Serine acetyltransferase from Neisseria gonorrhoeae; structural and biochemical basis of inhibition.

Serine acetyltransferase (SAT) catalyzes the first step in the two-step pathway to synthesize l-cysteine in bacteria and plants. SAT synthesizes O-acetylserine from substrates l-serine and acetyl coenzyme A and is a key enzyme for regulating cellular cysteine levels by feedback inhibition of l-cysteine, and its involvement in the cysteine synthase complex. We have performed extensive structural and kinetic characterization of the SAT enzyme from the antibiotic-resistant pathogen Neisseria gonorrhoeae. Using X-ray crystallography, we have solved the structures of NgSAT with the non-natural ligand, l-malate (present in the crystallization screen) to 2.01 Šand with the natural substrate l-serine (2.80 Å) bound. Both structures are hexamers, with each monomer displaying the characteristic left-handed parallel ß-helix domain of the acyltransferase superfamily of enzymes. Each structure displays both extended and closed conformations of the C-terminal tail. l-malate bound in the active site results in an interesting mix of open and closed active site conformations, exhibiting a structural change mimicking the conformation of cysteine (inhibitor) bound structures from other organisms. Kinetic characterization shows competitive inhibition of l-cysteine with substrates l-serine and acetyl coenzyme A. The SAT reaction represents a key point for the regulation of cysteine biosynthesis and controlling cellular sulfur due to feedback inhibition by l-cysteine and formation of the cysteine synthase complex. Data presented here provide the structural and mechanistic basis for inhibitor design and given this enzyme is not present in humans could be explored to combat the rise of extensively antimicrobial resistant N. gonorrhoeae.

Structures and kinetics for plant nucleoside triphosphate diphosphohydrolases support a domain motion catalytic mechanism.

Extracellular nucleoside triphosphate diphosphohydrolases (NTPDases) are enzymes that hydrolyze extracellular nucleotides to the respective monophosphate nucleotides. In the past 20 years, NTPDases belonging to mammalian, parasitic and prokaryotic domains of life have been discovered, cloned and characterized. We reveal the first structures of NTPDases from the legume plant species Trifolium repens (7WC) and Vigna unguiculata subsp. cylindrica (DbLNP). Four crystal structures of 7WC and DbLNP were determined at resolutions between 1.9 and 2.6 Å. For 7WC, structures were determined for an -apo form (1.89 Å) and with the product AMP (2.15 Å) and adenine and phosphate (1.76 Å) bound. For DbLNP, a structure was solved with phosphate and manganese bound (2.60 Å). Thorough kinetic data and analysis is presented. The structure of 7WC and DbLNP reveals that these NTPDases can adopt two conformations depending on the molecule and co-factor bound in the active site. A central hinge region creates a "butterfly-like" motion of the domains that reduces the width of the inter-domain active site cleft upon molecule binding. This phenomenon has been previously described in Rattus norvegicus and Legionella pneumophila NTPDaseI and Toxoplasma gondii NTPDaseIII suggesting a common catalytic mechanism across the domains of life.

Bactericidal activity of macrophages against Streptococcus uberis is different in mammary gland secretions of lactating and drying off cows.

The aim of this study was to compare the ability of milk macrophages and macrophages from the mammary gland secretions during the mid-dry period for their interaction with the mastitis-causing Streptococcus uberis. We also aimed to determine if S. uberis induced the release of the cytokine tumour necrosis alpha (TNF-alpha) and the bactericidal moiety nitric oxide (NO) from milk macrophages of lactating cows and macrophages from the mammary gland secretions at the mid-dry period. Macrophages were isolated from the mammary gland secretions of cows during the mid-lactation or mid-dry period, and compared with blood monocytes for their interaction with the important mastitis-causing pathogen S. uberis. When infected in vitro with S. uberis, milk macrophages from lactating cows with S. uberis released modest amounts of the cytokine tumour necrosis factor alpha (TNF-alpha) (139 pg/ml) and the bactericidal moiety nitric oxide (NO) (3-4 microM of nitrite). Blood monocytes from lactating cows released significantly higher amounts of TNF-alpha (345 +/- 143 pg/ml) and NO (7 +/- 2 microM of nitrite) after interaction with S. uberis, compared to milk macrophages (P < 0.01 for both TNF-alpha and NO). Stimulation of blood monocytes with the cytokine interferon-gamma (IFN-gamma) enhanced significantly the release of NO and TNF-alpha, but IFN-gamma did not significantly enhance the production of NO and TNF-alpha by milk macrophages from lactating cows. Milk macrophages from all lactating cows failed to kill S. uberis efficiently, and this lack of killing was unaffected by prior treatment with gamma interferon (IFN-gamma) (P > 0.05). Rather, S. uberis multiplied significantly inside infected milk macrophages from lactating cows, with a two-fold increase in bacterial numbers at 2 h post-infection. Milk macrophages from lactating cows were able however, to kill a significant proportion (50-60%, P < 0.01) of phagocytosed Staphylococcus aureus. Blood monocytes from all cows were found to exert significant bactericidal activity against S. uberis. There were no significant differences in the bactericidal activity of milk macrophages obtained from lactating cows with low somatic cell counts (SCC; < 10(5) ml(-1)) compared with those with a mildly elevated SCC (> 10(5) ml(-1)) (P > 0.05). In contrast, mammary gland secretion macrophages isolated from the same cows in the mid-dry period killed a significant proportion of phagocytosed S. uberis (50-65% of ingested S. uberis killed, P < 0.01) although cytokine production in response to in vitro bacterial infection was low. We conclude that the bactericidal activity of mammary gland secretion macrophages against a virulent strain of S. uberis is low during the lactation period. In addition, our data indicate that S. uberis is not a strong inducer of NO and TNF-alpha in macrophages from the milk or mammary gland secretions of cows during the drying off period. Finally, IFN-gamma does not activate milk macrophages or macrophages from cows during the lactating period or mammary gland secretions during the drying off period.

The structure of a glycoside hydrolase 29 family member from a rumen bacterium reveals unique, dual carbohydrate-binding domains.

Glycoside hydrolase (GH) family 29 consists solely of α-L-fucosidases. These enzymes catalyse the hydrolysis of glycosidic bonds. Here, the structure of GH29_0940, a protein cloned from metagenomic DNA from the rumen of a cow, has been solved, which reveals a multi-domain arrangement that has only recently been identified in bacterial GH29 enzymes. The microbial species that provided the source of this enzyme is unknown. This enzyme contains a second carbohydrate-binding domain at its C-terminal end in addition to the typical N-terminal catalytic domain and carbohydrate-binding domain arrangement of GH29-family proteins. GH29_0940 is a monomer and its overall structure consists of an N-terminal TIM-barrel-like domain, a central ß-sandwich domain and a C-terminal ß-sandwich domain. The TIM-barrel-like catalytic domain exhibits a (ß/α)8/7 arrangement in the core instead of the typical (ß/α)8 topology, with the `missing' α-helix replaced by a long meandering loop that `closes' the barrel structure and suggests a high degree of structural flexibility in the catalytic core. This feature was also noted in all six other structures of GH29 enzymes that have been deposited in the PDB. Based on sequence and structural similarity, the residues Asp162 and Glu220 are proposed to serve as the catalytic nucleophile and the proton donor, respectively. Like other GH29 enzymes, the GH29_0940 structure shows five strictly conserved residues in the catalytic pocket. The structure shows two glycerol molecules in the active site, which have also been observed in other GH29 structures, suggesting that the enzyme catalyses the hydrolysis of small carbohydrates. The two binding domains are classed as family 32 carbohydrate-binding modules (CBM32). These domains have residues involved in ligand binding in the loop regions at the edge of the ß-sandwich. The predicted substrate-binding residues differ between the modules, suggesting that different modules bind to different groups on the substrate(s). Enzymes that possess multiple copies of CBMs are thought to have a complex mechanism of ligand recognition. Defined electron density identifying a long 20-amino-acid hydrophilic loop separating the two CBMs was observed. This suggests that the additional C-terminal domain may have a dynamic range of movement enabled by the loop, allowing a unique mode of action for a GH29 enzyme that has not been identified previously.

The structure of the oligomerization domain of Lsr2 from Mycobacterium tuberculosis reveals a mechanism for chromosome organization and protection.

Lsr2 is a small DNA-binding protein present in mycobacteria and related actinobacteria that regulates gene expression and influences the organization of bacterial chromatin. Lsr2 is a dimer that binds to AT-rich regions of chromosomal DNA and physically protects DNA from damage by reactive oxygen intermediates (ROI). A recent structure of the C-terminal DNA-binding domain of Lsr2 provides a rationale for its interaction with the minor groove of DNA, its preference for AT-rich tracts, and its similarity to other bacterial nucleoid-associated DNA-binding domains. In contrast, the details of Lsr2 dimerization (and oligomerization) via its N-terminal domain, and the mechanism of Lsr2-mediated chromosomal cross-linking and protection is unknown. We have solved the structure of the N-terminal domain of Lsr2 (N-Lsr2) at 1.73 Å resolution using crystallographic ab initio approaches. The structure shows an intimate dimer of two ß-ß-a motifs with no close homologues in the structural databases. The organization of individual N-Lsr2 dimers in the crystal also reveals a mechanism for oligomerization. Proteolytic removal of three N-terminal residues from Lsr2 results in the formation of an anti-parallel ß-sheet between neighboring molecules and the formation of linear chains of N-Lsr2. Oligomerization can be artificially induced using low concentrations of trypsin and the arrangement of N-Lsr2 into long chains is observed in both monoclinic and hexagonal crystallographic space groups. In solution, oligomerization of N-Lsr2 is also observed following treatment with trypsin. A change in chromosomal topology after the addition of trypsin to full-length Lsr2-DNA complexes and protection of DNA towards DNAse digestion can be observed using electron microscopy and electrophoresis. These results suggest a mechanism for oligomerization of Lsr2 via protease-activation leading to chromosome compaction and protection, and concomitant down-regulation of large numbers of genes. This mechanism is likely to be relevant under conditions of stress where cellular proteases are known to be upregulated.