Insights into the genomic and functional divergence of NAT gene family to serve microbial secondary metabolism.

Microbial NAT enzymes, which employ acyl-CoA to acylate aromatic amines and hydrazines, have been well-studied for their role in xenobiotic metabolism. Some homologues have also been linked to secondary metabolism, but this function of NAT enzymes is not as well-known. For this comparative study, we surveyed sequenced microbial genomes to update the list of formally annotated NAT genes, adding over 4000 new sequences (mainly bacterial, but also archaeal, fungal and protist) and portraying a broad but not universal distribution of NATs in the microbiocosmos. Localization of NAT sequences within microbial gene clusters was not a rare finding, and this association was evident across all main types of biosynthetic gene clusters (BGCs) implicated in secondary metabolism. Interrogation of the MIBiG database for experimentally characterized clusters with NAT genes further supports that secondary metabolism must be a major function for microbial NAT enzymes and should not be overlooked by researchers in the field. We also show that NAT sequences can be associated with bacterial plasmids potentially involved in horizontal gene transfer. Combined, our computational predictions and MIBiG literature findings reveal the extraordinary functional diversification of microbial NAT genes, prompting further research into their role in predicted BGCs with as yet uncharacterized function.

Fusarium verticillioides NAT1 (FDB2) N-malonyltransferase is structurally, functionally and phylogenetically distinct from its N-acetyltransferase (NAT) homologues.

Fusarium endophytes damage cereal crops and contaminate produce with mycotoxins. Those fungi overcome the main chemical defence of host via detoxification by a malonyl-CoA-dependent enzyme homologous to xenobiotic metabolizing arylamine N-acetyltransferase (NAT). In Fusarium verticillioides (teleomorph Gibberella moniliformis, GIBMO), this N-malonyltransferase activity is attributed to (GIBMO)NAT1, and the fungus has two additional isoenzymes, (GIBMO)NAT3 (N-acetyltransferase) and (GIBMO)NAT2 (unknown function). We present the crystallographic structure of (GIBMO)NAT1, also modelling other fungal NAT homologues. Monomeric (GIBMO)NAT1 is distinctive, with access to the catalytic core through two "tunnel-like" entries separated by a "bridge-like" helix. In the quaternary arrangement, (GIBMO)NAT1 monomers interact in pairs along an extensive interface whereby one entry of each monomer is covered by the N-terminus of the other monomer. Although monomeric (GIBMO)NAT1 apparently accommodates acetyl-CoA better than malonyl-CoA, dimerization changes the active site to allow malonyl-CoA to reach the catalytic triad (Cys110, His158 and Asp173) via the single uncovered entry, and anchor its terminal carboxyl-group via hydrogen bonds to Arg109, Asn157 and Thr261. Lacking a terminal carboxyl-group, acetyl-CoA cannot form such stabilizing interactions, while longer acyl-CoAs enter the active site but cannot reach catalytic Cys. Other NAT isoenzymes lack such structural features, with (GIBMO)NAT3 resembling bacterial NATs and (GIBMO)NAT2 adopting a structure intermediate between (GIBMO)NAT1 and (GIBMO)NAT3. Biochemical assays confirmed differential donor substrate preference of (GIBMO)NAT isoenzymes, with phylogenetic analysis demonstrating evolutionary separation. Given the role of (GIBMO)NAT1 in enhancing Fusarium pathogenicity, unravelling the structure and function of this enzyme may benefit research into more targeted strategies for pathogen control.

Lung cancer, treatment and nutritional status.

Lung cancer is often diagnosed at inoperable advanced stages, and most patients exhibit cancer cachexia. The nutritional status of patients has been previously observed to serve a key role in cancer survival and cancer surgery. The aim of the current study was to collect information regarding the treatment of patients and associate them with different nutritional measurements. A total of 82 patients with non-small cell lung cancer were included in the present study. Several parameters were assessed, such as body mass index (BMI), Mediterranian diet score, number of years spent smoking, basic metabolsim (RMR; kcal/day), VO2 (ml/min), ventilation (lt/min) and physical activity. All the aforementioned parameters were associated with patient treatment, nutritional status and survival. Two-way ANOVA was conducted and pairwise group mean differences were tested using Fisher's LSD and Tukey tests. Normality and variance homogeneity was checked in all cases. The results revealed that RMR and oxygen consumption were negatively affected by the survival status of patients (P=0.012 and P=0.043, respectively). The mean fat difference was higher in patients treated with immunotherapy, and lower in those treated with chemotherapy in addition to immunotherapy, as demonstrated by Tukey comparisons. The survival of 25 patients were affected by the treatment they received (P=0.006). Chemotherapy administered in addition to immunotherapy prolonged patient life almost two-fold when compared with the individual effects of the two treatments, which became equal according to Fisher's LSD comparisons. In conclusion, the nutritional status of patients was associated with the administration of chemotherapy in addition to immunotherapy, and patient survival. Increased metabolism and fat mass were also associated with prolonged survival.

Folding Simulations of a Nuclear Receptor Box-Containing Peptide Demonstrate the Structural Persistence of the LxxLL Motif Even in the Absence of Its Cognate Receptor.

Regulation of nuclear receptors by their coactivators involves the recognition and binding of a specific sequence motif contained in the coactivator sequence. This motif is known as the nuclear receptor (NR) box and contains a conserved LxxLL subsequence, where L is leucine and x is any amino acid residue. Crystallographic studies have shown that the LxxLL motifs adopt an α-helical conformation when bound to their cognate nuclear receptors. Here we use an extensive set of folding molecular dynamics simulations to examine whether the α-helical conformation demonstrated by the LxxLL motifs in the bound state may represent a persistent structural preference of these peptides even in the absence of their cognate receptors. To this end, we have performed a grand total of 35 µs of adaptive tempering folding simulations of an NR-box-containing peptide derived from Drosophila's fushi tarazu segmentation gene product. Our simulations-performed using full electrostatics and an explicit representation of two different solvents (water and a TFE/water mixture)-clearly indicate the presence of a persistent helical preference of the LxxLL motif with a concomitant native-like structure and contacts between the motif's leucine residues. To lend further support to our findings, we compare the simulation-derived peptide dynamics with experimental NMR-derived nuclear Overhauser effect (NOE) measurements that had been previously obtained for the same peptide in the same two solvents. The comparison demonstrates a quantitative agreement between simulation and experiment with average upper bound NOE violations of less than 0.084 Å, thus independently validating our main conclusion concerning the intrinsic preference of NR-box motifs to form helical structures even in the absence of their cognate receptors.