Versatility and Complexity: Common and Uncommon Facets of LysR-Type Transcriptional Regulators.

LysR-type transcriptional regulators (LTTRs) form one of the largest families of bacterial regulators. They are widely distributed and contribute to all aspects of metabolism and physiology. Most are homotetramers, with each subunit composed of an N-terminal DNA-binding domain followed by a long helix connecting to an effector-binding domain.

Overproduction, purification, and transcriptional activity of recombinant Acinetobacter baylyi ADP1 RNA polymerase holoenzyme.

Acinetobacter baylyi is an interesting model organism to investigate bacterial metabolism due to its vast repertoire of metabolic enzymes and ease of genetic manipulation. However, the study of gene expression in vitro is dependent on the availability of its RNA polymerase (RNAp), an essential enzyme in transcription. In this work, we developed a convenient method of producing the recombinant A.

Engineered citrate synthase alters Acetate Accumulation in Escherichia coli.

Metabolic engineering is used to improve titers, yields and generation rates for biochemical products in host microbes such as Escherichia coli. A wide range of biochemicals are derived from the central carbon metabolite acetyl-CoA, and the largest native drain of acetyl-CoA in most microbes including E. coli is entry into the tricarboxylic acid (TCA) cycle via citrate synthase (coded by the gltA gene).

Overproduction and purification of highly active recombinant Pseudomonas aeruginosa str. PAO1 RNA polymerase holoenzyme complex.

The bacterial RNA polymerase (RNAP) is a large, complex molecular machine that is the engine of gene expression. Despite global conservation in their structures and function, RNAPs from different bacteria can have unique features in promoter and transcription factor recognition. Therefore, availability of purified RNAP from different bacteria is key to understanding these species-specific aspects and will be valuable for antibiotic drug discovery.

Engineering CatM, a LysR-Type Transcriptional Regulator, to Respond Synergistically to Two Effectors.

The simultaneous response of one transcriptional regulator to different effectors remains largely unexplored. Nevertheless, such interactions can substantially impact gene expression by rapidly integrating cellular signals and by expanding the range of transcriptional responses. In this study, similarities between paralogs were exploited to engineer novel responses in CatM, a regulator that controls benzoate degradation in ADP1.

Accurate prediction of human miRNA targets via graph modeling of the miRNA-target duplex.

miRNAs are involved in many critical cellular activities through binding to their mRNA targets, e.g. in cell proliferation, differentiation, death, growth control, and developmental timing.

Malonate degradation in ADP1: operon organization and regulation by MdcR.

Transcriptional regulators in the LysR or GntR families are typically encoded in the genomic neighbourhood of bacterial genes for malonate degradation. While these arrangements have been evaluated using bioinformatics methods, experimental studies demonstrating co-transcription of predicted operons were lacking. Here, transcriptional regulation was characterized for a cluster of genes that enable a soil bacterium, ADP1, to use malonate as a carbon source.

The DNA-binding domain of BenM reveals the structural basis for the recognition of a T-N11-A sequence motif by LysR-type transcriptional regulators.

LysR-type transcriptional regulators (LTTRs) play critical roles in metabolism and constitute the largest family of bacterial regulators. To understand protein-DNA interactions, atomic structures of the DNA-binding domain and linker-helix regions of a prototypical LTTR, BenM, were determined by X-ray crystallography. BenM structures with and without bound DNA reveal a set of highly conserved amino acids that interact directly with DNA bases.

Defying stereotypes: the elusive search for a universal model of LysR-type regulation.

LysR-type transcriptional regulators (LTTRs) compose the largest family of homologous regulators in bacteria. Considering their prevalence, it is not surprising that LTTRs control diverse metabolic functions. Arguably, the most unexpected aspect of LTTRs is the paucity of available structural information.

Full-length structures of BenM and two variants reveal different oligomerization schemes for LysR-type transcriptional regulators.

BenM, a LysR-type transcriptional regulator (LTTR) from the bacterium Acinetobacter baylyi, responds synergistically to benzoate and cis,cis-muconate. With these effectors, BenM activates gene expression during benzoate consumption. Without effectors, BenM represses transcription.

Mechanism of Hg-C protonolysis in the organomercurial lyase MerB.

Demethylation is a key reaction in global mercury cycling. The bacterial organomercurial lyase, MerB, catalyzes the demethylation of a wide range of organomercurials via Hg-C protonolysis. Two strictly conserved cysteine residues in the active site are required for catalysis, but the source of the catalytic proton and the detailed reaction mechanism have not been determined.

Inducer responses of BenM, a LysR-type transcriptional regulator from Acinetobacter baylyi ADP1.

BenM and CatM control transcription of a complex regulon for aromatic compound degradation. These Acinetobacter baylyi paralogues belong to the largest family of prokaryotic transcriptional regulators, the LysR-type proteins. Whereas BenM activates transcription synergistically in response to two effectors, benzoate and cis,cis-muconate, CatM responds only to cis,cis-muconate.

The crystal structure of the Pseudomonas dacunhae aspartate-beta-decarboxylase dodecamer reveals an unknown oligomeric assembly for a pyridoxal-5'-phosphate-dependent enzyme.

The Pseudomonas dacunhael-aspartate-beta-decarboxylase (ABDC, aspartate 4-decarboxylase, aspartate 4-carboxylyase, E.C. 4.

Crystal structure of the Homo sapiens kynureninase-3-hydroxyhippuric acid inhibitor complex: insights into the molecular basis of kynureninase substrate specificity.

Homo sapiens kynureninase is a pyridoxal-5'-phosphate dependent enzyme that catalyzes the hydrolytic cleavage of 3-hydroxykynurenine to yield 3-hydroxyanthranilate and L-alanine as part of the tryptophan catabolic pathway leading to the de novo biosynthesis of NAD(+). This pathway results in quinolinate, an excitotoxin that is an NMDA receptor agonist. High levels of quinolinate have been correlated with the etiology of neurodegenerative disorders such as AIDS-related dementia and Alzheimer's disease.

Oligomerization of BenM, a LysR-type transcriptional regulator: structural basis for the aggregation of proteins in this family.

LysR-type transcriptional regulators comprise the largest family of homologous regulatory DNA-binding proteins in bacteria. A problematic challenge in the crystallization of LysR-type regulators stems from the insolubility and precipitation difficulties encountered with high concentrations of the full-length versions of these proteins. A general oligomerization scheme is proposed for this protein family based on the structures of the effector-binding domain of BenM in two different space groups, P4(3)22 and C222(1).

Crystal structure of Homo sapiens kynureninase.

Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography.

Distinct effector-binding sites enable synergistic transcriptional activation by BenM, a LysR-type regulator.

BenM, a bacterial transcriptional regulator, responds synergistically to two effectors, benzoate and cis,cis-muconate. CatM, a paralog with overlapping function, responds only to muconate. Structures of their effector-binding domains revealed two effector-binding sites in BenM.

Optimization of a mouse recombinant antibody fragment for efficient production from Escherichia coli.

A mutagenized mouse recombinant antibody fragment (rFab) that recognized HIV capsid protein was isolated from Escherichia coli at a level of 12 mg per liter of culture using standard shake flask methods. This is one of the highest yields of a modified antibody fragment obtained using non-fermentor-based methods. Recombinant Fab was isolated directly from the culture medium, which lacked complex materials such as tryptone and yeast extract.

The Aspergillus fumigatus cell wall is organized in domains that are remodelled during polarity establishment.

Aspergillus fumigatus is a life-threatening and increasingly frequent pathogen of the immunocompromised. Like other filamentous fungi A. fumigatus grows in a highly polar manner, adding new cell wall to the apical region of hyphae.

Benzoate decreases the binding of cis,cis-muconate to the BenM regulator despite the synergistic effect of both compounds on transcriptional activation.

Fluorescence emission spectroscopy was used to investigate interactions between two effectors and BenM, a transcriptional regulator of benzoate catabolism. BenM had a higher affinity for cis,cis-muconate than for benzoate as the sole effector. However, the presence of benzoate increased the apparent dissociation constant (reduced the affinity) of the protein for cis,cis-muconate.

Three-dimensional structure of kynureninase from Pseudomonas fluorescens.

Kynureninase [E.C. 3.

Crystallization of the effector-binding domains of BenM and CatM, LysR-type transcriptional regulators from Acinetobacter sp. ADP1.

BenM, a member of the LysR-type family of transcriptional regulators, controls genes for benzoate degradation in the Gram-negative bacterium Acinetobacter sp. strain ADP1. Recent studies show that BenM activates benABCDE expression synergistically in response to two effector ligands: cis,cis-muconate (CCM) and benzoate.

SwoHp, a nucleoside diphosphate kinase, is essential in Aspergillus nidulans.

The temperature-sensitive swoH1 mutant of Aspergillus nidulans was previously identified in a screen for mutants with defects in polar growth. In the present work, we found that the swoH1 mutant swelled, lysed, and did not produce conidia during extended incubation at the restrictive temperature. When shifted from the permissive to the restrictive temperature, swoH1 showed the temperature-sensitive swelling phenotype only after 8 h at the higher temperature.

Generation of a phagemid mouse recombinant antibody fragment library by multisite-directed mutagenesis.

A nonimmune phagemid recombinant antibody fragment (rFab) library was generated with a nominal diversity of 1.16 x 10(7) using the QuikChange Multi Site-Directed Mutagenesis kit. Two degenerate primers spanning the third complementarity-determining region (CDR) loops of the antibody fragment light and heavy chain were mutated such that eight or nine amino acids were randomly changed per CDR loop.

Aspergillus nidulans swoF encodes an N-myristoyl transferase.

Polar growth is a fundamental process in filamentous fungi and is necessary for disease initiation in many pathogenic systems. Previously, swoF was identified in Aspergillus nidulans as a single-locus, temperature-sensitive (ts) mutant aberrant in both polarity establishment and polarity maintenance. The swoF gene was cloned by complementation of the ts phenotype and sequenced.