Journal of Molecular Biology
Regular ArticleMain-chain Bond Lengths and Bond Angles in Protein Structures
Abstract
The main-chain bond lengths and bond angles of protein structures are analysed as a function of resolution. Neither the means nor standard deviations of these parameters show any correlation with resolution over the resolution range investigated. This is as might be expected as bond lengths and bond angles are likely to be heavily influenced by the geometrical restraints applied during structure refinement. The size of this influence is then investigated by performing an analysis of variance on the mean values across the five most commonly used refinement methods. The differences in means are found to be highly statistically significant, suggesting that the difference target values used by the different methods leave their imprint on the structures they refine. This has implications concerning the actual target values used during refinement and stresses the importance of the values being not only accurate but also consistent from one refinement method to another.
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Jack bean urease
2024, UreasesUrease (urea amidohydrolase, EC 3.5.1.5) is a seed protein common to most leguminosae, it also occurs in many bacteria, fungi, and several species of yeast. Urease allows organisms to use exogenous and internally generated urea as a nitrogen source by catalyzing the hydrolysis of urea to ammonia and carbon dioxide. Plant ureases especially “jack bean urease (JBU)” hold a very special place in history; JBU was the first enzyme to be crystallized back in 1926 by James B. Sumner and the authors were the first to report its structure in 2013, 83 years later. In 1975, it was discovered that JBU contains nickel ions at its active site and suggested its biological significance in the catalytic activity of the enzyme. Plant ureases exert insecticidal activity through interaction with cell membrane lipids, which evokes a strong interest in exploring them further. It has been almost a century since the crystallization of JBU was reported by Sumner. In this review, the authors discuss its history, significance, purification, crystallization, structure determination, oligomeric assembly, active site architecture of native and fluoride-inhibited JBU, and insecticidal activity. This chapter aims to inspire and motivate the readers to further unravel the mystery of this moonlighting enzyme.
Larvicidal activity, enzyme inhibitory effect, and molecular docking by essential oil, hydrolate, aqueous extract, and major compounds from the leaves of Eugenia uniflora against Aedes aegypti
2023, Industrial Crops and ProductsEugenia uniflora is a plant native to Brazil that has a wide geographical distribution and produces a high content of essential oils. The aim of the present study was to investigate the larvicidal activity of the hydrolate (Eu-HYD), aqueous extract (Eu-AE), essential oil (Eu-EO), and major compounds obtained from E. uniflora leaves against Aedes aegypti as well as assess the effects on the digestive enzymes of the larvae. Eu-HYD and Eu-AE were obtained from the leaves as byproducts of the EO isolation by hydrodistillation. Selina-1,3,7(11)-trien-8-one (1, 57.55 %) and oxidoselina-1,3,7(11)-trien-8-one (2, 21.18 %) were the major chemical compounds in the EO, as determined by gas chromatography with flame ionization detection and gas chromatography coupled to mass spectrometry. Eu-EO exhibited the most promising larvicidal activity (LC50 = 35.9 ± 1.02 mg/L), followed by Eu-AE (LC50 = 12.205 ± 1.04 mg/L) and Eu-HYD (LC50 = 42.4 ± 1.02 mg/L), whereas compounds 1 and 2 had an LC50 of 77.8 ± 1.06 and 41.2 ± 1.06 mg/L, respectively. Moreover, Eu-EO, Eu-AE, and Eu-HYD inhibited α-amylase activity in the Ae. aegypti larvae. Docking studies on the altered enzymes indicated that the affinity of compound 2 for Ae. aegypti α-amylase is slightly higher than that of compound 1. These results suggest notable larvicidal activity for all products of Eugenia uniflora leaves, making such products bioactive candidates for interrupting the reproduction cycle of Ae. aegypti, which is the main vector of arboviruses.
Crystal structures of non-uracil ring fragments in complex with Mycobacterium tuberculosis uracil DNA glycosylase (MtUng) as a starting point for novel inhibitor design: A case study with the barbituric acid fragment
2023, European Journal of Medicinal ChemistryUracil DNA glycosylase (UDG or Ung) is a key enzyme involved in uracil excision from the DNA as a repair mechanism. Designing Ung inhibitors is thus a promising strategy to treat different cancers and infectious diseases. The uracil ring and its derivatives have been shown to inhibit Mycobacterium tuberculosis Ung (MtUng), resulting from specific and strong binding with the uracil-binding pocket (UBP). To design novel MtUng inhibitors, we screened several non-uracil ring fragments hypothesised to occupy MtUng UBP due to their high similarity to the uracil structural motif. These efforts have resulted in the discovery of novel MtUng ring inhibitors. Here we report the co-crystallised poses of these fragments, confirming their binding within the UBP, thus providing a robust structural framework for the design of novel lead compounds. We selected the barbituric acid (BA) ring as a case study for further derivatisation and SAR analysis. The modelling studies predicted the BA ring of the designed analogues to interact with the MtUng UBP much like the uracil ring. The synthesised compounds were screened in vitro using radioactivity and a fluorescence-based assay. These studies led to a novel BA-based MtUng inhibitor 18a (IC50 = 300 μM) displaying ∼24-fold potency over the uracil ring.
Membrane-active and DNA binding related double-action antimycobacterial mechanism of antimicrobial peptide W3R6 and its synthetic analogs
2023, Biochimica et Biophysica Acta - General SubjectsThe emergence of multidrug- or extremely drug-resistant M. tuberculosis strains has made very few drugs available for current tuberculosis treatment. Antimicrobial peptides can be employed as a promising alternative strategy for TB treatment. Here, we designed and synthesized a series of peptide sequences based on the structure-activity relationships of natural sequences of antimicrobial peptides. The peptide W3R6 and its analogs were screened and found to have potent antimycobacterial activity against M. smegmatis, and no hemolytic activity against human erythrocytes. The evidence from the mechanism of action study indicated that W3R6 and its analogs can interact with the mycobacterial membrane in a lytic manner and form pores on the outer membrane of M. smegmatis. Significant colocalization of D-W3R6 with mycobacterial DNA was observed by confocal laser scanning microscopy and DNA retardation assays, which suggested that the antimycobacterial mechanism of action of the peptide was associated with the unprotected genomic DNA of M. smegmatis. In general, W3R6 and its analogs act on not only the mycobacterial membrane but also the genomic DNA in the cytoplasm, which makes it difficult for mycobacteria to generate resistance due to the peptides having two targets. In addition, the peptides can effectively eliminate M. smegmatis cells from infected macrophages. Our findings indicated that the antimicrobial peptide W3R6 could be a novel lead compound to overcome the threat from drug-resistant M. tuberculosis strains in the development of potent AMPs for TB therapeutic applications.
A set of closely related methyltransferases for site-specific tailoring of anthraquinone pigments
2023, StructureModification of the polyketide anthraquinone AQ-256 in the entomopathogenic Photorhabdus luminescens involves several O-methylations, but the biosynthetic gene cluster antA-I lacks corresponding tailoring enzymes. We here describe the identification of five putative, highly homologous O-methyltransferases encoded in the genome of P. luminescens. Activity assays in vitro and deletion experiments in vivo revealed that three of them account for anthraquinone tailoring by producing three monomethylated and two dimethylated species of AQ-256. X-ray structures of all five enzymes indicate high structural and mechanistic similarity. As confirmed by structure-based mutagenesis, a conserved histidine at the active site likely functions as a general base for substrate deprotonation and subsequent methyl transfer in all enzymes. Eight complex structures with AQ-256 as well as mono- and dimethylated derivatives confirm the substrate specificity patterns found in vitro and visualize how single amino acid differences in the active-site pockets impact substrate orientation and govern site-specific methylation.
Recognition mechanism of endocellulase for β-glucan containing β(1 → 3),(1 → 4) mixed-linkages
2022, Carbohydrate ResearchGlycoside hydrolase family 12 endocellulase (GH family12) plays a key role in the degradation of β-glucan and cellulose. Hyperthermostable GH family 12 endocellulase from the archaeon Pyrococcus furiosus (EGPf) catalyzes the hydrolysis of β(1 → 4) glucosidic linkages in cellulose and β-glucan containing β(1 → 3),(1 → 4) mixed-linkages. Therefore, in the combination with the hyperthermophilic β-glucosidase from P. furiosus (BGLPf), non-crystalline cellulose and β-glucan can be degraded to glucose completely by EGPf at high temperature. X-ray crystallography and protein engineering were used to reveal how the β(1 → 4) and β(1 → 3) linkages in β-glucan substrates are recognized by the enzyme. Structural and functional analyses clarified that the active site of EGPf consists of six subsites: the reducing end subsites (+1 and + 2) recognize both β(1 → 4) and β(1 → 3) linkages of various substrates in a productive binding mode, and recognition is controlled by Trp121 and Gln208 located at subsite +2. It was also revealed that the deep cleft in subsite −4 can accommodate the torsion angles of substrates consisting of β(1 → 3),(1 → 4) mixed-linkages due to the changing tilt of the Trp62 side chain. From the structural similarity, it is proposed that the substrate specificity of family 12 endocellulases towards β(1 → 3),(1 → 4) mixed-linkage substrates are controlled by the subsites (+1, +2, and −4). Furthermore, the function of family 12 endocellulase could be improved by protein engineering method using the information of the analysis.