Poly(ethylene terephthalate) (PET) is a class of plastic material widely used in modern society, but large amounts of PET waste cause severe environmental problems. Obtained from a PET‐consuming ...bacterium Ideonella sakaiensis, the enzyme PETase exhibits superb hydrolytic activity and substrate preference toward PET. Here, we summarize some recent advances in the crystallographic analysis of PETase. These reports uncover structural features of PETase that are involved in its catalytic activity. In comparison to homologous enzymes, PETase contains an additional disulfide bond as well as an extended β8‐α6 loop. More importantly, the crystal structures of PETase in complex with substrate and product analogs provide critical information for understanding the mechanism of action of PETase. In particular, the wobbling conformation of W156 is closely related to the binding of substrate and product. These new findings are of great importance for further in‐depth research and engineering development of PETase, and should advance the implementation of plastic biodegradation strategy.
Poly(ethylene terephthalate) (PET) plastic material waste causes severe environmental burden worldwide. PET biological decomposition, mediated by a specific enzyme called PETase from a bacterium which can utilize PET as a carbon source, has recently attracted much attention. In this review, the crystal structure of the novel PETase reported from several recent advanced studies is summarized.
PET hydrolase (PETase), which hydrolyzes polyethylene terephthalate (PET) into soluble building blocks, provides an attractive avenue for the bioconversion of plastics. Here we present the structures ...of a novel PETase from the PET-consuming microbe Ideonella sakaiensis in complex with substrate and product analogs. Through structural analyses, mutagenesis, and activity measurements, a substrate-binding mode is proposed, and several features critical for catalysis are elucidated.
Acute hepatopancreatic necrosis disease (AHPND) is a severe, newly emergent penaeid shrimp disease caused by Vibrio parahaemolyticus that has already led to tremendous losses in the cultured shrimp ...industry. Until now, its disease-causing mechanism has remained unclear. Here we show that an AHPND-causing strain of V. parahaemolyticus contains a 70-kbp plasmid (pVA1) with a postsegregational killing system, and that the ability to cause disease is abolished by the natural absence or experimental deletion of the plasmid-encoded homologs of the Photorhabdus insect-related (Pir) toxins PirA and PirB. We determined the crystal structure of the V. parahaemolyticus PirA and PirB (PirA(vp) and PirB(vp)) proteins and found that the overall structural topology of PirA(vp)/PirB(vp) is very similar to that of the Bacillus Cry insecticidal toxin-like proteins, despite the low sequence identity (<10%). This structural similarity suggests that the putative PirAB(vp) heterodimer might emulate the functional domains of the Cry protein, and in particular its pore-forming activity. The gene organization of pVA1 further suggested that pirAB(vp) may be lost or acquired by horizontal gene transfer via transposition or homologous recombination.
Mammalian innate immune sensor STING (STimulator of INterferon Gene) was recently found to originate from bacteria. During phage infection, bacterial STING sense c-di-GMP generated by the CD-NTase ...(cGAS/DncV-like nucleotidyltransferase) encoded in the same operon and signal suicide commitment as a defense strategy that restricts phage propagation. However, the precise binding mode of c-di-GMP to bacterial STING and the specific recognition mechanism are still elusive. Here, we determine two complex crystal structures of bacterial STING/c-di-GMP, which provide a clear picture of how c-di-GMP is distinguished from other cyclic dinucleotides. The protein-protein interactions further reveal the driving force behind filament formation of bacterial STING. Finally, we group the bacterial STING into two classes based on the conserved motif in β-strand lid, which dictate their ligand specificity and oligomerization mechanism, and propose an evolution-based model that describes the transition from c-di-GMP-dependent signaling in bacteria to 2'3'-cGAMP-dependent signaling in eukaryotes.
Cytochrome P450 monooxygenases are versatile heme-thiolate enzymes that catalyze a wide range of reactions. Self-sufficient cytochrome P450 enzymes contain the redox partners in a single polypeptide ...chain. Here, we present the crystal structure of full-length CYP116B46, a self-sufficient P450. The continuous polypeptide chain comprises three functional domains, which align well with the direction of electrons traveling from FMN to the heme through the 2Fe-2S cluster. FMN and the 2Fe-2S cluster are positioned closely, which facilitates efficient electron shuttling. The edge-to-edge straight-line distance between the 2Fe-2S cluster and heme is approx. 25.3 Å. The role of several residues located between the 2Fe-2S cluster and heme in the catalytic reaction is probed in mutagenesis experiments. These findings not only provide insights into the intramolecular electron transfer of self-sufficient P450s, but are also of interest for biotechnological applications of self-sufficient P450s.
Feline infectious peritonitis virus (FIPV) is an alphacoronavirus that causes a nearly 100% mortality rate without effective treatment. Here we report a 3.3-Å cryoelectron microscopy (cryo-EM) ...structure of the serotype I FIPV spike (S) protein, which is responsible for host recognition and viral entry. Mass spectrometry provided site-specific compositions of densely distributed high-mannose and complex-type N-glycans that account for 1/4 of the total molecular mass; most of the N-glycans could be visualized by cryo-EM. Specifically, the N-glycans that wedge between 2 galectin-like domains within the S1 subunit of FIPV S protein result in a unique propeller-like conformation, underscoring the importance of glycosylation in maintaining protein structures. The cleavage site within the S2 subunit responsible for activation also showed distinct structural features and glycosylation. These structural insights provide a blueprint for a better molecular understanding of the pathogenesis of FIP.
Abstract
Purine-containing nucleotide second messengers regulate diverse cellular activities. Cyclic di-pyrimidines mediate anti-phage functions in bacteria; however, the synthesis mechanism remains ...elusive. Here, we determine the high-resolution structures of cyclic di-pyrimidine-synthesizing
c
GAS/
D
ncV-like
n
ucleotidyl
t
ransferases (CD-NTases) in clade E (CdnE) in its apo, substrate-, and intermediate-bound states. A conserved (R/Q)xW motif controlling the pyrimidine specificity of donor nucleotide is identified. Mutation of Trp or Arg from the (R/Q)xW motif to Ala rewires its specificity to purine nucleotides, producing mixed purine-pyrimidine cyclic dinucleotides (CDNs). Preferential binding of uracil over cytosine bases explains the product specificity of cyclic di-pyrimidine-synthesizing CdnE to cyclic di-UMP (cUU). Based on the intermediate-bound structures, a synthetic pathway for cUU containing a unique 2’3’-phosphodiester linkage through intermediate pppU3’−5’pU is deduced. Our results provide a framework for pyrimidine selection and establish the importance of conserved residues at the C-terminal loop for the specificity determination of CD-NTases.
Summary
Phytoplasmas are bacterial plant pathogens which can induce severe symptoms including dwarfism, phyllody and virescence in an infected plant. Because phytoplasmas infect many important crops ...such as peanut and papaya they have caused serious agricultural losses. The phytoplasmal effector causing phyllody 1 (PHYL1) is an important phytoplasmal pathogenic factor which affects the biological function of MADS transcription factors by interacting with their K (keratin‐like) domain, thus resulting in abnormal plant developments such as phyllody. Until now, lack of information on the structure of PHYL1 has prevented a detailed understanding of the binding mechanism between PHYL1 and the MADS transcription factors. Here, we present the crystal structure of PHYL1 from peanut witches’‐broom phytoplasma (PHYL1PnWB). This protein was found to fold into a unique α‐helical hairpin with exposed hydrophobic residues on its surface that may play an important role in its biological function. Using proteomics approaches, we propose a binding mode of PHYL1PnWB with the K domain of the MADS transcription factor SEPALLATA3 (SEP3_K) and identify the residues of PHYL1PnWB that are important for this interaction. Furthermore, using surface plasmon resonance we measure the binding strength of PHYL1PnWB proteins to SEP3_K. Lastly, based on confocal images, we found that α‐helix 2 of PHYL1PnWB plays an important role in PHYL1‐mediated degradation of SEP3. Taken together, these results provide a structural understanding of the specific binding mechanism between PHYL1PnWB and SEP3_K.
Significance Statement
Phytoplasmal effector causing phyllody 1 (PHYL1) is a pathogenic factor that affects the biological functions of the plant's MADS transcription factor. We present here the crystal structure of PHYL1PnWB , and use it to explain the interaction between PHYL1PnWB and SEPALLATA3 MADS transcription factor. This work extends our understanding of how PHYL1PnWB affects plants, and helps furthering the development of a potential control strategy for phytoplasma diseases, such as inhibitor design against the PHYL1PnWB/SEP3_K interactions.
We report the first X‐ray crystallographic structure of the “head‐to‐middle” prenyltransferase, isosesquilavandulyl diphosphate synthase, involved in biosynthesis of the merochlorin class of ...antibiotics. The protein adopts the ζ or cis‐prenyl transferase fold but remarkably, unlike tuberculosinol adenosine synthase and other cis‐prenyl transferases (e.g. cis‐farnesyl, decaprenyl, undecaprenyl diphosphate synthases), the large, hydrophobic side chain does not occupy a central hydrophobic tunnel. Instead, it occupies a surface pocket oriented at 90° to the hydrophobic tunnel. Product chain‐length control is achieved by squeezing out the ligand from the conventional allylic S1 binding site, with proton ion being achieved using a diphosphate‐Asn‐Ser relay. The structures revise and unify our thinking as to the mechanism of action of many other prenyl transferases and may also be of use in engineering new merochlorin‐class antibiotics.
Enzyme catalysis: Structures of a novel “head‐to‐middle” isoprenoid biosynthesis enzyme reveal a new mechanism of action for many enzymes involved in isoprenoid biosynthesis. This mechanism involves proton ion through phosphate groups and a proton relay.
Found recently in stignomatales, the Stig cyclases catalyze the Cope rearrangement and intramolecular cyclization to produce complex indole alkaloids. Five crystal structures were solved of subfamily ...1 and 2 Stig cyclases, which adopt a β‐sandwich fold like the non‐catalytic carbohydrate‐binding motif. Several complex structures were also determined of indole‐based compounds, which are bound to the hydrophobic terminal cavity, where a conserved Asp residue makes an H‐bond to the indole N and triggers the acid‐catalyzed Cope rearrangement. Through analyzing the enzyme–ligand interactions and mutagenesis experiments, several aromatic residues were found important in catalysis. Apart from a common substrate binding mode and catalytic mechanism, potential subfamily variations that may attribute to the different product specificity are implicated. These results shall expand our scope of enzymology, in particular for further investigation of the biosynthetic Cope rearrangement.
Complex structure elucidations of Stig cyclases reveal a common substrate‐binding mode and mechanism of action of these enzymes. Located near the bottom of a terminal cavity in the β‐sandwich, a strictly conserved aspartate plays a vital role in catalysis, while the surrounding active‐site residues including those in the overhanging loop may determine the type of cyclization and the final product.