Altered Escherichia coli membrane protein assembly machinery allows proper membrane assembly of eukaryotic protein vitamin K epoxide reductase.

Functional overexpression of polytopic membrane proteins, particularly when in a foreign host, is often a challenging task. Factors that negatively affect such processes are poorly understood. Using the mammalian membrane protein vitamin K epoxide reductase (VKORc1) as a reporter, we describe a genetic selection approach allowing the isolation of Escherichia coli mutants capable of functionally expressing this blood-coagulation enzyme. The isolated mutants map to components of membrane protein assembly and quality control proteins YidC and HslV. We show that changes in the VKORc1 sequence and in the YidC hydrophilic groove along with the inactivation of HslV promote VKORc1 activity and dramatically increase its expression level. We hypothesize that such changes correct for mismatches in the membrane topogenic signals between E. coli and eukaryotic cells guiding proper membrane integration. Furthermore, the obtained mutants allow the study of VKORc1 reaction mechanisms, inhibition by warfarin, and the high-throughput screening for potential anticoagulants.

Compounds targeting disulfide bond forming enzyme DsbB of Gram-negative bacteria.

In bacteria, disulfide bonds confer stability on many proteins exported to the cell envelope or beyond. These proteins include numerous bacterial virulence factors, and thus bacterial enzymes that promote disulfide bond formation represent targets for compounds inhibiting bacterial virulence. Here, we describe a new target- and cell-based screening methodology for identifying compounds that inhibit the disulfide bond-forming enzymes Escherichia coli DsbB (EcDsbB) or Mycobacterium tuberculosis VKOR (MtbVKOR), which can replace EcDsbB, although the two are not homologs. Initial screening of 51,487 compounds yielded six specifically inhibiting EcDsbB. These compounds share a structural motif and do not inhibit MtbVKOR. A medicinal chemistry approach led us to select related compounds, some of which are much more effective DsbB inhibitors than those found in the screen. These compounds inhibit purified DsbB and prevent anaerobic growth of E. coli. Furthermore, these compounds inhibit all but one of the DsbBs of nine other Gram-negative pathogenic bacteria tested.

Dioxygen Activation and Methane Hydroxylation by Soluble Methane Monooxygenase: A Tale of Two Irons and Three Proteins A list of abbreviations can be found in Section 7.

Methanotrophic bacteria are capable of using methane as their sole source of carbon and energy. The first step in methane metabolism, the oxidation of methane to methanol, is catalyzed by a fascinating enzyme system called methane monooxygenase (MMO). The selective oxidation of the very stable C-H bond in methane under ambient conditions is a remarkable feat that has not yet been repeated by synthetic catalysts and has attracted considerable scientific and commercial interest. The best studied MMO is a complex enzyme system that consists of three soluble protein components, all of which are required for efficient catalysis. Dioxygen activation and subsequent methane hydroxylation are catalyzed by a hydroxylase enzyme that contains a non-heme diiron site. A reductase protein accepts electrons from NADH and transfers them to the hydroxylase where they are used for the reductive activation of O(2). The third protein component couples electron and dioxygen consumption with methane oxidation. In this review we examine different aspects of catalysis by the MMO proteins, including the mechanisms of dioxygen activation at the diiron site and substrate hydroxylation by the activated oxygen species. We also discuss the role of complex formation between the different protein components in regulating various aspects of catalysis.

Dioxygen Activation and Methane Hydroxylation by Soluble Methane Monooxygenase: A Tale of Two Irons and Three Proteins.

Methanotrophic bacteria are capable of using methane as their sole source of carbon and energy. The first step in methane metabolism, the oxidation of methane to methanol, is catalyzed by a fascinating enzyme system called methane monooxygenase (MMO). The selective oxidation of the very stable C-H bond in methane under ambient conditions is a remarkable feat that has not yet been repeated by synthetic catalysts and has attracted considerable scientific and commercial interest. The best studied MMO is a complex enzyme system that consists of three soluble protein components, all of which are required for efficient catalysis. Dioxygen activation and subsequent methane hydroxylation are catalyzed by a hydroxylase enzyme that contains a non-heme diiron site. A reductase protein accepts electrons from NADH and transfers them to the hydroxylase where they are used for the reductive activation of O2 . The third protein component couples electron and dioxygen consumption with methane oxidation. In this review we examine different aspects of catalysis by the MMO proteins, including the mechanisms of dioxygen activation at the diiron site and substrate hydroxylation by the activated oxygen species. We also discuss the role of complex formation between the different protein components in regulating various aspects of catalysis.

Intermolecular electron-transfer reactions in soluble methane monooxygenase: a role for hysteresis in protein function.

Electron transfer from reduced nicotinamide adenine dinucleotide (NADH) to the hydroxylase component (MMOH) of soluble methane monooxygenase (sMMO) primes its non-heme diiron centers for reaction with dioxygen to generate high-valent iron intermediates that convert methane to methanol. This intermolecular electron-transfer step is facilitated by a reductase (MMOR), which contains [2Fe-2S] and flavin adenine dinucleotide (FAD) prosthetic groups. To investigate interprotein electron transfer, chemically reduced MMOR was mixed rapidly with oxidized MMOH in a stopped-flow apparatus, and optical changes associated with reductase oxidation were recorded. The reaction proceeds via four discrete kinetic phases corresponding to the transfer of four electrons into the two dinuclear iron sites of MMOH. Pre-equilibrating the hydroxylase with sMMO auxiliary proteins MMOB or MMOD severely diminishes electron-transfer throughput from MMOR, primarily by shifting the bulk of electron transfer to the slowest pathway. The biphasic reactions for electron transfer to MMOH from several MMOR ferredoxin analogues are also inhibited by MMOB and MMOD. These results, in conjunction with the previous finding that MMOB enhances electron-transfer rates from MMOR to MMOH when preformed MMOR-MMOH-MMOB complexes are allowed to react with NADH [Gassner, G. T.; Lippard, S. J. Biochemistry 1999, 38, 12768-12785], suggest that isomerization of the initial ternary complex is required for maximal electron-transfer rates. To account for the slow electron transfer observed for the ternary precomplex in this work, a model is proposed in which conformational changes imparted to the hydroxylase by MMOR are retained throughout the catalytic cycle. Several electron-transfer schemes are discussed with emphasis on those that invoke multiple interconverting MMOH populations.

Expression and characterization of ferredoxin and flavin adenine dinucleotide binding domains of the reductase component of soluble methane monooxygenase from Methylococcus capsulatus (Bath).

Soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath) catalyzes the selective oxidation of methane to methanol, the first step in the primary catabolic pathway of methanotrophic bacteria. A reductase (MMOR) mediates electron transfer from NADH through its FAD and [2Fe-2S] cofactors to the dinuclear non-heme iron sites housed in a hydroxylase (MMOH). The structurally distinct [2Fe-2S], FAD, and NADH binding domains of MMOR facilitated division of the protein into its functional ferredoxin (MMOR-Fd) and FAD/NADH (MMOR-FAD) component domains. The 10.9 kDa MMOR-Fd (MMOR residues 1-98) and 27.6 kDa MMOR-FAD (MMOR residues 99-348) were expressed and purified from recombinant Escherichia coli systems. The Fd and FAD domains have absorbance spectral features identical to those of the [2Fe-2S] and flavin components, respectively, of MMOR. Redox potentials, determined by reductive titrations that included indicator dyes, for the [2Fe-2S] and FAD cofactors in the domains are as follows: -205.2 +/- 1.3 mV for [2Fe-2S](ox/red), -172.4 +/- 2.0 mV for FAD(ox/sq), and -266.4 +/- 3.5 mV for FAD(sq/hq). Kinetic and spectral properties of intermediates observed in the reaction of oxidized MMOR-FAD (FAD(ox)) with NADH at 4 degrees C were established with stopped-flow UV-visible spectroscopy. Analysis of the influence of pH on MMOR-FAD optical spectra, redox potentials, and NADH reaction kinetics afforded pK(a) values for the semiquinone (FAD(sq)) and hydroquinone (FAD(hq)) MMOR-FAD species and two protonatable groups near the flavin cofactor. Electron transfer from MMOR-FAD(hq) to oxidized MMOR-Fd is extremely slow (k = 1500 M(-1) s(-1) at 25 degrees C, compared to 90 s(-1) at 4 degrees C for internal electron transfer between cofactors in MMOR), indicating that cofactor proximity is essential for efficient interdomain electron transfer.

Domain engineering of the reductase component of soluble methane monooxygenase from Methylococcus capsulatus (Bath).

Soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath) is a three-component enzyme system that catalyzes the conversion of methane to methanol. A reductase (MMOR), which contains [2Fe-2S] and FAD cofactors, facilitates electron transfer from NADH to the hydroxylase diiron active sites where dioxygen activation and substrate hydroxylation take place. By separately expressing the ferredoxin (MMORFd, MMOR residues 1-98) and FAD/NADH (MMOR-FAD, MMOR residues 99-348) domains of the reductase, nearly all biochemical properties of full-length MMOR are retained, except for interdomain electron transfer rates. To investigate the extent to which rapid electron transfer between domains might be restored and further to explore the modularity of MMOR, MMOR-Fd and MMOR-FAD were connected in a non-native fashion. Four different linker sequences were employed to create MMOR reversed-domain (MMOR-RD) constructs, MMOR(99-342)-linker-MMOR(2-98), with a domain connectivity observed in other homologous oxidoreductases. The optical, redox, and electron transfer properties of the four MMOR-RD proteins were characterized and compared with those of wild-type MMOR. The linker sequence plays a key role in controlling solvent accessibility to the FAD cofactor, as evidenced by perturbed flavin optical spectra, decreased FADox/FADsq redox potentials, and increased steady-state oxidase activities in three of the constructs. Stopped-flow optical spectroscopy revealed slow interdomain electron transfer (k < 0.04 s(-1) at 4 degrees C, compared with 90 s(-1) for wild-type MMOR) for all three MMOR-RD proteins with 7-residue linkers. A long (14-residue), flexible linker afforded much faster electron transfer between the FAD and [2Fe-2S] cofactors (k = 0.9 s(-1) at 4 degrees C).