Structural studies of AraR from B. thetaiotaomicron

The NrtR family of bacterial transcription factors is characterized by an N-terminal Nudix hydrolase-like effector binding domain and a C-terminal DNA binding domain. A bioinformatics analysis of the NrtR family represented by uncharacterized protein BT0354 in Bacteroides thetaiotaomicron suggests that these regulators control the catabolic pathways for L-arabinose. Many bacteria use L-arabinose as the sole source of carbon energy. The L-arabinose utilization pathway and its transcriptional regulation have been studied for a long time in several model microorganisms. Here we provide biochemical and structural characterization of the novel arabinose-responsive regulator of NrtR family protein BT0354, L-arabinose regulator from B. thetaiotaomicron (BtAraR). The BtAraR DNA binding and the role of effector molecule L-arabinose were confirmed using electrophoretic mobility shift assays. We have solved the crystal structures of BtAraR for two apo forms, and complexes with L-arabinose and double-stranded DNA target. The apo-1 form was solved as two dimers/AU in the R3 space group at 2.35 Å, while the apo-2 form was solved as one monomer/AU in the I213 space group at 2.56 Å resolution. The L-arabinose and DNA complex structures were solved as a dimer/AU in the P21 space group at 1.95 Å resolution and the P23 space group at 3.05 Å resolution, respectively. The biological unit of this protein is a dimer while the N-terminal ligand binding domain of the monomer adopts a Nudix hydrolase-like fold and the C-terminal DNA binding domain is a winged helix-turn-helix. The DNA binding-releasing mechanism can be rationalized through the comparison and analyses of these structures. The apo and DNA bound structures are more similar compared to the L-arabinose-bound structure. The r.m.s. deviation for the apo and DNA bound structures is 1.13 Å, while that for apo and the L-arabinose-bound structures is 4.54 Å. Details about the DNA binding mode, L-arabinose binding and L-arabinose induced structural change will be presented. This work was supported by National Institutes of Health grant GM094585 and by the U. S. Department of Energy, Office of Biological and Environmental Research, under contract DE-AC02-06CH11357.

Structure and specificity of novel aminopeptidase from marine sediment Archaea

The Earth's microbial diversity remained largely unexplored until recent developments in DNA sequencing. Novel methods enabled us to access genomic information of uncultured microbial organisms and create hypotheses about their metabolic capabilities. These predictions primarily rely on the sequence similarity between a novel protein and characterized proteins. Such an approach introduces a "culture" bias: the well-understood proteins come from a set of laboratory-grown bacteria, while novel microbial proteins are obtained from a variety of environments, including the most extreme. One such niche is ocean sediment – an unexplored ecosystem that plays important roles in geochemical cycles. Single-cell genomics targeting sedimentary populations identified four new archaeons encoding putative intra- and extra-cellular proteases [1]. This discovery suggests that heterotrophic marine Archaea evolved to degrade detrital proteins and might contribute to global carbon cycling. The novel proteases share some sequence similarity with well-known protein-degrading enzymes, but generally are distant homologs. Thus, functional screening is necessary to validate sequence-based predictions. One of the proteases shares sequence similarity with S15 peptidases, cocaine esterases and α-amino acid ester hydrolases (AEH). Phylogeny indicates that the gene is of bacterial origin. Enzymatic assays reveal α-aminopeptidase activity towards dipeptides with a preference for a small, L-configured hydrophobic residue at the N-terminus. The crystal structure shows a homotetrameric, self-compartmentalizing enzyme with four independent active sites localized inside the oligomeric assembly accessible from the internal channel. The active site contains a serine protease triad (Ser-His-Asp) and a cluster of negatively charged residues that bind the N-terminal NH3+ group of the substrate molecule. Therefore, the observed activity suggests that the enzyme (designated as AP TA1) may act on di- or tri-peptides produced during extracellular degradation and subsequently imported to the cell. As a close homolog of AEHs, it is also possible that AP TA1 might participate in the synthesis of yet-to-be-discovered secondary metabolites. Supported by NIH GM094585, DOE/BER DE-AC02-06CH11357 & C-DEBI 36202823 & 157595.

Carbapenemase NDM-1, Structural Analysis of the Catalytic Mechanism

The New Delhi Metallo β-lactamase (NDM-1), first identified in Klebsiella pneumoniae has been shown to hydrolyze nearly all clinical β-lactam antibiotics including carbapenems, considered "last resort" antibiotics. Its gene resides on mobile plasmids that move between different strains of bacteria posing a serious global threat to human health. There have also been reports of several variants, up to NDM-9, some with increased carbapenemase activity. As part of the NIGMS PSI:Biology effort, the Midwest Center for Structural Genomics (MCSG) together with the Structures of Mtb Proteins Conferring Susceptibility to Known Mtb Inhibitors partnership, made significant progress in investigating the enzyme atomic structure and catalytic mechanism. A large number of protein constructs as well as mutants were made and a number of high-resolution structures of NDM-1 (no Zn, one Zn, two Zn, two Mn or Cd, and complexed with antibiotics) and NDM-1 variants, NDM-2, NDM-3, NDM-4, NDM-5 and NDM-6 have been determined. We have determined the two structures of Michaelis complex: NDM-1 with two cadmium ions and a mixture of hydrolyzed and unhydrolyzed ampicillin (1.50 Å) and one with two cadmium ions and partly hydrolyzed faropenem (2.00 Å). The crystal structures revealed a ligand-binding pocket consisting of several flexible loops capable of accommodating many β-lactam substrates of different sizes and shapes. The structures with various metals suggest that the distance between the two metal atoms is closely correlated with substrate binding efficiency and hydrolysis and the pH-dependency of catalytic activity. For better understanding of catalytic mechanism of NDM-1, particularly the dynamics of substrate binding and the energy surfaces along the suggested reaction pathways, molecular dynamics calculations and hybrid classical/quantum (QM/MM) calculations were performed. This work was supported by NIH Grant GM094585 and by the U.S. DOE, OBER contract DE-AC02-06CH11357