Biology subjects

Li, D.-F.

Publications and source records attributed to Li, D.-F..

5 recordsLinked to original sources

bioRxiv2024

Discovery of the 1-naphthylamine biodegradation pathway reveals an enzyme that catalyzes 1-naphthylamine glutamylation

1-Naphthylamine (1NA), which is harmful to human and aquatic animals, has been used widely in the manufacturing of dyes, pesticides, and rubber antioxidants. Nevertheless, little is known about its environmental behavior and no bacteria have been reported to use it as the growth substrate. Herein, we describe a pathway for 1NA degradation in isolate Pseudomonas sp. strain JS3066, determine the structure and mechanism of the enzyme NpaA1 that catalyzes the initial reaction, and reveal how the pathway evolved. From genetic and enzymatic analysis, a cluster of 5 genes encoding a dioxygenase system was determined to be responsible for the initial steps in 1NA degradation through glutamylation of 1NA. The {gamma}-glutamylated 1NA was subsequently oxidized to 1,2-dihydroxynaphthalene which was further degraded by the well-established pathway of naphthalene degradation via catechol. Enzymatic analysis showed that NpaA1 catalyzed conversion of various anilines and naphthylamine derivatives. Structural and biochemical studies of NpaA1 revealed that the broad substrate specificity of NpaA1 is due to a large hydrophobic pocket, which is different from type I glutamine synthetase (GSI). The findings enhance understanding of degrading polycyclic aromatic amines, and will also enable the application of bioremediation at naphthylamine contaminated sites.

microbiology↗

bioRxiv2023

Attractant and repellent induce opposing changes in the chemoreceptor four-helix bundle ligand-binding domain

Motile bacteria navigate toward favorable conditions and away from unfavorable environments using chemotaxis. Mechanisms of sensing attractants are well understood, however molecular aspects of how bacteria sense repellents have not been established. Here, we identified malate as a repellent recognized by the MCP2021 chemoreceptor in a bacterium Comamonas testosteroni and showed that it binds to the same site as an attractant citrate. Binding determinants for a repellent and an attractant had only minor differences, and a single amino acid substitution in the binding site inverted the response to malate from a repellent to an attractant. We found that malate and citrate affect the oligomerization state of the ligand-binding domain in opposing way. We also observed opposing effects of repellent and attractant binding on the orientation of an alpha helix connecting the sensory domain to the transmembrane helix. We propose a model to illustrate how positive and negative signals are generated and transduced across the membrane and built chimera proteins to illustrate a universal nature of the transmembrane signaling by the repellent.

microbiology↗

bioRxiv2022

Mobile stamens enhance pollen dispersal by scaring floral visitors away

Animal-pollinated plants have to get pollen to a conspecific stigma while protecting it from getting eaten. We provide experimental evidence that touch-sensitive stamens function in (i) enhancing pollen export and (ii) reducing pollen loss to thieves. Stamens of Berberis and Mahonia are inserted between paired nectar glands and when touched by an insects tongue rapidly snap forward so that their valvate anthers press pollen on the insects tongue or face. We immobilized the stamens in otherwise unmodified flowers and studied pollen transfer in the field and under enclosed conditions. On flowers with immobilized stamens, the commonest bee visitor stayed up to 3.6x longer, yet removed 1.3x fewer pollen grains and deposited 2.1x fewer grains on stigmas per visit. Self-pollen from a single stamen hitting the stigma amounted to 6% of the grains received from single bee visits. Bees discarded pollen passively placed on their bodies, likely because of its berberine content; nectar has no berberine. Syrphid flies fed on both nectar and pollen, taking more when stamens were immobilized. Pollen-tracking experiments in two species showed that mobile-stamen-flowers donate pollen to many more recipients. These results demonstrate another mechanism by which plants simultaneously meter out their pollen and reduce pollen theft. HighlightsO_LIStamens that snap forward when triggered by a flower visitor may serve to meter out pollen, scare away pollen thieves, or place pollen more accurately. C_LIO_LIWe tested these hypotheses by experimentally immobilizing all six stamens in numerous flowers of Berberis and Mahonia species in the field and under enclosed conditions. C_LIO_LIIn flowers with immobilized stamens, the commonest bee species stayed up to 3.6x longer, yet removed 1.3x fewer pollen grains and deposited 2.1x fewer grains on stigmas per visit. Mobile stamens exported their pollen to significantly more neighboring flowers. C_LI Graphic abstractBehaviour and pollen transfer after flower visitors received a beating on the tongue or in the face by the forward-snapping stamens of Berberis. Stamens only snap forward if their filament basis is touched by an insect tongue. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=184 SRC="FIGDIR/small/498951v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@1dff517org.highwire.dtl.DTLVardef@53f369org.highwire.dtl.DTLVardef@1918627org.highwire.dtl.DTLVardef@fe9447_HPS_FORMAT_FIGEXP M_FIG C_FIG

ecology↗

bioRxiv2020

A novel oxidase from Alcaligenes sp. HO-1 oxidizes hydroxylamine to N2

Hydroxylamine is a key intermediate of microbial ammonia oxidation and plays an important role in the biogeochemical cycling of N-compounds. Hydroxylamine is oxidized to NO or N2O by hydroxylamine oxidases or cytochrome P460 from heterotrophic or autotrophic bacteria, but its enzymatic oxidation to N2 has not yet been observed. Here, we report on the discovery of a novel oxidase that converts hydroxylamine to N2 from the newly isolated heterotrophic nitrifier Alcaligenes strain HO-1. Strain HO-1 accumulated hydroxylamine and produced N2 from ammonia oxidation. Using transcriptome analysis and heterologous expression via fosmid library screening, we identified three genes (dnfABC) of strain HO-1 that enabled E. coli cells not only to produce hydroxylamine from 15N-labelled ammonium but also to further convert it to 15N2. The three genes were individually cloned and expressed, and their translational products DnfA, DnfB, and DnfC were purified. In vitro DnfA bound to hydroxylamine and catalyzed the conversion of hydroxylamine to N2 in the presence of FAD, NADH and O2. Thus, DnfA was identified as a novel hydroxylamine oxidase and catalyzed a previously unknown N-N bond forming reaction with a yet-to-be discovered mechanism. DnfA homologs were detected in different bacterial groups, suggesting that hydroxylamine oxidation to nitrogen might occur in additional microbial taxa.

microbiology↗

bioRxiv2019

Biochemical and structural characterization of a highly active branched-chain amino acid aminotransferase from Pseudomonas sp. for efficient biosynthesis of chiral amino acids

Aminotransferases (ATs) are important biocatalysts for the synthesis of chiral amines because of their capability of introducing amino group into ketones or keto acids as well as their high enantioselectivity, high regioselectivity and no requirement of external addition of cofactor. Among all ATs, branched-chain amino acid aminotransferase (BCAT) can reversibly catalyse branched-chain amino acids (BCAAs), including L-valine, L-leucine, and L-isoleucine, with -ketoglutaric acid to form the corresponding ketonic acids and L-glutamic acid. Alternatively, BCATs have been used for the biosynthesis of unnatural amino acids, such as L-tert-leucine. In the present study, the BCAT from Pseudomonas sp. (PsBCAT) was cloned and expressed in Escherichia coli for biochemical and structural analyses. The optimal reaction temperature and pH of PsBCAT were 40 {degrees}C and 8.5, respectively. PsBCAT exhibited a comparatively broader substrate spectrum, and showed remarkably high activity with L-leucine, L-valine, L-isoleucine and L-methionine with activities of 105 U/mg, 127 U/mg, 115 U/mg and 98 U/mg, respectively. Additionally, PsBCAT had activities with aromatic L-amino acids, L-histidine, L-lysine, and L-threonine. To analyse the catalytic mechanism of PsBCAT with the broad substrate spectrum, the crystal structure of PsBCAT was also determined. Finally, conjugated with the ornithine aminotransferase (OrnAT) from Bacillus subtilis, the coupled system was applied to the preparation of L-tert-leucine with 83% conversion, which provided an approximately 2.7-fold higher yield than the single BCAT reaction. IMPORTANCEDespite the enormous potential of BCATs, the vast majority of enzymes still lack suitably broad substrate scope and activity, thus new sources and novel enzymes are currently being investigated. Here, we described a previously uncharacterized PsBCAT, which showed a surprisingly wide substrate range and was more active towards BCAAs. This substrate promiscuity is unique for the BCAT family and could prove useful in industrial applications. Based on the determined crystal structure, we found some differences in the organization of the substrate binding cavity, which may influence the substrate specificity of the enzyme. Moreover, we demonstrated efficient biocatalytic asymmetric synthesis of L-tert-leucine using a coupling system, which can be used to remove the inhibitory by-product, and to shift the reaction equilibrium towards the product formation. In summary, the structural and functional characteristics of PsBCAT were analysed in detail, and this information will play an important role in the synthesis of chiral amino acids and will be conducive to industrial production of enantiopure chiral amines by aminotransferase.

biochemistry↗