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DNA polymerase III sliding clamp (DnaN) was recently validated as a new anti-tuberculosis target employing griselimycins. Three (2 S,4 R)−4-methylproline moieties of methylgriselimycin play significant roles in target binding and metabolic stability. Here, we identify the mycoplanecin biosynthetic gene cluster by genome mining using bait genes from the 4-methylproline pathway. We isolate and structurally elucidate four mycoplanecins comprising scarce homo-amino acids and 4-alkylprolines. Evaluating mycoplanecin E against Mycobacterium tuberculosis surprisingly reveals an excitingly low minimum inhibition concentration at 83 ng/mL, thus outcompeting griselimycin by approximately 24-fold. We show that mycoplanecins bind DnaN with nanomolar affinity and provide a co-crystal structure of mycoplanecin A-bound DnaN. Additionally, we reconstitute the biosyntheses of the unusual l-homoleucine, l-homonorleucine, and (2 S,4 R)−4-ethylproline building blocks by characterizing in vitro the full set of eight enzymes involved. The biosynthetic study, bioactivity evaluation, and drug target validation of mycoplanecins pave the way for their further development to tackle multidrug-resistant mycobacterial infections. Mycoplanecins show promising activity against tuberculosis. Here, the authors identify and study mycoplanecins' biosynthesis, antibacterial effects, and binding mechanism to DnaN, suggesting potential for fighting multidrug-resistant tuberculosis. [ABSTRACT FROM AUTHOR]

cis-Aconitate decarboxylase (ACOD1, IRG1) converts cis-aconitate to the immunomodulatory and antibacterial metabolite itaconate. Although the active site residues of human and mouse ACOD1 are identical, the mouse enzyme is about fivefold more active. Aiming to identify the cause of this difference, we mutated positions near the active site in human ACOD1 to the corresponding residues of mouse ACOD1 and measured resulting activities in vitro and in transfected cells. Interestingly, Homosapiens is the only species with methionine instead of isoleucine at residue 154 and introduction of isoleucine at this position increased the activity of human ACOD1 1.5-fold in transfected cells and 3.5-fold in vitro. Enzyme activity of gorilla ACOD1, which is almost identical to the human enzyme but has isoleucine at residue 154, was similar to the mouse enzyme in vitro. Met154 in human ACOD1 forms a sulfur-π bond to Phe381, which is positioned to impede access of the substrate to the active site. It appears that the ACOD1 sequence has changed at position 154 during human evolution, resulting in a pronounced decrease in activity. This change might have offered a selective advantage in diseases such as cancer. [ABSTRACT FROM AUTHOR]

Box C/D ribonucleoprotein complexes are RNA-guided methyltransferases that methylate the ribose 2'-OH of RNA. The central 'guide RNA' has box C and D motifs at its ends, which are crucial for activity. Archaeal guide RNAs have a second box C'/D' motif pair that is also essential for function. This second motif is poorly conserved in eukaryotes and its function is uncertain. Conflicting literature data report that eukaryotic box C'/D' motifs do or do not bind proteins specialized to recognize box C/D-motifs and are or are not important for function. Despite this uncertainty, the architecture of eukaryotic 2'-O-methylation enzymes is thought to be similar to that of their archaeal counterpart. Here, we use biochemistry, X-ray crystallography and mutant analysis to demonstrate the absence of functional box C'/D' motifs in more than 80% of yeast guide RNAs. We conclude that eukaryotic Box C/D RNPs have two non-symmetric protein assembly sites and that their three-dimensional architecture differs from that of archaeal 2'-O-methylation enzymes. [ABSTRACT FROM AUTHOR]

Penicillin G acylase (PGA) catalyzes the hydrolysis of penicillin G to 6-aminopenicillanic acid and phenylacetic acid, which provides the precursor for most semisynthetic penicillins. Most applications rely on PGAs from Gram-negative bacteria. Here we describe the first three crystal structures for PGAs from Gram-positive Bacilli and their utilization in protein engineering experiments for the manipulation of their thermostability. PGAs from Bacillus megaterium (BmPGA, Tm = 56.0 °C), Bacillus thermotolerans (BtPGA, Tm = 64.5 °C), and Bacillus sp. FJAT-27231 (FJAT-PGA, Tm = 74.3 °C) were recombinantly produced with B. megaterium, secreted, purified to apparent heterogeneity, and crystallized. Structures with resolutions of 2.20 Å (BmPGA), 2.27 Å (BtPGA), and 1.36 Å (FJAT-PGA) were obtained. They revealed high overall similarity, reflecting the high identity of up to approx. 75%. Notably, the active center displays a deletion of more than ten residues with respect to PGAs from Gram-negatives. This enlarges the substrate binding site and may indicate a different substrate spectrum. Based on the structures, ten single-chain FJAT-PGAs carrying artificial linkers were produced. However, in all cases, complete linker cleavage was observed. While thermostability remained in the wild-type range, the enzymatic activity dropped between 30 and 60%. Furthermore, four hybrid PGAs carrying subunits from two different enzymes were successfully produced. Their thermostabilities mostly lay between the values of the two mother enzymes. For one PGA increased, enzyme activity was observed. Overall, the three novel PGA structures combined with initial protein engineering experiments provide the basis for establishment of new PGA-based biotechnological processes. [ABSTRACT FROM AUTHOR]

Mutations in the gene encoding smooth muscle cell alpha actin (ACTA2) have recently been shown to cause familial thoracic aortic aneurysms leading to type A dissections (TAAD) and predispose to premature stroke and coronary artery disease. In order to further explore the role of ACTA2 variations in the pathogenesis of TAAD, we sequenced the coding regions of this gene in 40 unrelated German patients with TAAD (with (n=21) or without (n=19) clinical features suggestive of Marfan syndrome). All patients had previously tested negative for mutations in the FBN1 and TGFBR2 genes. We identified three novel ACTA2 mutations and mapped them on a three-dimensional model of actin. Two mutations affect residues within (M49V) or adjacent to (R39C), the DNAse-I-binding loop within subdomain 2 of alpha actin. They were observed in families with recurrent aortic aneurysm (R39C) or aortic dissection (M49V). The third mutation causes an exchange in the vicinity of the ATP-binding site (G304R) in a patient thought to have isolated TAAD. None of the affected individuals had clinical features typical for Marfan syndrome, and no case of premature stroke or coronary artery disease was reported from the affected families. In conclusion, we underscore the role of ACTA2 mutations in nonsyndromic TAAD and suggest that ACTA2 should be included in the genes routinely investigated for syndromic and nonsyndromic TAAD. Detailed clinical investigations of additional families are warranted to further explore the full range of phenotypic signs associated with the three novel mutations described here. [ABSTRACT FROM AUTHOR]

The phenazines are colored secondary metabolites that many bacterial species produce in such large quantities that they are visible to the naked eye. Phenazines are redox-active, enabling them to act as broad-specificity antibiotics and as virulence/survival factors as in the case of the blue pyocyanin produced by the opportunistic pathogen Pseudomonas aeruginosa. Here, we review the current understanding of their biosynthesis from chorismic acid. [ABSTRACT FROM AUTHOR]

Protein prenylation is a widespread phenomenon in eukaryotic cells that affects many important signaling molecules. We describe the structure-guided design of engineered protein prenyltransferases and their universal synthetic substrate, biotin-geranylpyrophosphate. These new tools allowed us to detect femtomolar amounts of prenylatable proteins in cells and organs and to identify their cognate protein prenyltransferases. Using this approach, we analyzed the in vivo effects of protein prenyltransferase inhibitors. Whereas some of the inhibitors displayed the expected activities, others lacked in vivo activity or targeted a broader spectrum of prenyltransferases than previously believed. To quantitate the in vivo effect of the prenylation inhibitors, we profiled biotin-geranyl–tagged RabGTPases across the proteome by mass spectrometry. We also demonstrate that sites of active vesicular transport carry most of the RabGTPases. This approach enables a quantitative proteome-wide analysis of the regulation of protein prenylation and its modulation by therapeutic agents. [ABSTRACT FROM AUTHOR]

Schizophyllan (SCH) is a high molecular weight homopolysaccharide composed of a β-(1,3)-D-glucan main chain with branching β-(1,6)-bound D-glucose residues. It forms triple helices that are highly stable towards heat and extreme pH, which provides SCH with interesting properties for industrial and medical applications. The recombinant anti-SCH antibody JoJ48C11 recognizes SCH and related β-(1,6)-branched β-(1,3)-D-glucans, but details governing its specificity are not known. Here, we fill this gap by determining crystal structures of the antigen binding fragment (Fab) of JoJ48C11 in the apo form and in complex with the unbranched β-(1,3)-D-glucose hexamer laminarihexaose 3.0 and 2.4 Å resolution, respectively. Together with docking studies, this allowed construction of a JoJ48C11/triple-helical SCH complex, leading to the identification of eight amino acid residues of JoJ48C11 (Tyr27H, His35H, Trp47H, Trp100H, Asp105H; Asp49L, Lys52L, Trp90L) that contribute to the recognition of glucose units from all three chains of the SCH triple helix. The importance of these amino acids was confirmed by mutagenesis and ELISA-based analysis. Our work provides an explanation for the specific recognition of triple-helical β-(1,6)-branched β-(1,3)-D-glucans by JoJ48C11 and provides another structure example for anti-carbohydrate antibodies. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance is a global threat that kills at least 75,000 people every year worldwide and causes extended hospital stays. In  the coming 10 years, antimicrobial resistance is projected to have huge health and economic burden on countries, and the scarcity of available antibiotics further worsens the situation. Antimicrobial resistance results mainly from indiscriminate antibiotic usage in humans, animals and agriculture, and from the rapid emergence and dissemination of resistant pathogens. This issue is challenging for antibiotic stewardship, strict regulations on antibiotics usage, large-scale surveillance and responsible public behavior. This demands international cooperation and integrated efforts under the ‘one-health’ strategy. Here, we review antimicrobial resistance and the one-health strategy. We discuss the historical issue of using antibiotics. We highlight the effectiveness of hygiene in livestock rearing, careful antibiotic usage and large-scale surveillance of animals, humans and environment domains. We present strategies for mitigation of antimicrobial resistance, exemplified by the successful ban of triclosan which induced a significant decline of resistant pathogens. We emphasize the benefits of the global antibiotic resistance partnership and of the one-health participation of stakeholders from public, healthcare professionals and government to mitigate antimicrobial resistance. [ABSTRACT FROM AUTHOR]