Overexpression and mutagenesis of the lipoamide dehydrogenase of Escherichia coli.
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ABSTRACT:
SUBMITTER: Allison N
PROVIDER: S-EPMC1135478 | biostudies-other | 1988 Dec
REPOSITORIES: biostudies-other
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Project description:Tuberculosis remains a global health emergency that calls for treatment regimens directed at new targets. Here we explored lipoamide dehydrogenase (Lpd), a metabolic and detoxifying enzyme in Mycobacterium tuberculosis (Mtb) whose deletion drastically impairs Mtb's ability to establish infection in the mouse. Upon screening more than 1.6 million compounds, we identified N-methylpyridine 3-sulfonamides as potent and species-selective inhibitors of Mtb Lpd affording >1000-fold selectivity versus the human homologue. The sulfonamides demonstrated low nanomolar affinity and bound at the lipoamide channel in an Lpd-inhibitor cocrystal. Their selectivity could be attributed, at least partially, to hydrogen bonding of the sulfonamide amide oxygen with the species variant Arg93 in the lipoamide channel. Although potent and selective, the sulfonamides did not enter mycobacteria, as determined by their inability to accumulate in Mtb to effective levels or to produce changes in intracellular metabolites. This work demonstrates that high potency and selectivity can be achieved at the lipoamide-binding site of Mtb Lpd, a site different from the NAD?/NADH pocket targeted by previously reported species-selective triazaspirodimethoxybenzoyl inhibitors.
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Project description:Mycobacterium tuberculosis (Mtb) remains the leading single cause of death from bacterial infection. Here we explored the possibility of species-selective inhibition of lipoamide dehydrogenase (Lpd), an enzyme central to Mtb's intermediary metabolism and antioxidant defense. High-throughput screening of combinatorial chemical libraries identified triazaspirodimethoxybenzoyls as high-nanomolar inhibitors of Mtb's Lpd that were noncompetitive versus NADH, NAD(+), and lipoamide and >100-fold selective compared to human Lpd. Efficacy required the dimethoxy and dichlorophenyl groups. The structure of an Lpd-inhibitor complex was resolved to 2.42 A by X-ray crystallography, revealing that the inhibitor occupied a pocket adjacent to the Lpd NADH/NAD(+) binding site. The inhibitor did not overlap with the adenosine moiety of NADH/NAD(+) but did overlap with positions predicted to bind the nicotinamide rings in NADH and NAD(+) complexes. The dimethoxy ring occupied a deep pocket adjacent to the FAD flavin ring where it would block coordination of the NADH nicotinamide ring, while the dichlorophenyl group occupied a more exposed pocket predicted to coordinate the NAD(+) nicotinamide. Several residues that are not conserved between the bacterial enzyme and its human homologue were predicted to contribute both to inhibitor binding and to species selectivity, as confirmed for three residues by analysis of the corresponding mutant Mtb Lpd proteins. Thus, nonconservation of residues lining the electron-transfer tunnel in Mtb Lpd can be exploited for development of species-selective Lpd inhibitors.
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Project description:A high-throughput method has been developed for the systematic mutagenesis of the Escherichia coli genome. The system is based on in vitro transposition of a modified Tn5 element, the Sce-poson, into linear fragments of each open reading frame. The transposon introduces both positive (kanamycin resistance) and negative (I-SceI recognition site) selectable markers for isolation of mutants and subsequent allele replacement, respectively. Reaction products are then introduced into the genome by homologous recombination via the lambdaRed proteins. The method has yielded insertion alleles for 1976 genes during a first pass through the genome including, unexpectedly, a number of known and putative essential genes. Sce-poson insertions can be easily replaced by markerless mutations by using the I-SceI homing endonuclease to select against retention of the transposon as demonstrated by the substitution of amber and/or in-frame deletions in six different genes. This allows a Sce-poson-containing gene to be specifically targeted for either designed or random modifications, as well as permitting the stepwise engineering of strains with multiple mutations. The promiscuous nature of Tn5 transposition also enables a targeted gene to be dissected by using randomly inserted Sce-posons as shown by a lacZ allelic series. Finally, assessment of the insertion sites by an iterative weighted matrix algorithm reveals that these hyperactive Tn5 complexes generally recognize a highly degenerate asymmetric motif on one end of the target site helping to explain the randomness of Tn5 transposition.
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Project description:Uropathogenic Escherichia coli (UPEC), which accounts for 85% of urinary tract infections (UTI), assembles biofilms in diverse environments, including the host. Besides forming biofilms on biotic surfaces and catheters, UPEC has evolved an intracellular pathogenic cascade that culminates in the formation of biofilm-like intracellular bacterial communities (IBCs) within bladder epithelial cells. Rapid bacterial replication during IBC formation augments a build-up in bacterial numbers and persistence within the host. Relatively little is known about factors mediating UPEC biofilm formation and how these overlap with IBC formation. To address this gap, we screened a UPEC transposon mutant library in three in vitro biofilm conditions: Luria broth (LB)-polyvinyl chloride (PVC), YESCA (yeast extract-Casamino Acids)-PVC, and YESCA-pellicle that are dependent on type 1 pili (LB) and curli (YESCA), respectively. Flagella are important in all three conditions. Mutants were identified that had biofilm defects in all three conditions but had no significant effects on the expression of type 1 pili, curli, or flagella. Thus, this approach uncovered a comprehensive inventory of novel effectors and regulators that are involved in UPEC biofilm formation under multiple conditions. A subset of these mutants was found to be dramatically attenuated and unable to form IBCs in a murine model of UTI. Collectively, this study expands our insights into UPEC multicellular behavior that may provide insights into IBC formation and virulence.
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Project description:Dehydrogenase pathway, one of diaminopimelate pathway, is important to the biosynthesis of L-lysine and peptidoglycan via one single reaction catalyzed by meso-diaminopimelate dehydrogenase (DapDH). In this study, the thermostable DapDH was introduced into diaminopimelate pathway that increased the final titer (from 71.8 to 119.5 g/L), carbon yield (from 35.3% to 49.1%) and productivity (from 1.80 to 2.99 g/(L∙h)) of L-lysine by LATR12-2∆rpiB::ddhSt in fed-batch fermentation. To do this, the kinetic properties and the effects of different DapDHs on L-lysine production were investigated, and the results indicated that overexpression of StDapDH in LATR12-2 was beneficial to construct an L-lysine producer with good productive performance because it exhibited the best of kinetic characteristics and optimal temperature as well as thermostability in reductive amination. Furthermore, ammonium availability was optimized, and found that 20 g/L of (NH4)2SO4 was the optimal ammonium concentration for improving the efficiency of L-lysine production by LATR12-2∆rpiB::ddhSt. Metabolomics analysis showed that introducing the StDapDH significantly enhanced carbon flux into pentose phosphate pathway and L-lysine biosynthetic pathway, thus increasing the levels of NADPH and precursors for L-lysine biosynthesis. This is the first report of a rational modification of diaminopimelate pathway that improves the efficiency of L-lysine production through overexpression of thermostable DapDH in E. coli.
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Project description:The Escherichia coli aroE gene encoding shikimate dehydrogenase was sequenced. The deduced amino acid sequence was confirmed by N-terminal amino acid sequencing and amino acid analysis of the overproduced protein. The complete polypeptide chain has 272 amino acid residues and has a calculated Mr of 29,380. E. coli shikimate dehydrogenase is homologous to the shikimate dehydrogenase domain of the fungal arom multifunctional enzymes and to the catabolic quinate dehydrogenase of Neurospora crassa.
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Project description:A simple generic method for optimizing membrane protein overexpression in Escherichia coli is still lacking. We have studied the physiological response of the widely used "Walker strains" C41(DE3) and C43(DE3), which are derived from BL21(DE3), to membrane protein overexpression. For unknown reasons, overexpression of many membrane proteins in these strains is hardly toxic, often resulting in high overexpression yields. By using a combination of physiological, proteomic, and genetic techniques we have shown that mutations in the lacUV5 promoter governing expression of T7 RNA polymerase are key to the improved membrane protein overexpression characteristics of the Walker strains. Based on this observation, we have engineered a derivative strain of E. coli BL21(DE3), termed Lemo21(DE3), in which the activity of the T7 RNA polymerase can be precisely controlled by its natural inhibitor T7 lysozyme (T7Lys). Lemo21(DE3) is tunable for membrane protein overexpression and conveniently allows optimizing overexpression of any given membrane protein by using only a single strain rather than a multitude of different strains. The generality and simplicity of our approach make it ideal for high-throughput applications.
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Project description:Escherichia coli (strain K-12, substrain MG1655) glycerol dehydrogenase (GldA) is required to catalyze the first step in fermentative glycerol metabolism. The protein was expressed and purified to homogeneity using a simple combination of heat-shock and chromatographic methods. The high yield of the protein (?250?mg per litre of culture) allows large-scale production for potential industrial applications. Purified GldA exhibited a homogeneous tetrameric state (?161?kDa) in solution and relatively high thermostability (Tm = 65.6°C). Sitting-drop sparse-matrix screens were used for protein crystallization. An optimized condition with ammonium sulfate (2?M) provided crystals suitable for diffraction, and a binary structure containing glycerol in the active site was solved at 2.8?Å resolution. Each GldA monomer consists of nine ?-strands, thirteen ?-helices, two 310-helices and several loops organized into two domains, the N- and C-terminal domains; the active site is located in a deep cleft between the two domains. The N-terminal domain contains a classic Rossmann fold for NAD+ binding. The O1 and O2 atoms of glycerol serve as ligands for the tetrahedrally coordinated Zn2+ ion. The orientation of the glycerol within the active site is mainly stabilized by van der Waals and electrostatic interactions with the benzyl ring of Phe245. Computer modeling suggests that the glycerol molecule is sandwiched by the Zn2+ and NAD+ ions. Based on this, the mechanism for the relaxed substrate specificity of this enzyme is also discussed.
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Project description:The Escherichia coli isocitrate dehydrogenase (ICDH) is one of the tricarboxylic acid cycle enzymes, playing key roles in energy production and carbon flux regulation. E. coli ICDH was the first bacterial enzyme shown to be regulated by reversible phosphorylation. However, the effect of lysine acetylation on E. coli ICDH, which has no sequence similarity with its counterparts in eukaryotes, is still unclear. Based on previous studies of E. coli acetylome and ICDH crystal structures, eight lysine residues were selected for mutational and kinetic analyses. They were replaced with acetyllysine by the genetic code expansion strategy or substituted with glutamine as a classic approach. Although acetylation decreased the overall ICDH activity, its effects were different site by site. Deacetylation tests demonstrated that the CobB deacetylase could deacetylate ICDH both in vivo and in vitro, but CobB was only specific for lysine residues at the protein surface. On the other hand, ICDH could be acetylated by acetyl-phosphate chemically in vitro. And in vivo acetylation tests indicated that the acetylation level of ICDH was correlated with the amounts of intracellular acetyl-phosphate. This study nicely complements previous proteomic studies to provide direct biochemical evidence for ICDH acetylation.
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