Structures of multidrug and toxic compound extrusion transporters and their mechanistic implications

Multidrug resistance poses grand challenges to the effective treatment of infectious diseases and cancers. Integral membrane proteins from the multidrug and toxic compound extrusion (MATE) family contribute to multidrug resistance by exporting a wide variety of therapeutic drugs across cell membranes. MATE proteins are conserved from bacteria to humans and can be categorized into the NorM, DinF and eukaryotic subfamilies. MATE transporters hold great appeal as potential therapeutic targets for curbing multidrug resistance, yet their transport mechanism remains elusive. During the past 5 years, X-ray structures of 4 NorM and DinF transporters have been reported and guided biochemical studies to reveal how MATE transporters extrude different drugs. Such advances, although substantial, have yet to be discussed collectively. Herein I review these structures and the unprecedented mechanistic insights that have been garnered from those structure-inspired studies, as well as lay out the outstanding questions that present exciting opportunities for future work.     

Kinetic and mechanistic studies of methylated liver alcohol dehydrogenase.

Reductive methylation of lysine residues activates liver alcohol dehydrogenase in the oxidation of primary alcohols, but decreases the activity of the enzyme towards secondary alcohols. The modification also desensitizes the dehydrogenase to substrate inhibition at high alcohol concentrations. Steady-state kinetic studies of methylated liver alcohol dehydrogenase over a wide range of alcohol concentrations suggest that alcohol oxidation proceeds via a random addition of coenzyme and substrate with a pathway for the formation of the productive enzyme-NADH-alcohol complex. To facilitate the analyses of the effects of methylation on liver alcohol dehydrogenase and factors affecting them, new operational kinetic parameters to describe the results at high substrate concentration were introduced. The changes in the dehydrogenase activity on alkylation were found to be associated with changes in the maximum velocities that are affected by the hydrophobicity of alkyl groups introduced at lysine residues. The desensitization of alkylated liver alcohol dehydrogenase to substrate inhibition is identified with a decrease in inhibitory Michaelis constants for alcohols and this is favoured by the steric effects of substituents at the lysine residues.     

Protocatechuate 3,4-dioxygenase. Inhibitor studies and mechanistic implications.

Protocatechuate 3,4-dioxygenase (EC 1.13.11.3) from Pseudomonas aeruginosa catalyzes the cleavage of 3,4-dihydroxybenzoate (protocatechuate) into beta-carboxy-cis,cis-muconate. The inhibition constants, Ki, of a series of substrate analogues were measured in order to assess the relative importance of the various functional groups on the substrate. Though important for binding, the carboxylate group is not essential for activity. Compounds with para hydroxy groups are better inhibitors than their meta isomers. Our studies of the enzyme-inhibitor complexes indicate that the 4-OH group of the substrate binds to the active-site iron. Taken together, M?ssbauer, EPR, and kinetic data suggest a mechanism where substrate reaction with oxygen is preceded by metal activation of substrate.     

Kinetics and interactions of molybdenum and iron-sulfur centers in bacterial enzymes of the xanthine oxidase family: mechanistic implications.

For isoquinoline 1-oxidoreductase (IsoOr), the reaction mechanism under turnover conditions was studied by EPR spectroscopy using rapid-freeze methods. IsoOr displays several EPR-active Mo(V) species including the "very rapid" component found also in xanthine oxidase (XanOx). For IsoOr, unlike XanOx or quinoline 2-oxidoreductase (QuinOr), this species is stable for about 1 h in the absence of an oxidizing substrate [Canne, C., Stephan, I., Finsterbusch, J., Lingens, F., Kappl, R., Fetzner, S., and Hüttermann, J. (1997) Biochemistry 36, 9780-9790]. Under rapid-freeze conditions in the presence of ferricyanide the very rapid species behaves as a kinetically competent intermediate present only during steady-state turnover. To explain the persistence of the very rapid species in IsoOr in the absence of an added oxidant, extremely slow product dissociation is required. This new finding that oxidative conditions facilitate decay of the very rapid signal for IsoOr supports the mechanism of substrate turnover proposed by Lowe, Richards, and Bray [Lowe, D. J., Richards, R. L., and Bray, R. C. (1997) Biochem. Soc. Trans. 25, 774-778]. Additional stopped-flow data reveal that alternative catalytic cycles occur in IsoOr and show that the product dissociates after transfer of a single oxidizing equivalent from ferricyanide. In rapid-freeze measurements magnetic interactions of the very rapid Mo(V) species and the iron-sulfur center FeSI of IsoOr and QuinOr were observed, proving that FeSI is located close to the molybdopterin cofactor in the two proteins. This finding is used to relate the two different iron-sulfur centers of the aldehyde oxidoreductase structure with the EPR-detectable FeS species of the enzymes.     

Bovine lens aldehyde dehydrogenase. Kinetics and mechanism.

Bovine lens cytoplasmic aldehyde dehydrogenase exhibits Michaelis-Menten kinetics with acetaldehyde, glyceraldehyde 3-phosphate, p-nitrobenzaldehyde, propionaldehyde, glycolaldehyde, glyceraldehyde, phenylacetylaldehyde and succinic semialdehyde as substrates. The enzyme was also active with malondialdehyde, and exhibited an esterase activity. Steady-state kinetic analyses show that the enzyme exhibits a compulsory-ordered ternary-complex mechanism with NAD+ binding before acetaldehyde. The enzyme was inhibited by disulfiram and by p-chloromercuribenzoate, and studies with with mercaptans indicated the involvement of thiol groups in catalysis.     

Behavioral implications of mechanistic ecology

Summary Mechanistic principles from engineering, meteorology, and soil physics are integrated with ecology and physiology to develop models for prediction of animal behavior. The Mojave Desert biome and the desert iguana are used to illustrate these principles.A transient energy balance model for animals in an outdoor environment is presented. The concepts and relationships have been tested in a wind tunnel, in a simulated desert, and in the field. The animal model requires anatomical information and knowledge of the thermoregulatory responses of the animal. The micrometeorological model requires only basic meteorological parameters and two soil physical properties as inputs. Tests of the model in the field show agreement between predicted and measured temperatures above and below the surface of about 2 to 3°C.The animal and micrometeorological models are combined to predict daily and seasonal activity patterns, available times for predator-prey interaction, and daily, seasonal and annual requirements for food and water. It is shown that food, water and the thermal environment can limit animal activity, and furthermore, the controlling limit changes with season. Actual observations of activity patterns and our predictions show close agreement, in many cases, and pose intriguing questions in those situations where agreement does not exist. This type of modeling can be used to further study predator-prey interactions, to study how changes in the environment might affect animal behavior, and to answer other important ecological and physiological questions.     

Kinetics of the reoxidation of succinate dehydrogenase.

The reoxidation phase of the catalytic cycle of succinate dehydrogenase was studied in Complex II preparations' by the rapid freeze-electron paramagnetic resonance (epr) technique. With the synthetic water-soluble Q₁ analog, 2,3-dimethoxy-5-methyl-6-pentyl-1, 4-benzoquinone (DPB), as the oxidant, the observed reoxidation of the epr-detectable components, previously reduced with dithionite or succinate, came to completion within a few milliseconds, well within the turnover time of the enzyme. Only ~80% of Fe-S center 1 and the HiPIP (the high-potential cluster) Fe-S center reacted rapidly with DPB, however; similarly incomplete reactions were observed previously in our studies of the reduction of the enzyme by succinate. The subsequent addition of ferricyanide, which appears to act as a chemical oxidant in these experiments, caused immediate reoxidation of the Fe-S centers and of the free radical. Ferricyanide and phenazine methosulfate (PMS) reoxidized all epr-detectable components in Complex II as well as in reconstitutively active, soluble preparations in' <6 ms, even at 0°C. Thus, reoxidation of the purified enzyme by PMS cannot be rate-limiting. Carboxamides and thenoyltrifluoroacetone inhibit strongly the reoxidation of the Fe-S center 1 and the HiPIP center by DPB, but not their reduction by succinate. These and other data suggest that these inhibitors block electron transport from the dehydrogenase to the Q pool on the O₂-side of the HiPIP center, but there is no evidence that they combine directly with the iron. A recent report that Wurster's blue reacts with soluble succinate dehydrogenase much more rapidly than does PMS could not be confirmed. The two oxidants react at equal rates with the purified soluble enzyme before and after it has been reincorporated into membranes.     

Kinetics and stability of immobilized chicken liver xanthine dehydrogenase.

Xanthine dehydrogenase (EC 1.2.1.37) was isolated from chicken livers and immobilized by adsorption to a Sepharose derivative, prepared by reaction of n-octylamine with CNBr-activated Sepharose 4B. Using a crude preparation of enzyme for immobilization it was observed that relatively more activity was adsorbed than protein, but the yield of immobilized activity increased as a purer enzyme preparation was used. As more activity and protein were bound, relatively less immobilized activity was recovered. This effect was probably due to blocking of active xanthine dehydrogenase by protein impurities. The kinetics of free and immobilized xanthine dehydrogenase were studied in the pH range 7.5-9.1. The Km and V values estimated for free xanthine dehydrogenase increase as the pH increase; the K'm and V values for the immobilized enzyme go through a minimum at pH 8.1. By varying the amount of enzyme activity bound per unit volume of gel, it was shown that K'm is larger than Km are result of substrate diffusion limitation in the pores of the support material. Both free and immobilized xanthine dehydrogenase showed substrate activation at low concentrations (up to 2 microM xanthine). Immobilized xanthine dehydrogenase was more stable than the free enzyme during storage in the temperature range of 4-50 degrees C. The operational stability of immobilized xanthine dehydrogenase at 30 degrees C was two orders of magnitude smaller than the storage stability, t 1/2 was 9 and 800 hr, respectively. The operational stability was, however, better than than of immobilized milk xanthine oxidase (t 1/2 = 1 hr). In addition, the amount of product formed per unit initial activity in one half-life, was higher for immobilized xanthine dehydrogenase than for immobilized xanthine oxidase. Unless immobilized milk xanthine oxidase can be considerable stabilized, immobilized chicken liver xanthine dehydrogenase is more promising for application in organic synthesis.     

Human liver akdehyde dehydrogenase. Kinetics of aldehyde oxidation.

Steady state initial velocity studies were carried out to determine the kinetic mechanism of human liver aldehyde dehydrogenase. Intersecting double reciprocal plots obtained in the absence of inhibitors demonstrated that the dehydrogenase reaction proceeded by sequential addition of both substrates prior to release of products. Dead end inhibition patterns obtained with coenzyme and substrate analogues (e.g. thionicotinamide-AD+ and chloral hydrate) indicated that NAD+ and aldehyde can bind in random fashion. The patterns of inhibition by the product NADH and of substrate inhibition by glyceraldehyde were also consistent with this mechanism. However, comparisons between kinetic constants associated with the dehydrogenase and esterase activities of this enzyme suggested that most of the dehydrogenase reaction flux proceeds via formation of an initial binary NAD+-enzyme complex over a wide range of substrate and coenzyme concentrations.     

Molecular structures of human argininosuccinate synthetase pseudogenes. Evolutionary and mechanistic implications

In the human genome there is one expressed gene for argininosuccinate synthetase and 14 pseudogenes. A cDNA coding for human argininosuccinate synthetase was used to screen a human genomic library. Twenty-five unique genomic clones were isolated and extensively characterized. At least seven clones represented processed argininosuccinate synthetase pseudogenes that lost the introns in the expressed gene. Restriction mapping demonstrated that these processed pseudogenes were located in distinct regions of the human genome. Complete nucleotide sequences of two processed pseudogenes, psi AS-1 and psi AS-3, and a partial sequence of psi AS-7 were determined. Both psi AS-1 and psi AS-3 had an adenine-rich region at their 3' end and were flanked by distinct imperfect direct repeats. A comparison of these pseudogene sequences to that of the cDNA demonstrated that psi AS-1 and psi AS-3 were 93% homologous to the cDNA, whereas psi AS-7 was 89% homologous to the cDNA. Therefore, it is estimated that psi AS-1 and psi AS-3 were created 10-11 million years ago, whereas psi AS-7 arose approximately 21 million years ago. We have estimated the evolutionary rate for the expressed argininosuccinate synthetase gene based on the sequences of psi AS-1 and psi AS-3. These data indicate that the expressed argininosuccinate synthetase gene is evolving at a rate similar to that of the beta-globin gene and much faster than the alpha-tubulin gene. Furthermore, a comparison of the sequences of psi AS-1 and psi AS-3 suggests the possibility that these pseudogenes arose from a common intermediate.     

pKa calculations for class A beta-lactamases: methodological and mechanistic implications.

Beta-lactamases are responsible for resistance to penicillins and related beta-lactam compounds. Despite numerous studies, the identity of the general base involved in the acylation step is still unclear. It has been proposed, on the basis of a previous pKa calculation and analysis of structural data, that the unprotonated Lys73 in the active site could act as the general base. Using a continuum electrostatic model with an improved treatment of the multiple titration site problem, we calculated the pKa values of all titratable residues in the substrate-free TEM-1 and Bacillus licheniformis class A beta-lactamases. The pKa of Lys73 in both enzymes was computed to be above 10, in good agreement with recent experimental data on the TEM-1 beta-lactamase, but inconsistent with the proposal that Lys73 acts as the general base. Even when the closest titratable residue, Glu166, is mutated to a neutral residue, the predicted downward shift of the pKa of Lys73 shows that it is unlikely to act as a proton abstractor in either enzyme. These results support a mechanism in which the proton of the active Ser70 is transferred to the carboxylate group of Glu166.     

Proline biosynthesis in Escherichia coli. Kinetic and mechanistic properties of glutamate semialdehyde dehydrogenase

The kinetics of the NADP+- and phosphate-dependent oxidation of glutamic acid 5-semialdehyde are consistent with a rapid-equilibrium random order mechanism. The Km for DL-pyrroline-5-carboxylic acid is 2.5 mM, for NADP+ is 0.05 mM and for phosphate is 0.35 mM. The Vmax is approx. 8.0 units per mg protein. The reaction is highly specific for the DL-pyrroline-5-carboxylic acid and NADP+, but a number of divalent anions can substitute for phosphate. NADPH is competitive with respect to all three substrates and an analog of gamma-glutamyl phosphate, 3-(phosphonoacetylamido)-L-alanine, is competitive with respect to DL-pyrroline-5-carboxylic acid and non-competitive with respect to NADP+ and phosphate, suggesting dead-end complex formation.     

Kinetics aspects of Gamma-hydroxybutyrate dehydrogenase

Two groups of metabolically related enzymes, the Group III family of Fe²⁺-dependent alcohol dehydrogenases (ADHs) and the separate subfamily of nucleoside diphosphates linked to x (nudix) hydrolases that activate Group III ADHs are under-characterized. Here we report the steady-state initial-velocity forward direction (alcohol → aldehyde) reaction of a Group III ADH, namely gamma-hydroxybutyrate dehydrogenase (GHBDH, UniProt: Q59104), cloned from Cupriavidus necator as a fusion protein. We also report the effects of nudix hydrolases on the GHBDH reaction. At optimal pH 9.0, the GHBDH reaction is activated ~2-fold by two different saturating purified nudix hydrolases, namely Bacillus methanolicus activator (ACT, UniProt: I3EA59) and Escherichia coli NudF (UniProt Q93K97) proteins. At physiological pH values of ~7.0, ACT activates by >3.5-fold. Initial-rate characterization at pH 9.0 of the forward direction un-activated and ACT-activated reactions show for both cases competitive inhibition by the product succinic semialdehyde versus GHB, and noncompetitive inhibitions by the three other substrate-product combinations. This pattern is consistent with NAD⁺ binding first in Mono-Iso Theorell-Chance kinetics. Mutants of some possibly important residues in GHBDH also were characterized. H265, conserved among all Group III ADHs and previously proposed to be a critical general base, is only ~4-fold helpful for GHBDH activity relevant to H265A. The four previously proposed conserved Fe²⁺ chelators (D193, H197, H261 and H280) each are essential for GHBDH activity. A 2-step explanation for cross-species stimulation by sub-stoichiometric ACT in the forward direction and confirmed lack of ACT stimulation in the reverse direction reaction is proposed.     

Kinetics and control of bovine adrenal glucose-6-phosphate dehydrogenase.

Michaelis-Menten kinetics are observed in studies of highly purified bovine adrenal glucose-6-phosphate dehydrogenase at pH8.0 in 0.1 M bicine. The Km for NADP+ is 3.8 muM and for glucose-6-phosphate, 61 muM. At pH 6.9 Km for NADP+ increases to 6.5 muM. The enzyme is inhibited by NADPH both at pH 6.8 and at 8.0 with a Kip of 2.36 muM at pH 8.0. Inhibition is competitive with respect to both substrates implying that addition of substrates is random ordered. The data are also interpreted in terms of "reducing charge", the mole fraction of coenzyme in the reduced form. This appears to be the major mechanism for regulation of the pentose shunt. D-glucose, oxidized by the enzyme at a very slow rate, is also a competitive inhibitor for the natural substrate with a Ki of 0.29 M. Phosphate is a competitive inhibitor for glucose-6-phosphate oxidation but both phosphate and sulfate accelerate glucose oxidation suggesting a common binding site for the two anions and the phosphate of the natural substrate. While binding of ACTH to our enzyme preparations has been observed, we have not been able, in spite of repeated attempts, to demonstrate augmentation of the activity of the enzyme by the addition of ACTH.     

Equilibrium dialysis study and mechanistic implications of coenzyme A binding to acetyl-CoA synthase/carbon monoxide dehydrogenase from Clostridium thermoaceticum

Clostridium thermoaceticum were determined by equilibrium dialysis. CoA bound to as-isolated native α ₂ β ₂ enzyme with K _D = 10 ± 8 μM and n = 0.2 ± 0.1 moles per αβ dimer, where K _D is the thermodynamic dissociation constant and n is the number of CoAs bound per αβ dimer of the enzyme. The enzyme is heterogeneous; for example, only  ∼ 30% of α subunits contain A-clusters with labile Ni ions (the remainder have nonlabile Ni ions and are nonfunctional). The observed n value suggests that CoA binds only to αβ units with Ni-labile A-clusters. The CoA binding properties of enzyme lacking labile Ni was essentially the same, indicating that CoA does not bind directly to the Ni of the A-cluster. This was further evidenced by the observation that bound CoA did not inhibit removal of the labile Ni by 1,10-phenanthroline. CoA did not bind CO-reduced enzyme, and the EPR signal exhibited by the one-electron reduced and CO-bound form of the A-cluster was unaffected by the presence of up to 200 μM CoA. In contrast, CoA did bind Ti(III)-citrate-reduced enzyme (K _D = 36 ± 16 μM, n = 0.16 ± 0.08). Implications of these results for the mechanism of catalysis are discussed. Received: 29 March 1999 / Accepted: 8 September 1999