Dan Ma

The accumulation of acetaminophen (APAP) in agricultural soils and water systems poses risks to ecosystems and public health. This study characterizes Acinetobacter sp. DL27, an APAP-degrading strain with broad temperature and pH adaptability, demonstrating its bioremediation potential in soil and wastewater. Through high-resolution mass spectrometry, we identified three novel metabolic intermediates, thereby refining the bacterial APAP degradation pathway. Multiomics analysis elucidated metabolic coordination and stress-tolerance mechanisms, leading to the identification of a novel amidase (AdA). Recombinant AdA exhibited activity over 10–60 °C and pH 4.0–10.0, with a Km of 8.96 ± 1.03 μM and a kcat/Km of 9.04 μM–1s–1, indicating higher catalytic efficiency than previously reported APAP amidases. Molecular dynamics simulations and site-directed mutagenesis confirmed that a Ser161–Ser185–Lys82 triad constitutes the catalytic center driving amide cleavage. These findings provide mechanistic insights into bacterial APAP biodegradation and highlight the practical application potential of strain DL27 and AdA.

Cefalexin (CFX), a prevalent β-lactam antibiotic, has increasingly been recognized as an emerging environmental contaminant,posing risks to both ecosystems and public health.In response to this challenge,researchers have isolated a novel bacterial strain,CEF13, from activated sludge,identified as Variovorax boronicumulans. This marks the first instance of a Variovorax species being reported to degrade CFX. Under optimal conditions—specifically, a temperature of 31°C, a pH of 8.3, and a 20% inoculum—strain CEF13 demonstrated remarkable efficiency, breaking down 94.61% of a 500 mg/L CFX solution within just 20 hours. Through LC-MS/MS analysis, twelve degradation intermediates were identified,shedding light on a degradation pathway involving the cleavage of the β-lactam ring,hydrolysis of amide bonds,and subsequent chemical modifications.Toxicity assays further confirmed the effectiveness of CEF13, showing that it completely neutralized the antibacterial properties of CFX. To delve deeper into the degradation mechanism, a multi-omics approach was adopted. The complete genome sequencing of CEF13,totaling 6,857,659 base pairs, revealed a diverse array of genes associated with catabolism and antibiotic resistance.Transcriptomic and proteomic analyses indicated that CEF13 responds to CFX stress by upregulating efflux pump systems,quorum sensing,and fatty acid metabolism pathways, while simultaneously downregulating ribosome synthesis and motility to conserve energy.Additionally,a pivotal β-lactamase gene, chr166 (designated blaVB1), was cloned and successfully expressed in a heterologous system. The purified BlaVB1 protein,approximately 70 kDa in size,exhibited optimal activity at 35°C and pH 7.0, directly hydrolyzing the β-lactam ring of CFX.Molecular docking studies pinpointed critical residues—ARG385, ARG392, and TRP488—responsible for substrate binding and catalysis, offering valuable insights for future drug design and bioremediation strategies.

Cephalosporin residues in agricultural soils, food systems, and aquatic environments pose severe threats to food safety, public health, and the ecological environment. While biodegradation represents a promising remediation strategy, the degradation mechanisms for most cephalosporins remain elusive. In this study, Pseudochrobactrum asaccharolyticum TY12 was isolated and demonstrated a broad-spectrum ability to degrade multiple cephalosporins. Notably, strain TY12 exhibited the highest cefixime (CFX) degradation efficiency, achieving a 93.87% removal of 100 mg/L CFX within 12 h. Ten degradation products of CFX were identified via high-resolution mass spectrometry, leading to a proposed degradation pathway. Notably, these products exhibited significantly attenuated antibacterial activity. Transcriptomic analysis and heterologous expression identified β-lactamase TY3865 as the key enzyme. Furthermore, molecular docking and site-directed mutagenesis revealed the residues critical for its catalytic activity. These findings provide mechanistic insights into antibiotic biodegradation and demonstrate the potential of TY12 for treating cephalosporin-contaminated wastewater.

The diversity and functions of metallothioneins (MTs) in Archaea remain poorly understood. This study identifies 180 archaeal MTs from 406 genomes, revealing distinct evolutionary lineages and structural diversity. Phylogenetic analysis suggests a novel class with divergent ancestry. AlphaFold3-based modeling highlights conserved Cys-rich motifs and irregular structures, potentially enhancing adaptation to extreme environments. Functional assays show that four MTs (MT334126, MT382228, MT114104, and MT282443) increase cadmium resistance, while MT334126 and MT382228 also enhance copper resistance. Targeted mutations at key sites boost metal adsorption, validated by structural modeling. This work provides the first comprehensive insight into archaeal MTs, laying the foundation for environmental and biotechnological applications.

Phthalate esters (PAEs) constitute a widespread class of xenobiotic contaminants characterized by environmental persistence and ecological recalcitrance. Under aerobic conditions, PAE mineralization proceeds via 4,5-dihydroxyphthalate (4,5-DHP) as a key metabolic intermediate, which undergoes stereospecific decarboxylation to protocatechuate (PCA), a versatile pharmacophore with broad therapeutic potential. Here, we elucidate the molecular architecture and catalytic determinants of a previously uncharacterized 4,5-DHP decarboxylase (DhpD) isolated from ultramicrobacteria. Enzymatic assays revealed that PAE-UM2851 exhibits peak activity at pH 7.5 and 45 °C, with a K_m of 911.5 μM. Functional characterization confirmed that PAE-UM2851 catalyzes the decarboxylation of 4,5-DHP to PCA, supported by FTIR spectral evidence showing reduced - CO (1640 cm^-1) and - OH (3450 cm^-1) vibrational modes. Structure-function analysis identified five evolutionarily conserved residues (Ser57, Arg84, Thr115, Lys150, and His223) essential for regulating regioselective decarboxylation. This study not only clarifies the catalytic mechanism of DhpD but also outlines a potential biosynthetic route for PCA.

Phthalates (PAEs), such as dibutyl phthalate (DBP), are emerging contaminants in agriculture that are commonly used in plastic films, pesticides, and soil amendments. Their accumulation in water threatens ecosystems and human health. Biodegradation offers a promising solution, but the DBP degradation mechanisms remain unclear. This study identified two 4,5-PCD isoenzymes (PCD34 and PCD67) in Curvibacter sp. PAE-UM. PCD34 showed higher affinity and broader substrate specificity, while PCD67 had better thermal stability and metal ion sensitivity. Both enzymes converted protocatechuate (PCA) to 4CHMS at 45% efficiency in 5 min. Molecular dynamics revealed the stable binding of PCD67, while mutagenesis enhanced PCD34 activity (124.4%) by reducing steric hindrance. These findings may aid in the engineering of strains for PAE bioremediation in agricultural wastewater.

Polyacrylamide (PAM) is a high-molecular-weight polymer with extensive applications. However, the inefficient natural degradation of PAM results in environmental accumulation of the polymer. Biodegradation is an environmentally friendly approach in the field of PAM treatment. The first phase of PAM biodegradation is the deamination of PAM, forming the product poly(acrylic acid) (PAA). The second phase of PAM biodegradation involves the cleavage of PAA into small molecules, which is a crucial step in the degradation pathway of PAM. However, the enzyme that catalyzes the degradation of PAA and the molecular mechanism remain unclear. Here, a novel monooxygenase PCX02514 is identified as the key enzyme for PAA degradation. Through biochemical experiments, the monooxygenase PCX02514 oxidizes PAA with the participation of NADPH, causing the cleavage of carbon chains and a decrease in the molecular weight of PAA. In addition, the crystal structure of the monooxygenase PCX02514 is solved at a resolution of 1.97 Å. The active pocket is in a long cavity that extends from the C-terminus of the TIM barrel to the protein surface and exhibits positive electrostatic potential, thereby causing the migration of oxygen-negative ions into the active pocket and facilitating the reaction between the substrates and monooxygenase PCX02514. Moreover, Arg10-Arg125-Ser186-Arg187-His253 are proposed as potential active sites in monooxygenase PCX02514. Our research characterizes the molecular mechanism of this monooxygenase, providing a theoretical basis and valuable tools for PAM bioremediation.