deficient muscles at the occipital and temporal region of the skull create mechanical strain against the galea aponeurotica. Key findings and conclusionsTaking all of this into consideration, treatment options for androgenetic alopecia should include a low cholesterol and low glycaemic index diet, improved glucose control, and fortification with magnesium. Furthermore, the current narrative does not endorse severe caloric restriction for obvious health reasons.

The relation between acute changes in the systemic inflammatory response and circulating thiamine and magnesium concentrations after elective knee arthroplasty

Thiamine diphosphate (TDP) and magnesium are co-factors for key enzymes in human intermediary metabolism. However, their role in the systemic inflammatory response (SIR) is not clear. Therefore, the aim of the present study was to examine the relation between acute changes in the SIR and thiamine and magnesium dependent enzyme activity in patients undergoing elective knee arthroplasty (a standard reproducible surgical injury in apparently healthy individuals). Patients (n = 35) who underwent elective total knee arthroplasty had venous blood samples collected pre- and post-operatively for 3 days, for measurement of whole blood TDP, serum and erythrocyte magnesium, erythrocyte transketolase activity (ETKA), lactate dehydrogenase (LDH), glucose and lactate concentrations. Pre-operatively, TDP concentrations, erythrocyte magnesium concentrations, ETKA and plasma glucose were within normal limits for all patients. In contrast, 5 patients (14%) had low serum magnesium concentrations (< 0.75 mmol/L). On post-operative day1, both TDP concentrations (p < 0.001) and basal ETKA (p < 0.05) increased and serum magnesium concentrations decreased (p < 0.001). Erythrocyte magnesium concentrations correlated with serum magnesium concentrations (rs = 0.338, p < 0.05) and remained constant during SIR. Post-operatively 14 patients (40%) had low serum magnesium concentrations. On day1 serum magnesium concentrations were directly associated with LDH (p < 0.05), WCC (p < 0.05) and neutrophils (p < 0.01). Whole blood TDP and basal ETKA increased while serum magnesium concentrations decreased, indicating increased requirement for thiamine and magnesium dependent enzyme activity during SIR. Therefore, thiamine and magnesium represent potentially modifiable therapeutic targets that may modulate the host inflammatory response. Erythrocyte magnesium concentrations are likely to be reliable measures of status, whereas serum magnesium concentrations and whole blood TDP may not.ClinicalTrials.gov: NCT03554668.

Structures of fungal and plant acetohydroxyacid synthases.

Acetohydroxyacid synthase (AHAS), also known as acetolactate synthase, is a flavin adenine dinucleotide-, thiamine diphosphate- and magnesium-dependent enzyme that catalyses the first step in the biosynthesis of branched-chain amino acids1. It is the target for more than 50 commercial herbicides2. AHAS requires both catalytic and regulatory subunits for maximal activity and functionality. Here we describe structures of the hexadecameric AHAS complexes of Saccharomyces cerevisiae and dodecameric AHAS complexes of Arabidopsis thaliana. We found that the regulatory subunits of these AHAS complexes form a core to which the catalytic subunit dimers are attached, adopting the shape of a Maltese cross. The structures show how the catalytic and regulatory subunits communicate with each other to provide a pathway for activation and for feedback inhibition by branched-chain amino acids. We also show that the AHAS complex of Mycobacterium tuberculosis adopts a similar structure, thus demonstrating that the overall AHAS architecture is conserved across kingdoms.

3,4‐dihydroxy‐2‐butanone‐4‐phosphate synthase (RibB) of riboflavin biosynthesis has a mononuclear magnesium active site

Riboflavin (vitamin B2) is a precursor for the synthesis of the cofactors FAD and FMN. Like other vitamins, riboflavin cannot be synthesized by mammals and must be obtained from the diet. Bacteria and plants synthesize riboflavin in five steps using the enzymes RibA‐E. The focus of this study is the 3,4‐dihydroxy‐2‐butanone‐4‐phosphate synthase, otherwise known as RibB. RibB is a magnesium dependent enzyme that catalyzes the conversion of the sugar ribulose‐5‐phosphate (Ru5P), a product of the pentose phosphate pathway, into 3,4‐dihydroxy‐2‐butanone‐4‐phosphate (DHBP). The reaction catalyzed by RibB is an unusual deformylation reaction in which the fourth carbon of the five carbon sugar is removed as formate. Bacher and colleagues1 have proposed that the mechanism is dependent on a skeletal rearrangement linking C3 and C5. We propose a fragmentation mechanism, in which the bond between carbons C3 and C4 is broken allowing formation of the new link between carbons C3 and C5 followed by release of formate. To test these competing hypotheses, we are using NMR and X‐ray crystallography to observe the intermediates formed during catalysis. We have produced 13C labeled Ru5P for use in NMR experiments and shown enzymatic formation of DHBP and formate. We also show the 1.1 Å structure of apo RibB. Previously reported product bound structures of RibB have been modeled with two metal ions in the active site2. However, we report here intrinsic tyrosine fluorescence and activity assay profiles that indicate that RibB requires a single magnesium for full activity.Support or Funding InformationWeaver Graduate Student Fellowship, the National Science Foundation CHE‐1403293, The University of Kansas 2018 General Research FundThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Y-Box-Binding Protein 1 Stimulates Abasic Site Cleavage.

Apurinic/apyrimidinic (AP) sites are among the most frequent DNA lesions. The first step in the AP site repair involves the magnesium-dependent enzyme AP endonuclease 1 (APE1) that catalyzes hydrolytic cleavage of the DNA phosphodiester bond at the 5' side of the AP site, thereby generating a single-strand DNA break flanked by the 3'-OH and 5'-deoxyribose phosphate (dRP) groups. Increased APE1 activity in cancer cells might correlate with tumor chemoresistance to DNA-damaging treatment. It has been previously shown that the multifunctional oncoprotein Y-box-binding protein 1 (YB-1) interacts with APE1 and inhibits APE1-catalyzed hydrolysis of AP sites in single-stranded DNAs. In this work, we demonstrated that YB-1 stabilizes the APE1 complex with double-stranded DNAs containing the AP sites and stimulates cleavage of these AP sites at low magnesium concentrations.

Distinct facilitated diffusion mechanisms by E. coli Type II restriction endonucleases.

The passive search by proteins for particular DNA sequences involving nonspecific DNA is essential for gene regulation, DNA repair, phage defense, and diverse epigenetic processes. Distinct mechanisms contribute to these searches, and it remains unresolved as to which mechanism or blend of mechanisms best suits a particular protein and, more importantly, its biological role. To address this, we compare the translocation properties of two well-studied bacterial restriction endonucleases (ENases), EcoRI and EcoRV. These dimeric, magnesium-dependent enzymes hydrolyze related sites (EcoRI ENase, 5'-GAATTC-3'; EcoRV ENase, 5'-GATATC-3'), leaving overhangs and blunt DNA segments, respectively. Here, we demonstrate that the extensive sliding by EcoRI ENase, involving sliding up to ∼600 bp prior to dissociating from the DNA, contrasts with a larger reliance on hopping mechanism(s) by EcoRV ENase. The mechanism displayed by EcoRI ENase results in a highly thorough search of DNA, whereas the EcoRV ENase mechanism results in an extended, yet less rigorous, interrogation of DNA sequence space. We describe how these mechanistic distinctions are complemented by other aspects of these endonucleases, such as the 10-fold higher in vivo concentrations of EcoRI ENase compared to that of EcoRV ENase. Further, we hypothesize that the highly diverse enzyme arsenal that bacteria employ against foreign DNA involves seemingly similar enzymes that rely on distinct but complementary search mechanisms. Our comparative approach reveals how different proteins utilize distinct site-locating strategies.

Why calcium inhibits magnesium-dependent enzyme phosphoserine phosphatase? A theoretical study

Phosphoserine phosphatase (PSP) utilizes one Mg2+ ion to catalyze the hydrolysis of phospho-l-serine. The displacement of Mg2+ by Ca2+ results in the loss of activity. The reaction mechanisms for the enzyme with both Mg2+ and Ca2+ bound were investigated using hybrid density functional theory. A large quantum chemical model abstracted from the X-ray crystal structure was employed in the calculations. Our calculations shed new insight into the catalytic mechanism of the natural enzyme and its lack of activity by Ca2+ substitution. For the catalytic reaction, our calculations showed that the whole reaction proceeds through two steps, namely dephosphorylation and phosphate hydrolysis. The associated barriers for these two steps are calculated to be 11.9 and 12.0 kcal mol−1, respectively. The Mg-bound Asp11 residue functions as a nucleophile to attack the phosphorus moiety, in concomitant with the departure of the leaving group, which takes a proton from the neutral Asp13 residue. In the subsequent step, the newly formed anionic Asp13 residue activates a water molecule to perform the reverse attack on the phosphoryl intermediate, affording the phosphate product. The substitution of Mg2+ by Ca2+ results in different metal coordination fashion, in which the Asp167 residue changes from bidentate to monodentate and a second water molecule becomes ligated to Ca2+. The calculated barriers for the hydrolysis are ca 8 kcal mol−1 higher than those in the native enzyme, which reconciles with the fact that Ca2+ inhibits the activity of PSP. Several possible reasons are discussed.

How mechanical energy of phosphorylating enzymes transforms into the energy of chemical bonds?

Recent discovery of huge magnesium isotope effect in the rate of enzymatic synthesis of ATP offers a new insight on the mechanochemistry of enzymes as the molecular machines. The activity of magnesium-dependent enzymes (ATPase, phosphocreatine, phosphoglycerate kinases), in which Mg2+ ion has a magnetic isotope nucleus 25Mg, was found to be two or three times higher than that of enzymes in which the Mg2+ ion has nonmagnetic, spinless isotopic nuclei 24Mg or 26Mg. This isotope effect demonstrates unambiguously that the ATP synthesis is a spin-dependent process in which the Mg2+ ion is a reagent. It transforms mechanical energy of unequilibrium conformations of enzyme macromolecule into the energy of the P–O bond in ATP.

Probing of HIV-1 Integrase/DNA Interactions Using Novel Analogs of Viral DNA

The specific activity of the human immunodeficiency virus, type 1 (HIV-1), integrase on the viral long terminal repeat requires the binding of the enzyme to certain sequences located in the U3 and U5 regions at the ends of viral DNA, but the determinants of this specific DNA-protein recognition are not yet completely understood. We synthesized DNA duplexes mimicking the U5 region and containing either 2'-modified nucleosides or 1,3-propanediol insertions and studied their interactions with HIV-1 integrase, using Mn2+ or Mg2+ ions as integrase cofactors. These DNA modifications had no strong effect on integrase binding to the substrate analogs but significantly affected 3'-end processing rate. The effects of nucleoside modifications at positions 5, 6, and especially 3 strongly depended on the cationic cofactor used. These effects were much more pronounced in the presence of Mg2+ than in the presence of Mn2+. Modifications of base pairs 7-9 affected 3'-end processing equally in the presence of both ions. Adenine from the 3rd bp is thought to form at least two hydrogen bonds with integrase that are crucial for specific DNA recognition. The complementary base, thymine, is not important for integrase activity. For other positions, our results suggest that integrase recognizes a fine structure of the sugar-phosphate backbone rather than heterocyclic bases. Integrase interactions with the unprocessed strand at positions 5-8 are more important than interactions with the processed strand for specific substrate recognition. Based on our results, we suggest a model for integrase interaction with the U5 substrate.

Magnesium magnetic isotope effect: A key to the mechanochemistry of phosphorylating enzymes as molecular machines

A discovery of the huge magnesium isotope effect in enzymatic ATP synthesis provides a new insight into mechanochemistry of enzymes as the molecular machines. It has been found that the catalytic activity values of ATPase, creatine kinase and phosphoglycerate kinase are 2 to 4-fold higher once their active sites contain magnetic (25Mg) not spinless, non-magnetic (24Mg, 26Mg), magnesium cation isotopes. This clearly proves that the ATP synthesis is a spin-selective process involving Mg2+ as the electron accepting reagent. The formation of ATP takes place in an ion-radical pair resulted by two partners, ATP oxyradical and Mg+. The magnesium bivalent cation is a key player in this process, this ion transforms the protein molecule mechanics into a mere chemistry. This ion is a most critical detail of structure of the magnesium dependent phosphorylation enzymes as the mechanochemical molecular machines.

SELECTIVITY AND UPTAKE OF LITHIUM

Lithium salts are widely utilized as psychopharmacological agents for the therapy of bipolar disorders and related illnesses. Although it is readily bioavailable and has just a single biologically relevant oxidation state, its biological activity remains poorly understood. This comment will examine several key issues in this area: 1) Given the chemical properties of lithium, how do ion transport and metal-dependent enzymes mediate the selectivity of lithium and other alkali ions? 2) Is it possible to predict which magnesium-dependent enzymes can be inhibited by lithium? Answers to these questions are now accessible because of the recent structural determinations of ion channels and of magnesium-dependent, lithium-sensitive inositol polyphosphate 1-phosphatase. This comment will attempt to synthesize the dispersed coordination chemistry data for lithium which can help us evaluate these potential targets for lithium therapy

5-Aminolaevulinic acid dehydratase: metals, mutants and mechanism.

5-Aminolaevulinic acid dehydratase catalyses the formation of porphobilinogen from two molecules of 5-aminolaevulinic acid. The studies described highlight the importance of a bivalent metal ion and two active-site lysine residues for the functioning of 5-aminolaevulinic acid dehydratase. Dehydratases fall into two main categories: zinc-dependent enzymes and magnesium-dependent enzymes. Mutations that introduced zinc-binding ligands into a magnesium-dependent enzyme conferred an absolute requirement for zinc. Mutagenesis of lysine residues 247 and 195 in the Escherichia coli enzyme lead to dramatic effects on enzyme activity, with lysine 247 being absolutely essential. Mutation of either lysine 247 or 195 to cysteine, and treatment of the mutant enzyme with 2-bromethylamine, resulted in the recovery of substantial enzyme activity. The effects of the site-directed alkylating inhibitor, 5-chlorolaevulinic acid, and 4,7-dioxosebacic acid, a putative intermediate analogue, were investigated by X-ray crystallography. These inhibitors reacted with both active-site lysine residues. The role of these two lysine residues in the enzyme mechanism is discussed.

Structural and catalytic chemistry of magnesium-dependent enzymes.

Structural and catalytic chemistry of magnesium-dependent enzymes.

Magnesium activation of nuclease enzymes—the importance of water

Enzymatic metal-mediated hydrolysis of phosphate esters may proceed through either inner- or outer-sphere pathways. Theoretical and experimental consideration of this problem suggests that outer-sphere pathways should predominate for magnesium-promoted reactions, where hydrogen bonding to metal-bound waters is the dominant stabilizing interaction, Comparison of a variety of magnesium-dependent enzymes reflects a requirement for differentially hydrated metal cofactor, which can only be achieved by limiting the number of protein ligands to the metal. As a result, these enzymes have evolved distinct mechanisms for control of metal binding affinities ( K D), all of which lie in the range of ∼0.5 mM. This article describes and discusses these issues, which have not previously been considered in detail.

2.1 Å structure of Serratia endonuclease suggests a mechanism for binding to double-stranded DNA

The crystal structure of Serratia endonuclease has been solved to 2.1 A by multiple isomorphous replacement. This magnesium-dependent enzyme is equally active against single- and double-stranded DNA, as well as RNA, without any apparent base preference. The Serratia endonuclease fold is distinct from that of other nucleases that have been solved by X-ray diffraction. The refined structure consists of a central layer containing six antiparallel beta-strands which is flanked on one side by a helical domain and on the opposite side by one dominant helix and a very long coiled loop. Electrostatic calculations reveal a strongly polarized molecular surface and suggest that a cleft between this long helix and loop, near His 89, may contain the active site of the enzyme.

Crystallization and preliminary crystallographic analysis of a novel nuclease from Serratia marcescens

Crystals have been obtained of the extracellular endonuclease from the bacterial pathogen Serratia marcescens. This magnesium-dependent enzyme is equally active against single and double-stranded DNA, as well as RNA, without any apparent base preference. The Serratia nuclease is not homologous with staphylococcal nuclease, the only other broad specificity endonuclease for which a structure exists, nor is it homologous with other nucleases that have been solved by X-ray diffraction. The structure of this enzyme should, therefore, provide new information about this class of enzyme. At present we have succeeded in obtaining large, high quality crystals using ammonium sulfate. They crystallize in the orthorhombic space group P2 12 12 1, with cell dimensions a = 106·7 A ̊ , b = 74·5 A ̊ , c = 68·9 A ̊ , and diffract to beyond 2 Å. Low-resolution native data sets have been recorded and a search is under way for heavy-atom derivatives.

A kinetic colorimetric procedure for quantifying magnesium in serum.

We have developed a kinetic colorimetric procedure for determination of magnesium in serum. The magnesium-dependent enzyme glycerol kinase is used to phosphorylate glycerol to glycerol 3-phosphate, the latter being oxidized to dihydroxyacetone phosphate and hydrogen peroxide by glycerophosphate oxidase. The generated hydrogen peroxide is then reduced by peroxidase with the simultaneous oxidative coupling of 4-aminoantipyrine and 2-hydroxy-3,5-dichlorobenzenesulfonate, producing a red reaction product with an absorption maximum at 510 nm. The rate of color production is proportional to the concentration of the Mg X ATP complex, which is, in turn, proportional to the magnesium concentration in serum. This method is rapid and precise, avoids the use of expensive instrumentation, is easily automated, and results compare well with those by the Du Pont aca and manual Magon sulfonate methods.

Action of amiodarone on rabbit muscle calcium-dependent adenosine triphosphatase

The effects of amiodarone [2-butyl-3-(3,5-diiodo-4-β-diethylaminoethoxy-benzoyl)-benzofuran hydrochloride], a potent antianginal and antiarrhythmic drug, have been studied in vitro on the ATP splitting activities of several rabbit muscle preparations. The “actomyosin” and myofibrillar heart and crural muscle preparations contained a calcium-dependent ATPase, which was considered as representative of the contractile system, together with other ATPases which could be kept inactive by omitting magnesium ions and by adding sodium azide (mitochondrial ATPase). Amiodarone inhibits the magnesium-dependent enzyme of all preparations in the same way as it acts on guinea pig heart sodium and potassium activated magnesium-dependent ATPase; 6 inhibition was shown, in the case of heart “actomyosin”, to be competitive with respect to ATP, not to magnesium. On the “contractile” calcium-dependent ATPase of all preparations, however, amiodarone failed to exert any effect at concentrations up to 0.6 mM. It is concluded that amiodarone is not likely to impair muscular and cardiac contractility.