Molecular characterization of novel pyridoxal‐5′‐phosphate‐dependent enzymes from the human microbiome

Pyridoxal‐5′‐phosphate or PLP, the active form of vitamin B6, is a highly versatile cofactor that participates in a large number of mechanistically diverse enzymatic reactions in basic metabolism. PLP‐dependent enzymes account for ～1.5% of most prokaryotic genomes and are estimated to be involved in ～4% of all catalytic reactions, making this an important class of enzymes. Here, we structurally and functionally characterize three novel PLP‐dependent enzymes from bacteria in the human microbiome: two are from Eubacterium rectale, a dominant, nonpathogenic, fecal, Gram‐positive bacteria, and the third is from Porphyromonas gingivalis, which plays a major role in human periodontal disease. All adopt the Type I PLP‐dependent enzyme fold and structure‐guided biochemical analysis enabled functional assignments as tryptophan, aromatic, and probable phosphoserine aminotransferases.     

Structure and Function of a Novel ld-Carboxypeptidase A Involved in Peptidoglycan Recycling

Approximately 50% of cell wall peptidoglycan in Gram-negative bacteria is recycled with each generation. The primary substrates used for peptidoglycan biosynthesis and recycling in the cytoplasm are GlcNAc-MurNAc(anhydro)-tetrapeptide and its degradation product, the free tetrapeptide. This complex process involves ∼15 proteins, among which the cytoplasmic enzyme ld-carboxypeptidase A (LdcA) catabolizes the bond between the last two l- and d-amino acid residues in the tetrapeptide to form the tripeptide, which is then utilized as a substrate by murein peptide ligase (Mpl). LdcA has been proposed as an antibacterial target. The crystal structure of Novosphingobium aromaticivorans DSM 12444 LdcA (NaLdcA) was determined at 1.89-Å resolution. The enzyme was biochemically characterized and its interactions with the substrate modeled, identifying residues potentially involved in substrate binding. Unaccounted electron density at the dimer interface in the crystal suggested a potential site for disrupting protein-protein interactions should a dimer be required to perform its function in bacteria. Our analysis extends the identification of functional residues to several other homologs, which include enzymes from bacteria that are involved in hydrocarbon degradation and destruction of coral reefs. The NaLdcA crystal structure provides an alternate system for investigating the structure-function relationships of LdcA and increases the structural coverage of the protagonists in bacterial cell wall recycling.     

Is an aggressive surgical approach worthwhile in biliary cancer?

Background

Biliary cancer includes cancer of the gallbladder as well as extrahepatic and intrahepatic cholangiocarcinoma. Surgery is the only curative treatment option available. Recently, much more aggressive surgical approaches have been employed. Therefore, we have investigated outcome of biliary cancer before and after establishment of an aggressive surgical approach.

Methods

Retrospective single-center analysis comparing two time periods of 5 years each. During the second period new surgical expertise and a much more aggressive surgical approach were used.

Results

In the first time period (5/1995–4/2000) only 29 patients with biliary cancer were treated at our institution, while a total of 85 patients were treated during the second time period (5/2000–4/2005). Surgical resection was attempted in 55% during the first period versus 62% in the second; resection was complete in 37.5% and 58.5%, respectively. Patients undergoing resection during the second time period were more likely to be without relapse compared with patients undergoing resection in the first time period. No patient from the first period is without evidence of disease, compared to 11 patients operated in the second period. Resected patients had better survival compared with unresected patients for all tumor locations (gallbladder, extrahepatic and intrahepatic cholangiocarcinomas). Overall survival of patients was not significantly different between patients treated during the first versus the second time period.

Conclusion

In patients with biliary cancer surgical resection should be attempted whenever possible. However, long-term survival can be achieved only when a complete resection is obtained.

     

N. elongata produces type IV pili that mediate interspecies gene transfer with N. gonorrhoeae

The genus Neisseria contains at least eight commensal and two pathogenic species. According to the Neisseria phylogenetic tree, commensals are basal to the pathogens. N. elongata, which is at the opposite end of the tree from N. gonorrhoeae, has been observed to be fimbriated, and these fimbriae are correlated with genetic competence in this organism. We tested the hypothesis that the fimbriae of N. elongata are Type IV pili (Tfp), and that Tfp functions in genetic competence. We provide evidence that the N. elongata fimbriae are indeed Tfp. Tfp, as well as the DNA Uptake Sequence (DUS), greatly enhance N. elongata DNA transformation. Tfp allows N. elongata to make intimate contact with N. gonorrhoeae and to mediate the transfer of antibiotic resistance markers between these two species. We conclude that Tfp functional for genetic competence is a trait of a commensal member of the Neisseria genus. Our findings provide a mechanism for the horizontal gene transfer that has been observed among Neisseria species.     

Structure of the pilus assembly protein TadZ from Eubacterium rectale: implications for polar localization

The tad (tight adherence) locus encodes a protein translocation system that produces a novel variant of type IV pili. The pilus assembly protein TadZ (called CpaE in Caulobacter crescentus) is ubiquitous in tad loci, but is absent in other type IV pilus biogenesis systems. The crystal structure of TadZ from Eubacterium rectale (ErTadZ), in complex with ATP and Mg²⁺, was determined to 2.1 Å resolution. ErTadZ contains an atypical ATPase domain with a variant of a deviant Walker‐A motif that retains ATP binding capacity while displaying only low intrinsic ATPase activity. The bound ATP plays an important role in dimerization of ErTadZ. The N‐terminal atypical receiver domain resembles the canonical receiver domain of response regulators, but has a degenerate, stripped‐down ‘active site’. Homology modelling of the N‐terminal atypical receiver domain of CpaE indicates that it has a conserved protein–protein binding surface similar to that of the polar localization module of the social mobility protein FrzS, suggesting a similar function. Our structural results also suggest that TadZ localizes to the pole through the atypical receiver domain during an early stage of pili biogenesis, and functions as a hub for recruiting other pili components, thus providing insights into the Tad pilus assembly process.     

Stable chromosome aberrations in the lymphocytes of a population living in the vicinity of the Semipalatinsk nuclear test site

SalomaTranslocation analysis using FISH (fluorescence in situ hybridization) chromosome painting was performed to evaluate the magnitude of exposure to ionizing radiation among the human population living close to the Semipalatinsk nuclear test site in Kazakhstan. We studied two generations of people living in villages that were in the path of the radioactive cloud from the first Soviet surface nuclear test performed in August 1949 and from later tests. The older generation (P(0)) lived in the area at the time of testing, and the younger generation (F(1)) was exposed to smaller doses from the residual fallout and later tests. In both P(0) and F(1) generations, similar translocation frequencies were observed in persons living in either the Semipalatinsk area or a noncontaminated area. Assuming translocation stability in peripheral blood lymphocytes over several decades, these findings suggest that on average, the magnitude of exposure of this cohort in the Semipalatinsk area has been considerably smaller than that reported in the literature. Previously reported doses of the order of 1-4.5 Gy (mean 2.9 Gy in the P(0) generation) cannot be confirmed by the present data.     

Changes in beta-endorphin content in discrete areas of the hypothalamus throughout proestrus and diestrus of the rat

The aim of the present study is to investigate changes in beta-endorphin content in the hypothalamus during different stages of the estrous cycle. Groups of 9 to 10 Sprague-Dawley rats were sacrificed every two hours on proestrus from 8.00 to 18.00 h and groups of 7 to 8 rats were sacrificed on diestrus at 8.00, 12.00, 14.00 and 18.00 h. Preoptic suprachiasmatic region, posterior hypothalamus, arcuate nucleus and median eminence were dissected and assayed for beta-endorphin. A significant increase in beta-endorphin content was detected in the arcuate nucleus during proestrus (9.00 h: 1.76 +/- .31; 14.00 h: 4.10 +/- .85 microgram/g tissue wet weight). Levels did not change during diestrus (1.18 +/- .06 microgram/g). The increase caused significant differences in beta-endorphin values between both days at 12.00, 14.00 and 18.00 h, while the concentrations at 8.00 h were similar. The opposite pattern was observed in the median eminence with significantly higher proestrous beta-endorphin levels at 8.00 h (11.24 +/- 3.1 vs 3.52 +/- .64 microgram/g) and nonsignificant differences for the rest of the day. No significant change in beta-endorphin concentration was seen in the preoptic suprachiasmatic region over the day of proestrus (1.35 +/- .09 microgram/g). Diestrous beta-endorphin concentrations in this region were higher during the morning (2.60 +/- .65 microgram/g) and lower at 18.00 h (0.94 +/- .12 microgram/g) when compared to proestrous values. This pattern was caused by a 50% increase in beta-endorphin during the afternoon of diestrus. No changes were observed in the posterior hypothalamus on either day with comparable levels of beta-endorphin except at 18.00 h, when values were significantly higher on proestrus (1.66 +/- .30 vs 0.83 +/- .06 microgram/g).