Structural and functional insights into the enzymatic activities of lipases from Burkholderia stagnalis and Burkholderia plantarii.

Lipases with high interesterification activity are important enzymes for industrial use. The lipase from Burkholderia stagnalis (BsL) exhibits higher interesterification activity than that from Burkholderia plantarii (BpL) despite their significant sequence similarity. In this study, we determined the crystal structure of BsL at 1.40 Å resolution. Utilizing structural insights, we have successfully augmented the interesterification activity of BpL by over twofold. This enhancement was achieved by substituting threonine with serine at position 289 through forming an expansive space in the substrate-binding site. Additionally, we discuss the activity mechanism based on the kinetic parameters. Our study sheds light on the structural determinants of the interesterification activity of lipase.

Juglone, a plant-derived 1,4-naphthoquinone, binds to hydroxylamine oxidoreductase and inhibits the electron transfer to cytochrome c554.

IMPORTANCE: Nitrification, the microbial conversion of ammonia to nitrate via nitrite, plays a pivotal role in the global nitrogen cycle. However, the excessive use of ammonium-based fertilizers in agriculture has disrupted this cycle, leading to groundwater pollution and greenhouse gas emissions. In this study, we have demonstrated the inhibitory effects of plant-derived juglone and related 1,4-naphthoquinones on the nitrification process in Nitrosomonas europaea. Notably, the inhibition mechanism is elucidated in which 1,4-naphthoquinones interact with hydroxylamine oxidoreductase, disrupting the electron transfer to cytochrome c554, a physiological electron acceptor. These findings support the notion that phytochemicals can impede nitrification by interfering with the essential electron transfer process in ammonia oxidation. The findings presented in this article offer valuable insights for the development of strategies aimed at the management of nitrification, reduction of fertilizer utilization, and mitigation of greenhouse gas emissions.

Enhanced affinity of racemic phosphorothioate DNA with transcription factor SATB1 arising from diastereomer-specific hydrogen bonds and hydrophobic contacts.

Phosphorothioate modification is commonly introduced into therapeutic oligonucleotides, typically as a racemic mixture in which either of the two non-bridging phosphate oxygens is replaced by sulfur, which frequently increases affinities with proteins. Here, we used isothermal titration calorimetry and X-ray crystallography to investigate the thermodynamic and structural properties of the interaction between the primary DNA-binding domain (CUTr1) of transcription factor SATB1 and dodecamer DNAs with racemic phosphorothioate modifications at the six sites known to contact CUTr1 directly. For both the modified and unmodified DNAs, the binding reactions were enthalpy-driven at a moderate salt concentration (50 mM NaCl), while being entropy-driven at higher salt concentrations with reduced affinities. The phosphorothioate modifications lowered this susceptibility to salt, resulting in a significantly enhanced affinity at a higher salt concentration (200 mM NaCl), although only some DNA molecular species remained interacting with CUTr1. This was explained by unequal populations of the two diastereomers in the crystal structure of the complex of CUTr1 and the phosphorothioate-modified DNA. The preferred diastereomer formed more hydrogen bonds with the oxygen atoms and/or more hydrophobic contacts with the sulfur atoms than the other, revealing the origins of the enhanced affinity.

Introducing a Gene Knockout Directly Into the Amastigote Stage of Trypanosoma cruzi Using the CRISPR/Cas9 System.

Trypanosoma cruzi is a pathogenic protozoan parasite that causes Chagas' disease mainly in Latin America. In order to identify a novel drug target against T. cruzi, it is important to validate the essentiality of the target gene in the mammalian stage of the parasite, the amastigote. Amastigotes of T. cruzi replicate inside the host cell; thus, it is difficult to conduct a knockout experiment without going through other developmental stages. Recently, our group reported a growth condition in which the amastigote can replicate axenically for up to 10 days without losing its amastigote-like properties. By using this temporal axenic amastigote culture, we successfully introduced gRNAs directly into the Cas9-expressing amastigote to cause gene knockouts and analyzed their phenotypes exclusively in the amastigote stage. In this report, we describe a detailed protocol to produce in vitro derived extracellular amastigotes, and to utilize the axenic culture in a CRISPR/Cas9-mediated knockout experiment. The growth phenotype of knockout amastigotes can be evaluated either by cell counts of the axenic culture, or by replication of intracellular amastigote after host cell invasion. This method bypasses the parasite stage differentiation normally involved in producing a transgenic or a knockout amastigote. Utilization of the temporal axenic amastigote culture has the potential to expand the experimental freedom of stage-specific studies in T. cruzi.

Utilization of proliferable extracellular amastigotes for transient gene expression, drug sensitivity assay, and CRISPR/Cas9-mediated gene knockout in Trypanosoma cruzi.

Trypanosoma cruzi has three distinct life cycle stages; epimastigote, trypomastigote, and amastigote. Amastigote is the replication stage in host mammalian cells, hence this stage of parasite has clinical significance in drug development research. Presence of extracellular amastigotes (EA) and their infection capability have been known for some decades. Here, we demonstrate that EA can be utilized as an axenic culture to aid in stage-specific study of T. cruzi. Amastigote-like property of axenic amastigote can be sustained in LIT medium at 37°C at least for 1 week, judging from their morphology, amastigote-specific UTR-regulated GFP expression, and stage-specific expression of selected endogenous genes. Inhibitory effect of benznidazole and nifurtimox on axenic amastigotes was comparable to that on intracellular amastigotes. Exogenous nucleic acids can be transfected into EA via conventional electroporation, and selective marker could be utilized for enrichment of transfectants. We also demonstrate that CRISPR/Cas9-mediated gene knockout can be performed in EA. Essentiality of the target gene can be evaluated by the growth capability of the knockout EA, either by continuation of axenic culturing or by host infection and following replication as intracellular amastigotes. By taking advantage of the accessibility and sturdiness of EA, we can potentially expand our experimental freedom in studying amastigote stage of T. cruzi.

NMR Biochemical Assay for Oxidosqualene Cyclase: Evaluation of Inhibitor Activities on Trypanosoma cruzi and Human Enzymes.

Oxidosqualene cyclase (OSC), a membrane-associated protein, is a key enzyme of sterol biosynthesis. Here we report a novel assay for OSC, involving reaction in aqueous solution, NMR quantification in organic solvent, and factor analysis of spectra. We evaluated one known and three novel inhibitors on OSC of Trypanosoma cruzi, a parasite causative of Chagas disease, and compared their effects on human OSC for selectivity. Among them, one novel inhibitor showed a significant parasiticidal activity.

Cloning and sequence analysis of poly(tetramethylene succinate) depolymerase from Acidovorax delafieldii strain BS-3.

A gene encoding poly(tetramethylene succinate), PBS, depolymerase, pbsA, has been cloned from Acidovorax delafieldii strain BS-3 chromosomal DNA. The clone expressed in Escherichia coli showed the ability to degrade both PBS and poly[(tetramethylene succinate)-co-adipate] that are kinds of biodegradable plastics. PBS depolymerase was considered to be a kind of lipase, since it also degrades olive oil. It had no apparent hydrophobic-amino-acid-rich region which exists in other known plastic-degrading enzymes. From the result of amino acid homology search, PbsA was found to have some similarities with lipases of Streptomyces sp. and Mollaxella sp. In the motif surrounding the active site Ser residue (Gly-X1-Ser-X2-Gly), PbsA was revealed to have a Trp residue in the X1 position instead of His which is most likely found in other bacterial lipases.