Structure-function analyses of the G729R 2-oxoadipate dehydrogenase genetic variant associated with a disorder of l-lysine metabolism.

2-Oxoadipate dehydrogenase (E1a, also known as DHTKD1, dehydrogenase E1, and transketolase domain-containing protein 1) is a thiamin diphosphate-dependent enzyme and part of the 2-oxoadipate dehydrogenase complex (OADHc) in l-lysine catabolism. Genetic findings have linked mutations in the DHTKD1 gene to several metabolic disorders. These include α-aminoadipic and α-ketoadipic aciduria (AMOXAD), a rare disorder of l-lysine, l-hydroxylysine, and l-tryptophan catabolism, associated with clinical presentations such as developmental delay, mild-to-severe intellectual disability, ataxia, epilepsy, and behavioral disorders that cannot currently be managed by available treatments. A heterozygous missense mutation, c.2185G→A (p.G729R), in DHTKD1 has been identified in most AMOXAD cases. Here, we report that the G729R E1a variant when assembled into OADHc in vitro displays a 50-fold decrease in catalytic efficiency for NADH production and a significantly reduced rate of glutaryl-CoA production by dihydrolipoamide succinyl-transferase (E2o). However, the G729R E1a substitution did not affect any of the three side-reactions associated solely with G729R E1a, prompting us to determine the structure-function effects of this mutation. A multipronged systematic analysis of the reaction rates in the OADHc pathway, supplemented with results from chemical cross-linking and hydrogen-deuterium exchange MS, revealed that the c.2185G→A DHTKD1 mutation affects E1a-E2o assembly, leading to impaired channeling of OADHc intermediates. Cross-linking between the C-terminal region of both E1a and G729R E1a with the E2o lipoyl and core domains suggested that correct positioning of the C-terminal E1a region is essential for the intermediate channeling. These findings may inform the development of interventions to counter the effects of pathogenic DHTKD1 mutations.

A multipronged approach unravels unprecedented protein-protein interactions in the human 2-oxoglutarate dehydrogenase multienzyme complex.

The human 2-oxoglutaric acid dehydrogenase complex (hOGDHc) plays a pivotal role in the tricarboxylic acid (TCA) cycle, and its diminished activity is associated with neurodegenerative diseases. The hOGDHc comprises three components, hE1o, hE2o, and hE3, and we recently reported functionally active E1o and E2o components, enabling studies on their assembly. No atomic-resolution structure for the hE2o component is currently available, so here we first studied the interactions in the binary subcomplexes (hE1o-hE2o, hE1o-hE3, and hE2o-hE3) to gain insight into the strength of their interactions and to identify the interaction loci in them. We carried out multiple physico-chemical studies, including fluorescence, hydrogen-deuterium exchange MS (HDX-MS), and chemical cross-linking MS (CL-MS). Our fluorescence studies suggested a strong interaction for the hE1o-hE2o subcomplex, but a much weaker interaction in the hE1o-hE3 subcomplex, and failed to identify any interaction in the hE2o-hE3 subcomplex. The HDX-MS studies gave evidence for interactions in the hE1o-hE2o and hE1o-hE3 subcomplexes comprising full-length components, identifying: (i) the N-terminal region of hE1o, in particular the two peptides 18YVEEM22 and 27ENPKSVHKSWDIF39 as constituting the binding region responsible for the assembly of the hE1o with both the hE2o and hE3 components into hOGDHc, an hE1 region absent in available X-ray structures; and (ii) a novel hE2o region comprising residues from both a linker region and from the catalytic domain as being a critical region interacting with hE1o. The CL-MS identified the loci in the hE1o and hE2o components interacting with each other.

Structure and function of the catalytic domain of the dihydrolipoyl acetyltransferase component in Escherichia coli pyruvate dehydrogenase complex.

The Escherichia coli pyruvate dehydrogenase complex (PDHc) catalyzing conversion of pyruvate to acetyl-CoA comprises three components: E1p, E2p, and E3. The E2p is the five-domain core component, consisting of three tandem lipoyl domains (LDs), a peripheral subunit binding domain (PSBD), and a catalytic domain (E2pCD). Herein are reported the following. 1) The x-ray structure of E2pCD revealed both intra- and intertrimer interactions, similar to those reported for other E2pCDs. 2) Reconstitution of recombinant LD and E2pCD with E1p and E3p into PDHc could maintain at least 6.4% activity (NADH production), confirming the functional competence of the E2pCD and active center coupling among E1p, LD, E2pCD, and E3 even in the absence of PSBD and of a covalent link between domains within E2p. 3) Direct acetyl transfer between LD and coenzyme A catalyzed by E2pCD was observed with a rate constant of 199 s(-1), comparable with the rate of NADH production in the PDHc reaction. Hence, neither reductive acetylation of E2p nor acetyl transfer within E2p is rate-limiting. 4) An unprecedented finding is that although no interaction could be detected between E1p and E2pCD by itself, a domain-induced interaction was identified on E1p active centers upon assembly with E2p and C-terminally truncated E2p proteins by hydrogen/deuterium exchange mass spectrometry. The inclusion of each additional domain of E2p strengthened the interaction with E1p, and the interaction was strongest with intact E2p. E2p domain-induced changes at the E1p active site were also manifested by the appearance of a circular dichroism band characteristic of the canonical 4'-aminopyrimidine tautomer of bound thiamin diphosphate (AP).

A Novel Small-Molecule Inhibitor of the Mycobacterium tuberculosis Demethylmenaquinone Methyltransferase MenG Is Bactericidal to Both Growing and Nutritionally Deprived Persister Cells.

Active tuberculosis (TB) and latent Mycobacterium tuberculosis infection both require lengthy treatments to achieve durable cures. This problem has partly been attributable to the existence of nonreplicating M. tuberculosis "persisters" that are difficult to kill using conventional anti-TB treatments. Compounds that target the respiratory pathway have the potential to kill both replicating and persistent M. tuberculosis and shorten TB treatment, as this pathway is essential in both metabolic states. We developed a novel respiratory pathway-specific whole-cell screen to identify new respiration inhibitors. This screen identified the biphenyl amide GSK1733953A (DG70) as a likely respiration inhibitor. DG70 inhibited both clinical drug-susceptible and drug-resistant M. tuberculosis strains. Whole-genome sequencing of DG70-resistant colonies identified mutations in menG (rv0558), which is responsible for the final step in menaquinone biosynthesis and required for respiration. Overexpression of menG from wild-type and DG70-resistant isolates increased the DG70 MIC by 4× and 8× to 30×, respectively. Radiolabeling and high-resolution mass spectrometry studies confirmed that DG70 inhibited the final step in menaquinone biosynthesis. DG70 also inhibited oxygen utilization and ATP biosynthesis, which was reversed by external menaquinone supplementation. DG70 was bactericidal in actively replicating cultures and in a nutritionally deprived persistence model. DG70 was synergistic with the first-line TB drugs isoniazid, rifampin, and the respiratory inhibitor bedaquiline. The combination of DG70 and isoniazid completely sterilized cultures in the persistence model by day 10. These results suggest that MenG is a good therapeutic target and that compounds targeting MenG along with standard TB therapy have the potential to shorten TB treatment duration.IMPORTANCE This study shows that MenG, which is responsible for the last enzymatic step in menaquinone biosynthesis, may be a good drug target for improving TB treatments. We describe the first small-molecule inhibitor (DG70) of Mycobacterium tuberculosis MenG and show that DG70 has characteristics that are highly desirable for a new antitubercular agent, including bactericidality against both actively growing and nonreplicating mycobacteria and synergy with several first-line drugs that are currently used to treat TB.

Microtrap modulated flame ionization detector for on-line monitoring of methane.

A microtrap for on-line analysis of trace methane was developed. Silicosteel tubing, 15 cm long, 1.02 mm i.d. and 1.59 mm o.d. was packed with Carbosphere 80/100 mesh and placed between the standard gas source and the flame ionization detector (FID) to be used as a preconcentrator and an injector. The sample stream was passed continuously through the microtrap that was heated at a fixed interval by an electric pulse. To achieve the highest adsorption and desorption efficiency, parameters affecting the response of the system were optimized, and adsorption temperature in the range of 25 to -50 degrees C was studied. The response from the microtrap as compared to that from direct-flame ionization detector was enhanced by as much as 260 times. The on-line microtrap system showed good linearity (r2 > 0.99), low detection limit (28.3 ppbv) and good long term stability (relative standard deviation, RSD of less than 5.0%). Therefore, this simple device is suitable for on-line analysis of trace methane and similar small molecules in the environment as in high purity gases.

Development of continuous on-line purge and trap analysis.

An on-line purge and trap system for continuous monitoring of Volatile Organic Compounds (VOC) is presented. The purge chamber was designed for continuous extraction of VOC from water with nitrogen. The analytes were preconcentrated on a microtrap prior to analysis by GC with flame ionization detection. The microtrap served as a fast injection device for carrying out analysis at high frequency. Continuous monitoring was accomplished by performing injections at fixed intervals. This system showed high sensitivity, high precision, detection limits at the ppb level, and stable response over long periods of continuous operation. Factors affecting system performance were studied. A predictive model based on gas-liquid partitioning is also presented.

Preconcentration of volatile organics on self-assembled, carbon nanotubes in a microtrap.

This paper reports the self-assembly of carbon nanotubes (CNTs) on the inside wall of a steel capillary to fabricate a microtrap for the adsorption/desorption of trace organics. The microtrap functioned as a nanoconcentrator and an injector for gas chromatography (GC). The CNTs were deposited as a thin film by catalytic chemical vapor deposition from either CO or C2H4 as the precursor. The sorbent film synthesized from C2H4-CVD (CVD = chemical vapor deposition) had higher CNT density and thus was a stronger sorbent. In general, the CNT microtraps showed high-capacity adsorption and fast quantitative desorption, and the process showed excellent precision. This study demonstrates that CNT films can be deposited quite easily in a steel capillary for use in different analytical applications, and CNT films can perform as efficiently as packed-bed carbon sorbents.