Huanting Liu

Host–pathogen interactions frequently depend on key components of the bacterial cell surface, such as capsules and lipopolysaccharides in Gram-negative bacteria. The first step in the synthesis of lipid-linked polysaccharides is the substitution of a uridine diphosphate (UDP)-sugar by a lipid monophosphate catalysed by a phosphoglycosyl transferase (PGT). We report the 3.0 Å cryo-electron microscopy apo-structure of the PGT enzyme WbaP from Escherichia coli, a UDP-galactose:undecaprenolphosphate galactose-1-phosphoryl transferase. The structure is a dimer with each monomer formed of four N-terminal transmembrane helices, a small α/β domain with a distinctive β-hairpin that inserts into the other monomer, and a catalytic domain, which sits perpendicular to the transmembrane domain. A complex of WbaP with the UDP-galactose substrate shows binding of the UDP moiety by R319 and R377. Mutations of R319 and R377, along with K331 and R401, highlighted the essential nature of these residues for the catalytic activity of the protein, as confirmed by an in vivo functional assay. Our results provide new insights into the PGT family of enzymes.

Host–pathogen co‐evolution shapes resistance (R) proteins and their recognition of pathogen avirulence factors. However, little attention has been paid to naturally occurring genetic diversity in R genes. In this study, 12 Solanum bulbocastanum accessions from the Commonwealth Potato Collection were screened for resistance to Phytophthora infestans, identifying 11 resistant and one susceptible accession. Targeted enrichment sequencing of nucleotide‐binding leucine‐rich repeat (NLR) genes using RenSeq, followed by diagnostic RenSeq (dRenSeq) analysis, revealed that all accessions except 7650 contained Rpi‐blb1/RB variants. Variants in accessions 7641 and 7648 were non‐functional, while three novel functional variants were identified. Cloning and functional analysis of Rpi‐blb1/RB variants assessed their recognition of the avirulence factor IPI‐O1. Three variants were functional, conferring resistance to P. infestans. Variants in accessions 7644 and 7647 also recognized IPI‐O4, confirmed in transgenic potatoes. Analysis of a non‐functional variant in S. bulbocastanum accession 7648 identified amino acid Ser347 in the nucleotide‐binding (NB‐ARC) domain as critical for cell‐death initiation following IPI‐O1 recognition. Predictions from the FunFOLD2 protein–ligand interaction model suggested that Ser347 is essential for ATP binding, suggesting potential inhibition on pentameric resistosome assembly. Western blot analysis revealed that the mutation of Ser347 to Asn markedly compromises the Rpi‐blb1/RB protein stability, and co‐immunoprecipitation assay further confirmed that this mutation severely disrupts the self‐association of CCNB, thereby preventing Rpi‐blb1/RB activation. Consistently, substituting Asn347 with serine restored function, underscoring its key role in Rpi‐blb1/RB activity. Cell biology experiments demonstrated that Rpi‐blb1/RB relocalizes to the plasma membrane in response to IPI‐O1. This relocalization depends on Ser347, further supporting the idea that its mutation affects resistosome formation, impairing resistance. This study provides an in‐depth functional analysis of natural Rpi‐blb1/RB diversity, offering insights into NLR protein evolution and resistance mechanisms in potatoes.

The enterobacterial common antigen (ECA) is conserved in Gram-negative bacteria of the Enterobacterales order although its function is debated. ECA biogenesis depends on the Wzx/Wzy-dependent strategy whereby the newly synthesized lipid-linked repeat units, lipid III, are transferred across the inner membrane by the lipid III flippase WzxE. WzxE is part of the Wzx family and required in many glycan assembly systems, but an understanding of its molecular mechanism is hindered due to a lack of structural evidence. Here, we present the first X-ray structures of WzxE from Escherichia coli in complex with nanobodies. Both inward- and outward-facing conformations highlight two pairs of arginine residues that move in a reciprocal fashion, enabling flipping. One of the arginine pairs coordinated to a glutamate residue is essential for activity along with the C-terminal arginine rich tail located close to the entrance of the lumen. This work helps understand the translocation mechanism of the Wzx flippase family.

EMPIAR, the Electron Microscopy Public Image Archive centered at EMBL-EBI, is a public resource for raw electron microscopy images related to EMDB, contains micrographs, particle sets and tilt-series.

C-C bond formation is at the heart of anabolism and organic chemistry, but relatively few enzymatic strategies for catalyzing this reaction are known. The enzyme ForT catalyzes C-C bond formation between 5’-phosphoribosyl-1’-pyrophosphate (PRPP) and 4-amino- 1H -pyrazole-3,5-dicarboxylate to make a key intermediate in the biosynthesis of the C-nucleotide formycin A 5’-phosphate; we now report the 2.5 Å resolution structure of the ForT/PRPP complex and thus locate the active site. Site-directed mutagenesis has identified those residues critical for PRPP recognition and catalysis. Structural conservation with GHMP kinases suggests that stabilization of the negatively charged pyrophosphate leaving group is crucial for catalysis in ForT. A mechanism for this new class of C-C bond forming enzymes is proposed. Entry for the Table of Contents: A new class of enzymes catalyse C-C bond formation by irreversible CO ₂ and pyrophosphate production.

ForI is a PLP-dependent enzyme from the biosynthetic pathway of the C-nucleoside antibiotic formycin.

The competition between viruses and hosts is played out in all branches of life. Many prokaryotes have an adaptive immune system termed ‘CRISPR’ (clustered regularly interspaced short palindromic repeats) which is based on the capture of short pieces of viral DNA. The captured DNA is integrated into the genomic DNA of the organism flanked by direct repeats, transcribed and processed to generate crRNA (CRISPR RNA) that is loaded into a variety of effector complexes. These complexes carry out sequence-specific detection and destruction of invading mobile genetic elements. In the present paper, we report the structure and activity of a Cas6 (CRISPR-associated 6) enzyme (Sso1437) from Sulfolobus solfataricus responsible for the generation of unit-length crRNA species. The crystal structure reveals an unusual dimeric organization that is important for the enzyme's activity. In addition, the active site lacks the canonical catalytic histidine residue that has been viewed as an essential feature of the Cas6 family. Although several residues contribute towards catalysis, none is absolutely essential. Coupled with the very low catalytic rate constants of the Cas6 family and the plasticity of the active site, this suggests that the crRNA recognition and chaperone-like activities of the Cas6 family should be considered as equal to or even more important than their role as traditional enzymes.

DinG (damage inducible gene G) is a bacterial superfamily 2 helicase with 5′→3′ polarity. DinG is related to the XPD (xeroderma pigmentosum complementation group D) helicase family, and they have in common an FeS (iron–sulfur)-binding domain that is essential for the helicase activity. In the bacilli and clostridia, the DinG helicase has become fused with an N-terminal domain that is predicted to be an exonuclease. In the present paper we show that the DinG protein from Staphylococcus aureus lacks an FeS domain and is not a DNA helicase, although it retains DNA-dependent ATP hydrolysis activity. Instead, the enzyme is an active 3′→5′ exonuclease acting on single-stranded DNA and RNA substrates. The nuclease activity can be modulated by mutation of the ATP-binding cleft of the helicase domain, and is inhibited by ATP or ADP, suggesting a modified role for the inactive helicase domain in the control of the nuclease activity. By degrading rather than displacing RNA or DNA strands, the S. aureus DinG nuclease may accomplish the same function as the canonical DinG helicase.

Bacterial pathogens need to scavenge iron from their host for growth and proliferation during infection. They have evolved several strategies to do this, one being the biosynthesis and excretion of small, high-affinity iron chelators known as siderophores. The biosynthesis of siderophores is an important area of study, not only for potential therapeutic intervention but also to illuminate new enzyme chemistries. Two general pathways for siderophore biosynthesis exist: the well-characterized nonribosomal peptide synthetase (NRPS)-dependent pathway and the NRPS-independent siderophore (NIS) pathway, which relies on a different family of sparsely investigated synthetases. Here we report structural and biochemical studies of AcsD from Pectobacterium (formerly Erwinia) chrysanthemi, an NIS synthetase involved in achromobactin biosynthesis. The structures of ATP and citrate complexes provide a mechanistic rationale for stereospecific formation of an enzyme-bound (3R)-citryladenylate, which reacts with L-serine to form a likely achromobactin precursor. AcsD is a unique acyladenylate-forming enzyme with a new fold and chemical catalysis strategy.