DAHP synthase and chorismate mutase catalyze key steps in the shikimate biosynthetic pathway en route to aromatic amino acids. In Mycobacterium tuberculosis, chorismate mutase (MtCM; Rv0948c), located at the branch point toward phenylalanine and tyrosine, has poor activity on its own. However, it is efficiently activated by the first enzyme of the pathway, DAHP synthase (MtDS; Rv2178c), through formation of a non-covalent MtCM-MtDS complex. Here, we show how MtDS serves as an allosteric platform for feedback regulation of both enzymes, using X-ray crystallography, small-angle X-ray scattering, size-exclusion chromatography, and multi-angle light scattering. Crystal structures of the fully inhibited MtDS and the allosterically down-regulated MtCM-MtDS complex, solved at 2.8 and 2.7Å, respectively, reveal how effector binding at the internal MtDS subunit interfaces regulates the activity of MtDS and MtCM. While binding of all three metabolic end products to MtDS shuts down the entire pathway, the binding of phenylalanine jointly with tyrosine releases MtCM from the MtCM-MtDS complex, hence suppressing MtCM activation by 'inter-enzyme allostery'. This elegant regulatory principle, invoking a transient allosteric enzyme interaction, seems to be driven by dynamics and is likely a general strategy used by nature.

For more than half a century, transition state theory has provided a useful framework for understanding the origins of enzyme catalysis. As proposed by Pauling, enzymes accelerate chemical reactions by binding transition states tighter than substrates, thereby lowering the activation energy compared with that of the corresponding uncatalyzed process. This paradigm has been challenged for chorismate mutase (CM), a well-characterized metabolic enzyme that catalyzes the rearrangement of chorismate to prephenate. Calculations have predicted the decisive factor in CM catalysis to be ground state destabilization rather than transition state stabilization. Using X-ray crystallography, we show, in contrast, that a sluggish variant of Bacillus subtilis CM, in which a cationic active-site arginine was replaced by a neutral citrulline, is a poor catalyst even though it effectively preorganizes chorismate for the reaction. A series of high-resolution molecular snapshots of the reaction coordinate, including the apo enzyme, and complexes with substrate, transition state analog and product, demonstrate that an active site, which is only complementary in shape to a reactive substrate conformer, is insufficient for effective catalysis. Instead, as with other enzymes, electrostatic stabilization of the CM transition state appears to be crucial for achieving high reaction rates.pericyclic reaction | catalysis | enzyme mechanism | near attack conformation | X-ray crystal structures

The demand for animal protein has increased considerably worldwide, especially in China, where large numbers of livestock and poultry are produced. Antibiotics have been widely applied to promote growth and prevent diseases. However, the overuse of antibiotics in animal feed has caused serious environmental and health risks, especially the wide spread of antimicrobial resistance (AMR), which seriously affects animal and human health, food safety, ecosystems, and the sustainable future development of animal protein production. Unfortunately, AMR has already become a worldwide challenge, so international cooperation is becoming more important for combatting it. China’s efforts and determination to restrict antibiotic usage through law enforcement and effective management are of significance. In this review, we address the pollution problems of antibiotics; in particular, the AMR in water, soil, and plants caused by livestock and poultry manure in China. The negative impact of widespread and intensive use of antibiotics in livestock production is discussed. To reduce and mitigate AMR problems, we emphasize in this review the development of antibiotic substitutes for the era of antibiotic prohibition.

To meet increasing demand for animal protein, swine have been raised in large Chinese farms widely, using antibiotics as growth promoter. However, improper use of antibiotics has caused serious environmental and health risks, in particular Antimicrobial resistance (AMR). This paper reviews the consumption of antibiotics in swine production as well as AMR and the development of novel antibiotics or alternatives in China. The estimated application of antibiotics in animal production in China accounted for about 84240 tons in 2013. Overuse and abuse of antibiotics pose a great health risk to people through food-borne antibiotic residues and selection for antibiotic resistance. China unveiled a national plan to tackle antibiotic resistance in August 2016, but more support is needed for the development of new antibiotics or alternatives like plant extracts. Antibiotic resistance has been a major global challenge, so international collaboration between China and Europe is needed.

Type III secretion (TTS) systems are used by many Gram-negative pathogens to inject virulence proteins into the cells of their hosts. Several of these virulence effectors require TTS chaperones that maintain them in a secretion-competent state. Whereas most chaperones bind only one effector, Spa15 from the human pathogen Shigella flexneri and homologous chaperones bind several seemingly unrelated effectors, and were proposed to form a special subgroup. Its 1.8 Å crystal structure confirms this specific classification, showing that Spa15 has the same fold as other TTS effector chaperones, but forms a different dimer. The presence of hydrophobic sites on the Spa15 surface suggests that the different Spa15 effectors all possess similar structural elements that can bind these sites. Furthermore, the Spa15 structure reveals larger structural differences between class I chaperones than previously anticipated, which does not support the hypothesis that chaperone-effector complexes are structurally conserved and function as three-dimensional secretion signals.

SummaryThe hepatitis C virus (HCV) is a major etiologic agent for severe liver diseases (e.g. cirrhosis, fibrosis and hepatocellular carcinoma). Approximately 140 million people have chronic HCV infections and about 500 000 die yearly from HCV‐related liver pathologies. To date, there is no licensed vaccine available to prevent HCV infection and production of a HCV vaccine remains a major challenge. Here, we report the successful production of the HCV E1E2 heterodimer, an important vaccine candidate, in an edible crop (lettuce, Lactuca sativa) using Agrobacterium‐mediated transient expression technology. The wild‐type dimer (E1E2) and a variant without an N‐glycosylation site in the E2 polypeptide (E1E2∆N6) were expressed, and appropriate N‐glycosylation pattern and functionality of the E1E2 dimers were demonstrated. The humoral immune response induced by the HCV proteins was investigated in mice following oral administration of lettuce antigens with or without previous intramuscular prime with the mammalian HEK293T cell‐expressed HCV dimer. Immunization by oral feeding only resulted in development of weak serum levels of anti‐HCV IgM for both antigens; however, the E1E2∆N6 proteins produced higher amounts of secretory IgA, suggesting improved immunogenic properties of the N‐glycosylation mutant. The mice group receiving the intramuscular injection followed by two oral boosts with the lettuce E1E2 dimer developed a systemic but also a mucosal immune response, as demonstrated by the presence of anti‐HCV secretory IgA in faeces extracts. In summary, our study demonstrates the feasibility of producing complex viral antigens in lettuce, using plant transient expression technology, with great potential for future low‐cost oral vaccine development.

Aldolases are enzymes with potential applications in biosynthesis, depending on their activity, specificity and stability. In the present study, the genomes of Sulfolobus species were screened for aldolases. Two new KDGA [2-keto-3-deoxygluconate (2-oxo-3-deoxygluconate) aldolases] from Sulfolobus acidocaldarius and Sulfolobus tokodaii were identified, overexpressed in Escherichia coli and characterized. Both enzymes were found to have biochemical properties similar to the previously characterized S. solfataricus KDGA, including the condensation of pyruvate and either D,L-glyceraldehyde or D,L-glyceraldehyde 3-phosphate. The crystal structure of S. acidocaldarius KDGA revealed the presence of a novel phosphate-binding motif that allows the formation of multiple hydrogen-bonding interactions with the acceptor substrate, and enables high activity with glyceraldehyde 3-phosphate. Activity analyses with unnatural substrates revealed that these three KDGAs readily accept aldehydes with two to four carbon atoms, and that even aldoses with five carbon atoms are accepted to some extent. Water-mediated interactions permit binding of substrates in multiple conformations in the spacious hydrophilic binding site, and correlate with the observed broad substrate specificity.

Summary Dengue fever is a mosquito ( Aedes aegypti ) ‐transmitted viral disease that is endemic in more than 125 countries around the world. There are four serotypes of the dengue virus ( DENV 1‐4) and a safe and effective dengue vaccine must provide protection against all four serotypes. To date, the first vaccine, Dengvaxia ( CYD ‐ TDV ), is available after many decades’ efforts, but only has moderate efficacy. More effective and affordable vaccines are hence required. Plants offer promising vaccine production platforms and food crops offer additional advantages for the production of edible human and animal vaccines, thus eliminating the need for expensive fermentation, purification, cold storage and sterile delivery. Oral vaccines can elicit humoural and cellular immunity via both the mucosal and humoral immune systems. Here, we report the production of tetravalent EDIII antigen ( EDIII ‐1‐4) in stably transformed lettuce chloroplasts. Transplastomic EDIII ‐1‐4‐expressing lettuce lines were obtained and homoplasmy was verified by Southern blot analysis. Expression of EDIII ‐1‐4 antigens was demonstrated by immunoblotting, with the EDIII ‐1‐4 antigen accumulating to 3.45% of the total protein content. Immunological assays in rabbits showed immunogenicity of EDIII ‐1‐4. Our in vitro gastrointestinal digestion analysis revealed that EDIII ‐1‐4 antigens are well protected when passing through the oral and gastric digestion phases but underwent degradation during the intestinal phase. Our results demonstrate that lettuce chloroplast engineering is a promising approach for future production of an affordable oral dengue vaccine.