Vladimir O. Popov

Catalytic mechanism and application of formate dehydrogenase

NAD+-dependent formate dehydrogenase (FDH) is an abundant enzyme that plays an important role in energysupply of methylotrophic microorganisms and in response to stress in plants. FDH belongs to the superfamily of D-specific 2-hydroxy acid dehydrogenases. FDH is widely accepted as a model enzyme to study the mechanism of hydride ion transfer in the active center of dehydrogenases because the reaction catalyzed by the enzyme is devoid of proton transfer steps and implies a substrate with relatively simple structure. FDH is also widely used in enzymatic syntheses of optically active compounds as a versatile biocatalyst for NAD(P)H regeneration consumed in the main reaction. This review covers the late developments in cloning genes of FDH from various sources, studies of its catalytic mechanism and physiological role, and its application for new chiral syntheses.

Structural Basis for Phototoxicity of the Genetically Encoded Photosensitizer KillerRed

KillerRed is the only known fluorescent protein that demonstrates notable phototoxicity, exceeding that of the other green and red fluorescent proteins by at least 1,000-fold. KillerRed could serve as an instrument to inactivate target proteins or to kill cell populations in photodynamic therapy. However, the nature of KillerRed phototoxicity has remained unclear, impeding the development of more phototoxic variants. Here we present the results of a high resolution crystallographic study of KillerRed in the active fluorescent and in the photobleached non-fluorescent states. A unique and striking feature of the structure is a water-filled channel reaching the chromophore area from the end cap of the β-barrel that is probably one of the key structural features responsible for phototoxicity. A study of the structure-function relationship of KillerRed, supported by structure-based, site-directed mutagenesis, has also revealed the key residues most likely responsible for the phototoxic effect. In particular, Glu68 and Ser119, located adjacent to the chromophore, have been assigned as the primary trigger of the reaction chain.

High-resolution structural analysis of a novel octaheme cytochrome c nitrite reductase from the haloalkaliphilic bacterium Thioalkalivibrio nitratireducens

Bacterial pentaheme cytochrome c nitrite reductases (NrfAs) are key enzymes involved in the terminal step of dissimilatory nitrite reduction of the nitrogen cycle. Their structure and functions are well studied. Recently, a novel octaheme cytochrome c nitrite reductase (TvNiR) has been isolated from the haloalkaliphilic bacterium Thioalkalivibrio nitratireducens. Here we present high-resolution crystal structures of the apoenzyme and its complexes with the substrate (nitrite) and the inhibitor (azide). Both in the crystalline state and in solution, TvNiR exists as a stable hexamer containing 48 hemes-the largest number of hemes accommodated within one protein molecule known to date. The subunit of TvNiR consists of two domains. The N-terminal domain has a unique fold and contains three hemes. The catalytic C-terminal domain hosts the remaining five hemes, their arrangement, including the catalytic heme, being identical to that found in NrfAs. The complete set of eight hemes forms a spatial pattern characteristic of other multiheme proteins, including structurally characterized octaheme cytochromes. The catalytic machinery of TvNiR resembles that of NrfAs. It comprises the lysine residue at the proximal position of the catalytic heme, the catalytic triad of tyrosine, histidine, and arginine at the distal side, channels for the substrate and product transport with a characteristic gradient of electrostatic potential, and, finally, two conserved Ca(2+)-binding sites. However, TvNiR has a number of special structural features, including a covalent bond between the catalytic tyrosine and the adjacent cysteine and the unusual topography of the product channels that open into the void interior space of the protein hexamer. The role of these characteristic structural features in the catalysis by this enzyme is discussed.

Biofuel Cell Based on Microscale Nanostructured Electrodes with Inductive Coupling to Rat Brain Neurons

Miniature, self-contained biodevices powered by biofuel cells may enable a new generation of implantable, wireless, minimally invasive neural interfaces for neurophysiological in vivo studies and for clinical applications. Here we report on the fabrication of a direct electron transfer based glucose/oxygen enzymatic fuel cell (EFC) from genuinely three-dimensional (3D) nanostructured microscale gold electrodes, modified with suitable biocatalysts. We show that the process underlying the simple fabrication method of 3D nanostructured electrodes is based on an electrochemically driven transformation of physically deposited gold nanoparticles. We experimentally demonstrate that mediator-, cofactor-, and membrane-less EFCs do operate in cerebrospinal fluid and in the brain of a rat, producing amounts of electrical power sufficient to drive a self-contained biodevice, viz. 7 μW cm−2 in vitro and 2 μW cm−2 in vivo at an operating voltage of 0.4 V. Last but not least, we also demonstrate an inductive coupling between 3D nanobioelectrodes and living neurons.

SITE-DIRECTED MUTAGENESIS OF THE FORMATE DEHYDROGENASE ACTIVE CENTRE : ROLE OF THE HIS332-GLN313 PAIR IN ENZYME CATALYSIS

Gln313 and His332 residues in the active centre of NAD+‐dependent formate dehydrogenase (EC 1.2.1.2, FDH) from the bacterium Pseudomonas sp. 101 are conserved in all FDHs and are equivalent to the glutamate‐histidine pair in active sites of d‐specific 2‐hydroxyacid dehydrogenases. Two mutants of formate dehydrogenase from Pseudomonas sp. 101, Gln313Glu and His332Phe, have been obtained and characterised. The Gln313Glu mutation shifts the pK of the group controlling formate binding from less than 5.5 in wild‐type enzyme to 7.6 thus indicating that Gln313 is essential for the broad pH affinity profile towards substrate. His332Phe mutation leads to a complete loss of enzyme activity. The His332Phe mutant is still able to bind coenzyme but not substrate or analogues. The role of histidine in the active centre of FDH is discussed. The protonation state of His332 is not critical for catalysis but vital for substrate binding. A partial positive charge on the histidine imidazole, required for substrate binding, is provided via tight H‐bond to the Gln313 carboxamide.

Conserved supersecondary structural motif in NAD-dependent dehydrogenases

l‐ and d‐specific nicotinamide adenine dinucleotide (NAD)‐dependent dehydrogenases map to the same structural protein superfamily as defined by the Structural Classification of Proteins (SCOP) and are based on the Rossmann fold type domains. A detailed classification of these domains is proposed using a novel diagnostic parameter of the rms per aligned pair. The catalytic domain in d‐specific dehydrogenases shows a strong structural homology to the coenzyme binding domain. A topologically conserved part within the dehydrogenase superfamily reveals a supersecondary structural motif comprising the 5‐stranded left‐handedly twisted parallel β‐sheet with one complete and one partial Rossmann fold units and two α‐helices, the long helix, adjacent to and running roughly parallel with the β‐sheet plane and the helix connecting two Rossmann folds.

Organization of metabolic pathways and molecular-genetic mechanisms of xenobiotic degradation in microorganisms: A review

Contemporary data on the mechanism of biodegradation of aromatic hydrocarbons and biodegradation genes (genomic organization and pathways of evolution) in diverse groups of microorganisms have been reviewed. Studies of this problem are topical, in view of the need in identification and construction of new strains degrading xenobiotics, particularly those halogenated. For this reason, emphasis is placed on specific features of explored metabolic pathways that can be used for constructing new enzymatic systems not present in nature. Sections on the mechanisms of genomic rearrangements involving biodegradation determinants are presented from the same standpoint. Part of the review is devoted to analyzing methods used for studying the population dynamics of bacterial communities involved in xenobiotic degradation in natural biotopes or industrial waste disposal plants. Particular attention is given to methods of gene systematics.

Hydrogenase from the hydrogen-oxidizing bacterium Alcaligenes eutrophus Z I: Catalytic activity-quaternary structure relationship

Abstract The relationship between the quaternary structure and catalytic activity of the soluble hydrogenase (hydrogen:NAD + oxidoreductase, EC 1.12.1.2) from the hydrogen-oxidizing bacterium. Alcaligenes eutrophus strain Z I, has been investigated. The enzyme specific activity depends on both the protein concentration and preincubation conditions. The hydrogenase activity with NAD or methyl viologen as substrates is more sensitive to dilution than the NADH-dehydrogenase activity of the enzyme. The molecular weight of hydrogenase determined by gel-filtration decreases from 160000 to 90000, while the enzyme concentration falls from 4.5 to 0.044 mg/ml. Coenzymes change both the apparent molecular weight and concentration dependence of the hydrogenase specific activity. Gel-filtration under dissociation conditions reveals an enzyme fragment with a molecular weight of 60000 which is active in NADH-dehydrogenase reaction, but does not catalyse the hydrogenase activity. Gradient polyacrylamide gel electrophoresis experiments reveal only one component with a molecular weight of 180 000-150 000 which stains for both hydrogenase and NADH-dehydrogenase activity. Under dissociation conditions the activity band splits into several bands corresponding to molecular weights from 6000 to 200000 only in the case of NADH-dehydrogenase. It is proposed that the native enzyme can undergo reversible dissociation. It is hypothesized that hydrogenase contains a special subunit with a molecular weight of about 60000, which may determine the NADH-dehydrogenase activity of the enzyme.

Characterization of a thermostable short-chain alcohol dehydrogenase from the hyperthermophilic archaeon Thermococcus sibiricus.

ABSTRACT Short-chain alcohol dehydrogenase, encoded by the gene Tsib_0319 from the hyperthermophilic archaeon Thermococcus sibiricus, was expressed in Escherichia coli, purified and characterized as an NADPH-dependent enantioselective oxidoreductase with broad substrate specificity. The enzyme exhibits extremely high thermophilicity, thermostability, and tolerance to organic solvents and salts.

Direct electron transfer between Alcaligenes eutrophus Z-1 hydrogenase and glassy carbon electrodes

Abstract In this work we report on the ability of the Alcaligenes eutrophus Z-1 hydrogenase immobilized on glassy carbon electrodes to support the electro-enzymatic reduction of NAD+ at −700 mV vs. standard calomel electrode without external promoters or electron mediators. The electron-transfer process seems to be coupled to a relaxation of the quaternary structure of the enzyme molecules in contact with the electrode surface.