Substitution Of Amino Acid Residues Research Articles

Rare missense variant in MSH4 associated with primary gonadal failure in both 46, XX and 46, XY individuals.

Can whole-exome sequencing (WES) reveal a shared pathogenic variant responsible for primary gonadal failure in both male and female patients from a consanguineous family? Patients with primary ovarian insufficiency (POI) and non-obstructive azoospermia (NOA) were homozygous for the rare missense variant p. S754L located in the highly conserved MSH4 MutS signature motif of the ATPase domain. An oligozoospermic patient was heterozygous for the variant. MSH4 is a meiosis-specific protein expressed at a certain level in the testes and ovaries. Along with its heterodimer partner MSH5, it is responsible for double-strand Holliday junction recognition and stabilization, to ensure accurate chromosome segregation during meiosis. Knockout male and female mice for Msh4 and Msh5 are reportedly infertile due to meiotic arrest. In humans, MSH4 is associated with male and female gonadal failure, with distinct variations in the MutS domain V. This was a retrospective genetics study of a consanguineous family with multiple cases of gonadal failure in both genders. The subject family was recruited in Iran, in 2018. The proband who is affected by POI, an NOA brother, a fertile sister and their parents were subjected to WES. The discovered variant was validated in these individuals, and the rest of the family was also genotyped by Sanger sequencing. The variant was not detected in 800 healthy Iranian individuals from the Iranome database nor in 30 sporadic NOA and 30 sporadic POI patients. Suggested effect in aberrant splicing was studied by RT-PCR. Moreover, protein homology modeling was used to further investigate the amino acid substitution in silico. The discovered variant is very rare and has never been reported in the homozygous state. It occurs in the ATPase domain at Serine 754, the first residue within the highly conserved MutS signature motif, substituting it with a Leucine. All variant effect prediction tools indicated this variant as deleterious. Since the substitution occurs immediately before the Walker B motif at position 755, further investigations based on protein homology were conducted. Considering the modeling results, the nature of the substituted amino acid residue and the distances between p. S754L variation and the residues of the Walker B motif suggested the possibility of conformational changes affecting the ATPase activity of the protein. We have submitted dbSNP entry rs377712900 to ClinVar under SCV001169709, SCV001169708 and SCV001142647 for oligozoospermia, NOA and POI, respectively. Studies in model organisms can shed more light on the role of this variant as our results were obtained by variant effect prediction tools and protein homology modeling. Identification of variants in meiotic genes should improve genetic counseling for both male and female infertility. Also, as two of our NOA patients underwent testicular sperm extraction (TESE) with no success, ruling out the existence of pathogenic variants in meiotic genes in such patients prior to TESE could prove useful. This study was financially supported by Royan Institute in Tehran, Iran, and Institut Pasteur in Paris, France. The authors declare no competing interests. N/A.

The role of active-site amino acid residues in the cleavage of DNA and RNA substrates by human apurinic/apyrimidinic endonuclease APE1

BackgroundHuman apurinic/apyrimidinic endonuclease APE1 is one of participants of the DNA base excision repair pathway. APE1 processes AP-sites and many other types of DNA damage via hydrolysis of the phosphodiester bond on the 5′ side of the lesion. APE1 also acts as an endoribonuclease, i.e., can cleave undamaged RNA. MethodsUsing pre-steady-state kinetic analysis we examined the role of certain catalytically important amino acids in APE1 enzymatic pathway and described their involvement in the mechanism of the target nucleotide recognition. ResultsComparative analysis of the cleavage efficiency of damaged DNAs containing an abasic site, 5,6-dihydrouridine, or α-anomer of adenosine as well as 3′-5′-exonuclease degradation of undamaged DNA and endonuclease hydrolysis of RNA substrates by mutant APE1 enzymes containing a substitution of an active-site amino acid residue (D210N, N212A, T268D, M270A, or D308A) was performed. Detailed pre–steady-state kinetics of conformational changes of the enzyme and of DNA substrate molecules during recognition and cleavage of the abasic site were studied. ConclusionsIt was revealed that substitution T268D significantly disturbed initial DNA binding, whereas Asn212 is critical for the DNA-bending stage and catalysis. Substitution D210N increased the binding efficacy and blocked the catalytic reaction, but D308A decreased the binding efficacy owing to disruption of Mg2+ coordination. Finally, the substitution of Met270 also destabilized the enzyme–substrate complex but did not affect the catalytic reaction. SignificanceIt was found that the tested substitutions of the active-site amino acid residues affected different stages of the complex formation process as well as the catalytic reaction.

The hydrophobicity of an amino acid residue in a flexible loop of KP-43 protease alters activity toward a macromolecule substrate

KP-43, a 43-kDa alkaline serine protease, is resistant to chemical oxidants and surfactants, making it suitable for use in laundry detergents. An amino acid residue at position 195, in a unique flexible loop that binds a Ca2+ ion, dramatically affects the proteolytic activity and thermal stability of KP-43. In the present study, we obtained 20 variants with substitutions at position 195 and investigated how these residues affect hydrolytic activity toward a macromolecular substrate (casein) and a synthetic tetra-peptide (AAPL). At pH 10, the variant with the highest caseinolytic activity, Tyr195Gln, exhibited 4.4-fold higher activity than the variant with the lowest caseinolytic activity, Tyr195Trp. A significant negative correlation was observed between the hydrophobicity of the residue at position 195 and caseinolytic activity at pH 8–10. At pH 7, the correlation became weak; at pH 6, the correlation reversed to positive. Unlike casein, in the case of hydrolysis of AAPL, no correlation was observed at pH 10 or pH 6. Because the amino acid residue at position 195 is located on the protein surface and considered sufficiently far from the active cleft, the variation in caseinolytic activity between the 20 variants was attributed to changes in interaction efficiency with different states of casein at different pH values. To improve the enzymatic activity, we propose substituting amino acid residues on the protein surface to change the efficiency of interaction with the macromolecular substrates.Key points• A single amino acid residue on the protein surface markedly changed enzyme activity.• The hydrophobicity of the amino acid residue and enzyme activity had a correlation.• The key amino acid residue for substrate recognition exists on the protein surface.

Membrane Binding Promotes Annexin A2 Oligomerization.

Annexin A2 (AnxA2) is a cytosolic Ca2+ regulated membrane binding protein that can induce lipid domain formation and plays a role in exocytosis and endocytosis. To better understand the mode of annexin-membrane interaction, we analyzed membrane-bound AnxA2 assemblies by employing a novel 3-armed chemical crosslinker and specific AnxA2 mutant proteins. Our data show that AnxA2 forms crosslinkable oligomers upon binding to membranes containing negatively charged phospholipids. AnxA2 mutants with amino acid substitutions in residues predicted to be involved in lateral protein–protein interaction show compromised oligomer formation, albeit still being capable of binding to negatively charged membranes in the presence of Ca2+. These results suggest that lateral protein–protein interactions are involved in the formation of AnxA2 clusters on a biological membrane.

Effect of Polar Amino Acid Residue Substitution by Site-Directed Mutagenesis in the N-terminal Domain of Pseudomonas sp. Phytase on Enzyme Activity.

The N-terminal domain of the Pseudomonas sp. FB15 phytase increases low-temperature activity and catalytic efficiency. In this study, the 3D structure of the N-terminal domain was predicted and substitutions for the amino acid residues of the region assumed to be the active site were made. The activity of mutants, in which alanine (A) was substituted for the original residue, was investigated at various temperatures and pH values. Significant differences in enzymatic activity were observed only in mutant E263A, suggesting that the amino acid residue at position 263 of the N-terminal domain is important in enzyme activity.

Insights into the Effect of Genetic Variation in CYP2C9 (*11) on Drug Binding.

Cytochrome P450s (CYP) are a family of membrane associated heme‐containing enzymes involved in xenobiotic metabolism with recent relevance in pharmacogenomics. The human CYP2C9 enzyme metabolizes up to 15–20% of drugs that include warfarin, losartan, ibuprofen, and tolbutamide. It is highly polymorphic with several genetic variations associated with significantly altered activity of the enzyme. The CYP2C9*11 allele occurs predominantly in African Americans but also in Caucasian and other ethnic populations with a frequency of around 0.05. The *11 genetic variation represents a substitution of amino acid residue at 335 from arginine to tryptophan (R335W) that has demonstrated decreased catalytic activity towards various substrates including S‐warfarin. The R335W was created in CYP2C9 wild‐type (WT) construct using site‐directed mutagenesis and co‐expressed in E. Coli alongside chaperone proteins (GroEL/ES). Generation of purified CYP2C9*11 protein enabled catalytic studies and crystallization in complex with the drug substrate losartan. The enzymatic assays revealed marked reduction in losartan turn‐over by CYP2C9*11 compared to the WT enzyme. Additionally, comparison of the amino acid sequence revealed that R335 is conserved across the CYP2C subfamily of enzymes. The R335 is located on the distal J‐J’ loop and interacts with multiple amino acid side chains on the J and J’ helices. The computational analysis suggests that the substitution with hydrophobic and bulkier tryptophan located on the surface region may result in destabilizing interactions with several helices including those located near the active site heme, affecting protein stability and function. Overall, these observations establish the effect of distal variation prompting further evaluation by structural and biophysical studies.Support or Funding InformationThe research was funded by the start‐up funds from the Albany College of Pharmacy and Health Sciences.

Genetic sequence changes related to the attenuation of avian infectious bronchitis virus strain TW2575/98.

The attenuated avian infectious bronchitis virus (IBV), derived from a wild strain (TW2575/98w) in chicken embryos after 75 passages, is designed as a commercial vaccine strain (TW2575/98vac) to control the disease in Taiwan. The differences in viral infectivity, replication efficiency, and genome sequences between TW2575/98w and TW2575/98vac were determined and compared. TW2575/98vac caused earlier death of chicken embryos and had higher viral replication efficiency. Thirty amino acid substitutions resulting from 44 mutated nucleotides in the viral genome were found in TW2575/98vac. All of the molecular variations lead to attenuation, found in TW2575/98, were not observed consistently in the other IBVs (TW2296/95, Ark/Ark-DPI/81, the Massachusetts strain, GA98/CWL0470/98, and CK/CH/LDL/97I) and vice versa. After further comparisons and evaluations from three aspects: (1) longitudinal analysis on the timing of variations appeared in specific homologous strain passages, (2) horizontal evaluations with the amino acid changes between wild and vaccine strains among the other 5 IBVs, and (3) inspection on alterations in the chemical characteristics of substituted amino acid residues in viral proteins, four amino acid substitutions [V342D in p87, S1493P and P2025S in HD1, as well as F2308Y in HD1(P41)] were selected as highly possible candidates for successful TW2575/98w attenuation. Our findings imply that molecular variations, which contribute to the successful attenuation of different IBVs, are diverse and not restricted to a fixed pattern or specific amino acid substitutions in viral proteins. In addition, four amino acid changes within the replicase gene-encoded proteins might be associated with TW2575/98 virus virulence.Electronic supplementary materialThe online version of this article (10.1007/s11262-020-01753-5) contains supplementary material, which is available to authorized users.

The mitochondrial inner membrane protein LETM1 modulates cristae organization through its LETM domain

LETM1 is a mitochondrial inner membrane protein that is required for maintaining the mitochondrial morphology and cristae structures, and regulates mitochondrial ion homeostasis. Here we report a role of LETM1 in the organization of cristae structures. We identified four amino acid residues of human LETM1 that are crucial for complementation of the growth deficiency caused by gene deletion of a yeast LETM1 orthologue. Substituting amino acid residues with alanine disrupts the correct assembly of a protein complex containing LETM1 and prevents changes in the mitochondrial morphology induced by exogenous LETM1 expression. Moreover, the LETM1 protein changes the shapes of the membranes of in vitro-reconstituted proteoliposomes, leading to the formation of invaginated membrane structures on artificial liposomes. LETM1 mutant proteins with alanine substitutions fail to facilitate the formation of invaginated membrane structures, suggesting that LETM1 plays a fundamental role in the organization of mitochondrial membrane morphology.

Super-activator variants of the cyanobacterial transcriptional regulator ChlR essential for tetrapyrrole biosynthesis under low oxygen conditions.

ChlR is a MarR-type transcriptional regulator that activates the transcription of the chlAII-ho2-hemN operon in response to low oxygen conditions in the cyanobacterium Synechocystis sp. PCC 6803. Upon exposure to low oxygen conditions, ChlR activates transcription of the operon that encodes enzymes critical to tetrapyrrole biosynthesis under low oxygen conditions. We previously identified a super-activator variant, D35H, of ChlR that constitutively activates transcription of the operon. To gain insight into the low-oxygen induced activation of ChlR, we obtained eight additional super-activator variants of ChlR including D35H from pseudorevertants of a chlAI-disrupted mutant. Most substitutions were located in the N-terminal region of ChlR. Mapping of the substituted amino acid residues provided valuable structural insights that uncovered the activation mechanism of ChlR.

Elucidating Binding Sites and Affinities of ERα Agonists and Antagonists to Human Alpha-Fetoprotein by In Silico Modeling and Point Mutagenesis.

Alpha-fetoprotein (AFP) is a major embryo- and tumor-associated protein capable of binding and transporting a variety of hydrophobic ligands, including estrogens. AFP has been shown to inhibit estrogen receptor (ER)-positive tumor growth, which can be attributed to its estrogen-binding ability. Despite AFP having long been investigated, its three-dimensional (3D) structure has not been experimentally resolved and molecular mechanisms underlying AFP–ligand interaction remains obscure. In our study, we constructed a homology-based 3D model of human AFP (HAFP) with the purpose of molecular docking of ERα ligands, three agonists (17β-estradiol, estrone and diethylstilbestrol), and three antagonists (tamoxifen, afimoxifene and endoxifen) into the obtained structure. Based on the ligand-docked scoring functions, we identified three putative estrogen- and antiestrogen-binding sites with different ligand binding affinities. Two high-affinity binding sites were located (i) in a tunnel formed within HAFP subdomains IB and IIA and (ii) on the opposite side of the molecule in a groove originating from a cavity formed between domains I and III, while (iii) the third low-affinity binding site was found at the bottom of the cavity. Here, 100 ns molecular dynamics (MD) simulation allowed us to study their geometries and showed that HAFP–estrogen interactions were caused by van der Waals forces, while both hydrophobic and electrostatic interactions were almost equally involved in HAFP–antiestrogen binding. Molecular mechanics/Generalized Born surface area (MM/GBSA) rescoring method exploited for estimation of binding free energies (ΔGbind) showed that antiestrogens have higher affinities to HAFP as compared to estrogens. We performed in silico point substitutions of amino acid residues to confirm their roles in HAFP–ligand interactions and showed that Thr132, Leu138, His170, Phe172, Ser217, Gln221, His266, His316, Lys453, and Asp478 residues, along with two disulfide bonds (Cys224–Cys270 and Cys269–Cys277), have key roles in both HAFP–estrogen and HAFP–antiestrogen binding. Data obtained in our study contribute to understanding mechanisms underlying protein–ligand interactions and anticancer therapy strategies based on ERα-binding ligands.

Substitutions of Amino Acid Residues in the Substrate Binding Site of Horse Liver Alcohol Dehydrogenase Have Small Effects on the Structures but Significantly Affect Catalysis of Hydrogen Transfer.

Previous studies showed that the L57F and F93W alcohol dehydrogenases catalyze the oxidation of benzyl alcohol with some quantum mechanical hydrogen tunneling, whereas the V203A enzyme has diminished tunneling. Here, steady-state kinetics for the L57F and F93W enzymes were studied, and microscopic rate constants for the ordered bi-bi mechanism were estimated from simulations of transient kinetics for the S48T, F93A, S48T/F93A, F93W, and L57F enzymes. Catalytic efficiencies for benzyl alcohol oxidation (V1/EtKb) vary over a range of ∼100-fold for the less active enzymes up to the L57F enzyme and are mostly associated with the binding of alcohol rather than the rate constants for hydride transfer. In contrast, catalytic efficiencies for benzaldehyde reduction (V2/EtKp) are ∼500-fold higher for the L57F enzyme than for the less active enzymes and are mostly associated with the rate constants for hydride transfer. Atomic-resolution structures (1.1 Å) for the F93W and L57F enzymes complexed with NAD+ and 2,3,4,5,6-pentafluorobenzyl alcohol or 2,2,2-trifluoroethanol are almost identical to previous structures for the wild-type, S48T, and V203A enzymes. Least-squares refinement with SHELXL shows that the nicotinamide ring is slightly strained in all complexes and that the apparent donor-acceptor distances from C4N of NAD to C7 of pentafluorobenzyl alcohol range from 3.28 to 3.49 Å (±0.02 Å) and are not correlated with the rate constants for hydride transfer or hydrogen tunneling. How the substitutions affect the dynamics of reorganization during hydrogen transfer and the extent of tunneling remain to be determined.

Molecular Mechanisms of Pathologies of Skeletal and Cardiac Muscles Caused by Point Mutations in the Tropomyosin Genes.

The review is devoted to tropomyosin (Tpm) - actin-binding protein, which plays a crucial role in the regulation of contraction of skeletal and cardiac muscles. Special attention is paid to myopathies and cardiomyopathies - severe hereditary diseases of skeletal and cardiac muscles associated with point mutations in Tpm genes. The current views on the molecular mechanisms of these diseases and the effects of such mutations on the Tpm structure and functions are considered in detail. Besides, some part of the review is devoted to analysis of the properties of Tpm homodimers and heterodimers with myopathic substitutions of amino acid residues in only one of the two chains of the Tpm dimeric molecule.

Comparison of Predicted Amino Acid Sequences of Allatotropin/Allatostatin Receptors from Solitary to Eusocial Bee Species (Hymenoptera, Apoidea)

Abstract An increasingly amount of evidence supports that the evolution of eusociality is accompanies by shifts in ancient molecular and physiological pathways. The juvenile hormone, one of the most important hormones in the post-embryonic development of insects, attracts the most attention in the context of social organization. Allatoregulatory neuropeptides (Allatotropin, Allatostatin-A and Allatostatin-C) are known to regulate juvenile hormone synthesis and release in insects. In order to clarify the transitions of juvenile hormone synthesis involved in eusocial evolution, the substitutions of amino acid residues and the complexity of post-translational modifications in allatoregulatory neuropeptide receptors were characterized. Both allatotropin and allatostatin receptors are identified in all examined bee species regardless if they are solitary or eusocial. Although the amino acid sequences are highly conserved, phylogenetic results are consistent with the eusocial status. The abundance of predicted post-translational modifications correlates with social complexity except for that in allatostatin-C receptors. Even though the consequences of these specific amino acid substitutions and various post-translational modification complexity have not been studied, they likely contribute to the localizing, binding and coupling characteristics of the receptor groups.

The Mystery of “Magic Blood” - Familial Macrothrombocytopenia Associated with a Novel Variant in GP1BA Gene

Introduction: Inherited macrothrombocytopenias comprise a heterogeneous group of rare inherited disorders characterized by decreased platelet count with enlarged platelet size. Glycoprotein Ib (GP Ib) is a platelet surface membrane glycoprotein that is encoded by GP1BA gene, and functions as a receptor for von Willebrand factor (VWF). Mutations in the GP1BA gene are seen in Bernard-Soulier syndrome (BSS). Here we describe a family with an isolated giant platelet disorder and a novel variant in the GP1BA gene following an autosomal dominant mode of inheritance. In our kindred this variant was not associated with clinical history or specific laboratory evidence of BSS. Case Presentation: 34-year-old female reported first being diagnosed with macrothrombocytopenia at the age of 13 on routine blood work. The patient and 11 of her relatives spanning 5 generations have reported asymptomatic macrothrombocytopenia (Figure 1), described as "Magic Blood" by her family. Her platelet count fell below 20,000/microL during one of her pregnancies. She was treated as idiopathic thrombocytopenic purpura (ITP) and was given corticosteroids to increase platelet count without improvement. On presentation, patient's complete blood count was significant for low platelet at 55,000/microL and macrothrombocytes were observed on blood smear. Methods/Results: VWF assays, including Factor VIII activity, VWF Ag, and Ristocetin Cofactor, were within normal limits. VWF multimer analysis revealed a normal pattern and distribution of bands. Patient had a normal platelet aggregation in response to ADP, Collagen, Epinephrine, Ristocetin and Arachidonate. Flow Cytometry detected normal GP Ib and GP IIb/ IIIa expression. Whole exome sequencing and copy number analysis of 29 genes associated with thrombocytopenia revealed a c.97T>G substitution in the GP1BA gene predicted to result in the amino acid substitution p.Cys33Gly (Figure 2). To our knowledge, this variant has not been reported in the literature or public databases. To confirm this novel variant is the cause of the familiar macrothrombocytopenia, two relatives with macrothrombocytopenia, a maternal uncle and a first cousin, also underwent genetic testing and were found to have the same variant. Discussion: Variants in GP1BA are associated with both autosomal dominant and recessive forms of BSS and with autosomal dominant platelet-type VWD. Our kindred is surprisingly asymptomatic given the location and specific amino acid substitution generated by the variant. Amino acid residue p.Cys33 resides in an extracellular N-terminal domain that is critical for VWF binding and proper assembly of the GP1B-IX complex. A heterozygous substitution involving the same amino acid residue defined as p.Cys33Arg was observed in two patients with macrothrombocytopenia with no reported bleeding complications (MC, unpublished data). Substitution of a nearby cysteine residue defined as p.Cys20Gly has been reported in a case of monoallelic chronic macrothrombocytopenia without bleeding diathesis similar to our patient. Amino acid residues p.Cys20 and p.Cyc33 are highly conserved among divergent species and substitution of these amino acid residues appears to not be tolerated. Substitution of other cysteine amino acid residues in the extracellular domain of the GP1BA protein (p.Cys81 and p.Cys225) has also been reported in patients with biallelic BSS, suggesting that perturbation of cysteine amino acid residues is likely to affect protein structure and function. Conclusion:We think the p.Cys33Gly substitution found in our patient and her relatives is likely to be a primary cause of monoallelic GP1BA-associated macrothrombocytopenia. It is important to distinguish inherited macrothrombocytopenia from ITP in order to avoid unnecessary and potentially toxic treatment. Disclosures No relevant conflicts of interest to declare.

Prothrombin R541W Mutation Impairs Protein C Activation and Constitutes a New Genetic Risk Factor for Venous Thrombosis

Defective protein C (PC) pathway predisposes patients to venous thromboembolism (VTE)and is mostly, but not exclusively, attributed tohereditary PC or protein S (PS) deficiencies and activated PC resistance caused by factor V Leiden mutation. In a patient with acute mesenteric venous thrombosis and positive family history of VTE associated with the impaired PC pathway function determined by thrombin generation test, we identified a novel heterozygous prothrombin mutation p.Arg541Trp. Two more patients with positive family history of VTE carrying the same mutation were identified in a cohort of another 373 unrelated patients, making an overall prevalence of 0.8%. Family investigation revealed 11 individuals in the three pedigrees harboring the heterozygous prothrombin p.Arg541Trp mutation, and 8 of them (72%) had experienced episodes of VTE. Functional studies indicated the mutation moderately decreased procoagulant activity of prothrombin and had mild impact on the inactivation of thrombin by its inhibitor antithrombin. However, the amino acid residue substitution significantly compromised PC activation by thrombin, both in the absence and presence of soluble thrombomodulin, and thus rendered prothrombin function procoagulant biased. In conclusion, the prothrombin p.Arg541Trp mutation constitutes a new genetic risk factor of VTE by impairing function of PC pathway and tilting thrombin's procoagulant activity over anticoagulant function. Figure Disclosures Ding: Shanghai General Hospital affiliated to Shanghai Jiao Tong University, Shanghai: Patents & Royalties: W.W, Q.D and X.W of the co-authors are named as inventors on a patent application related to this work.. Wang:Ruijin Hospital, Shanghai Jiao Tong University School of Medicine: Patents & Royalties: W.W, Q.D and X.W of the co-authors are named as inventors on a patent application related to this work.. WU:Ruijin Hospital, Shanghai Jiao Tong University School of Medicine: Patents & Royalties: W.W, Q.D and X.W of the co-authors are named as inventors on a patent application related to this work..

Bound manganese oxides capable of reducing the bacteriochlorophyll dimer of modified reaction centers from Rhodobacter sphaeroides

A biohybrid model system is described that interfaces synthetic Mn-oxides with bacterial reaction centers to gain knowledge concerning redox reactions by metal clusters in proteins, in particular the Mn4CaO5 cluster of photosystem II. The ability of Mn-oxides to bind to modified bacterial reaction centers and transfer an electron to the light-induced oxidized bacteriochlorophyll dimer, P+, was characterized using optical spectroscopy. The environment of P was altered to obtain a high P/P+ midpoint potential. In addition, different metal-binding sites were introduced by substitution of amino acid residues as well as extension of the C-terminus of the M subunit with the C-terminal region of the D1 subunit of photosystem II. The Mn-compounds MnO2, αMn2O3, Mn3O4, CaMn2O4, and Mn3(PO4)2 were tested and compared to MnCl2. In general, addition of the Mn-compounds resulted in a decrease in the amount of P+ while the reduced quinone was still present, demonstrating that the Mn-compounds can serve as secondary electron donors. The extent of P+ reduction for the Mn-oxides was largest for αMn2O3 and CaMn2O4 and smallest for Mn3O4 and MnO2. The addition of Mn3(PO4)2 resulted in nearly complete P+ reduction, similar to MnCl2. Overall, the activity was correlated with the initial oxidation state of the Mn-compound. Transient optical measurements showed a fast kinetic component, assigned to reduction of P+ by the Mn-oxide, in addition to a slow component due to charge recombination. The results support the conjecture that the incorporation of Mn-oxides by ancient anoxygenic phototrophs was a step in the evolution of oxygenic photosynthesis.

Symbiotic incompatibility between soybean and Bradyrhizobium arises from one amino acid determinant in soybean Rj2 protein.

Cultivated soybean (Glycine max) carrying the Rj2 allele restricts nodulation with specific Bradyrhizobium strains via host immunity, mediated by rhizobial type III secretory protein NopP and the host resistance protein Rj2. Here we found that the single isoleucine residue I490 in Rj2 is required for induction of symbiotic incompatibility. Furthermore, we investigated the geographical distribution of the Rj2-genotype soybean in a large set of germplasm by single nucleotide polymorphism (SNP) genotyping using a SNP marker for I490. By allelic comparison of 79 accessions in the Japanese soybean mini-core collection, we suggest substitution of a single amino acid residue (R490 to I490) in Rj2 induces symbiotic incompatibility with Bradyrhizobium diazoefficiens USDA 122. The importance of I490 was verified by complementation of rj2-soybean by the dominant allele encoding the Rj2 protein containing I490 residue. The Rj2 allele was also found in Glycine soja, the wild progenitor of G. max, and their single amino acid polymorphisms were associated with the Rj2-nodulation phenotype. By SNP genotyping against 1583 soybean accessions, we detected the Rj2-genotype in 5.4% of G. max and 7.7% of G. soja accessions. Distribution of the Rj2-genotype soybean plants was relatively concentrated in the temperate Asian region. These results provide important information about the mechanism of host genotype-specific symbiotic incompatibility mediated by host immunity and suggest that the Rj2 gene has been maintained by environmental conditions during the process of soybean domestication.

Two optimized antimicrobial peptides with therapeutic potential for clinical antibiotic-resistant Staphylococcus aureus

The rapid increase of Methicillin-resistant Staphylococcus aureus (MRSA) infections and the cross-resistance of MRSA to other antibiotics create an urgent demand for new therapeutic agents. Antimicrobial peptides (AMPs) are one of the most promising options for next-generation antibiotics. In this study, novel peptides were designed based on antimicrobial peptide fragments derived from Aristicluthys nobilia interferon-I to promote anti-MRSA activity and decrease adverse effects. Design strategies included substitutions of charged or hydrophobic amino acid residues for noncharged polar residues to promote amphipathicity. Two designed peptides, P5 (YIRKIRRFFKKLKKILKK-NH2) and P9 (SYERKINRHFKTLKKNLKKK-NH2), showed potent antimicrobial activities against both sensitive Staphylococcus aureus clinical isolates and MRSA strains without significant hemolysis or cytotoxicity to human hemocytes and renal epithelial cells. Scanning Electronic Microscopy (SEM) and qRT-PCR were employed to investigate the effects of P5 and P9 on S. aureus biofilm formation, morphology, and virulence-related gene expression. P5 and P9 significantly inhibited the biofilm and destroyed the cell membrane integrity, in addition to down-regulating several virulence factor genes and biofilm formation-related genes including spa, hld, and sdrC. P5 and P9 could be promising candidate antibacterial agents for the treatment of MRSA infections.

The impact of single-nucleotide polymorphisms of human apurinic/apyrimidinic endonuclease 1 on specific DNA binding and catalysis

Human apurinic/apyrimidinic (AP) endonuclease APE1 is a crucial enzyme of the base excision repair (BER) pathway, which is in charge of recognition and initiation of removal of AP-sites in DNA. It is known that some single-nucleotide polymorphism (SNP) variants of APE1 have a reduced activity as compared to wild-type APE1. It has been hypothesized that genetic variation in APE1 might be responsible for an increased risk of some types of cancer. In the present work, analysis of SNPs of the APE1 gene was performed to select the set of variants having substitutions of amino acid residues on the surface of the enzyme globule and in the DNA-binding site, thereby affecting protein–protein interactions or the catalytic reaction, respectively. For seven APE1 variants (R221C, N222H, R237A, G241R, M270T, R274Q, and P311S), conformational dynamics and catalytic activities were examined. The conformational changes in the molecules of APE1 variants and in a DNA substrate were recorded as fluorescence changes of Trp and 2-aminopurine residues, respectively, using the stopped-flow technique. The results made it possible to determine the kinetic mechanism underlying the interactions of the APE1 variants with DNA substrates, to calculate the rate constants of the elementary stages, and to identify the stages of the process affected by mutation.

Structural diversification during glucosinolate breakdown: mechanisms of thiocyanate, epithionitrile and simple nitrile formation.

SummarySecondary metabolism is characterized by an impressive structural diversity. Here, we have addressed the mechanisms underlying structural diversification upon damage‐induced activation of glucosinolates, a group of thioglucosides found in the Brassicales. The classical pathway of glucosinolate activation involves myrosinase‐catalyzed hydrolysis and rearrangement of the aglucone to an isothiocyanate. Plants of the Brassicaceae possess specifier proteins, i.e. non‐heme iron proteins that promote the formation of alternative products by interfering with this reaction through unknown mechanisms. We have used structural information available for the thiocyanate‐forming protein from Thlaspi arvense (TaTFP), to test the impact of loops protruding at one side of its β‐propeller structure on product formation using the allylglucosinolate aglucone as substrate. In silico loop structure sampling and semiempirical quantum mechanical calculations identified a 3L2 loop conformation that enabled the Fe2+ cofactor to interact with the double bond of the allyl side chain. Only this arrangement enabled the formation of allylthiocyanate, a specific product of TaTFP. Simulation of 3,4‐epithiobutane nitrile formation, the second known product of TaTFP, required an alternative substrate docking arrangement in which Fe2+ interacts with the aglucone thiolate. In agreement with these results, substitution of 3L2 amino acid residues involved in the conformational change as well as exchange of critical amino acid residues of neighboring loops affected the allylthiocyanate versus epithionitrile proportion obtained upon myrosinase‐catalyzed allylglucosinolate hydrolysis in the presence of TaTFP in vitro. Based on these insights, we propose that specifier proteins are catalysts that might be classified as Fe2+‐dependent lyases.