Enzymology
Identification and characterization of a noncanonical menaquinone-linked formate dehydrogenase
The molybdenum/tungsten—bis-pyranopterin guanine dinucleotide family of formate dehydrogenases (FDHs) plays roles in several metabolic pathways ranging from carbon fixation to energy harvesting because of their reaction with a wide variety of redox partners. Indeed, this metabolic plasticity results from the diverse structures, cofactor content, and substrates used by partner subunits interacting with the catalytic hub. Here, we unveiled two noncanonical FDHs in Bacillus subtilis, which are organized into two-subunit complexes with unique features, ForCE1 and ForCE2.Active site architecture reveals coordination sphere flexibility and specificity determinants in a group of closely related molybdoenzymes
MtsZ is a molybdenum-containing methionine sulfoxide reductase that supports virulence in the human respiratory pathogen Haemophilus influenzae (Hi). HiMtsZ belongs to a group of structurally and spectroscopically uncharacterized S-/N-oxide reductases, all of which are found in bacterial pathogens. Here, we have solved the crystal structure of HiMtsZ, which reveals that the HiMtsZ substrate-binding site encompasses a previously unrecognized part that accommodates the methionine sulfoxide side chain via interaction with His182 and Arg166.Carnitine metabolism in the human gut: characterization of the two-component carnitine monooxygenase CntAB from Acinetobacter baumannii
Bacterial formation of trimethylamine (TMA) from carnitine in the gut microbiome has been linked to cardiovascular disease. During this process, the two-component carnitine monooxygenase (CntAB) catalyzes the oxygen-dependent cleavage of carnitine to TMA and malic semialdehyde. Individual redox states of the reductase CntB and the catalytic component CntA were investigated based on mutagenesis and electron paramagnetic resonance (EPR) spectroscopic approaches. Protein ligands of the flavin mononucleotide (FMN) and the plant-type [2Fe-2S] cluster of CntB and also of the Rieske-type [2Fe-2S] cluster and the mononuclear [Fe] center of CntA were identified.Characterization of the nonheme iron center of cysteamine dioxygenase and its interaction with substrates
Cysteamine dioxygenase (ADO) has been reported to exhibit two distinct biological functions with a nonheme iron center. It catalyzes oxidation of both cysteamine in sulfur metabolism and N-terminal cysteine-containing proteins or peptides, such as regulator of G protein signaling 5 (RGS5). It thereby preserves oxygen homeostasis in a variety of physiological processes. However, little is known about its catalytic center and how it interacts with these two types of primary substrates in addition to O2.The structure and reactivity of the HoxEFU complex from the cyanobacterium Synechocystis sp. PCC 6803
Cyanobacterial Hox is a [NiFe] hydrogenase that consists of the hydrogen (H2)-activating subunits HoxYH, which form a complex with the HoxEFU assembly to mediate reactions with soluble electron carriers like NAD(P)H and ferredoxin (Fdx), thereby coupling photosynthetic electron transfer to energy-transforming catalytic reactions. Researchers studying the HoxEFUYH complex have observed that HoxEFU can be isolated independently of HoxYH, leading to the hypothesis that HoxEFU is a distinct functional subcomplex rather than an artifact of Hox complex isolation.Discovery of a functional, contracted heme-binding motif within a multiheme cytochrome
Anaerobic ammonium-oxidizing (anammox) bacteria convert nitrite and ammonium via nitric oxide (NO) and hydrazine into dinitrogen gas by using a diverse array of proteins, including numerous c-type cytochromes. Many new catalytic and spectroscopic properties of c-type cytochromes have been unraveled by studies on the biochemical pathways underlying the anammox process. The unique anammox intermediate hydrazine is produced by a multiheme cytochrome c protein, hydrazine synthase, through the comproportionation of ammonium and NO and the input of three electrons.The catalytic mechanism of electron-bifurcating electron transfer flavoproteins (ETFs) involves an intermediary complex with NAD+
Electron bifurcation plays a key role in anaerobic energy metabolism, but it is a relatively new discovery, and only limited mechanistic information is available on the diverse enzymes that employ it. Herein, we focused on the bifurcating electron transfer flavoprotein (ETF) from the hyperthermophilic archaeon Pyrobaculum aerophilum. The EtfABCX enzyme complex couples NADH oxidation to the endergonic reduction of ferredoxin and exergonic reduction of menaquinone. We developed a model for the enzyme structure by using nondenaturing MS, cross-linking, and homology modeling in which EtfA, -B, and -C each contained FAD, whereas EtfX contained two [4Fe-4S] clusters.Roles of distal aspartate and arginine of B-class dye-decolorizing peroxidase in heterolytic hydrogen peroxide cleavage
Dye-decolorizing peroxidases (DyPs) represent the most recently classified hydrogen peroxide–dependent heme peroxidase family. Although widely distributed with more than 5000 annotated genes and hailed for their biotechnological potential, detailed biochemical characterization of their reaction mechanism remains limited. Here, we present the high-resolution crystal structures of WT B-class DyP from the pathogenic bacterium Klebsiella pneumoniae (KpDyP) (1.6 Å) and the variants D143A (1.3 Å), R232A (1.9 Å), and D143A/R232A (1.1 Å).Sequential and differential interaction of assembly factors during nitrogenase MoFe protein maturation
Nitrogenases reduce atmospheric nitrogen, yielding the basic inorganic molecule ammonia. The nitrogenase MoFe protein contains two cofactors, a [7Fe-9S-Mo-C-homocitrate] active-site species, designated FeMo-cofactor, and a [8Fe-7S] electron-transfer mediator called P-cluster. Both cofactors are essential for molybdenum-dependent nitrogenase catalysis in the nitrogen-fixing bacterium Azotobacter vinelandii. We show here that three proteins, NafH, NifW, and NifZ, copurify with MoFe protein produced by an A.Function and maturation of the Fe–S center in dihydroxyacid dehydratase from Arabidopsis
Dihydroxyacid dehydratase (DHAD) is the third enzyme required for branched-chain amino acid biosynthesis in bacteria, fungi, and plants. DHAD enzymes contain two distinct types of active-site Fe–S clusters. The best characterized examples are Escherichia coli DHAD, which contains an oxygen-labile [Fe4S4] cluster, and spinach DHAD, which contains an oxygen-resistant [Fe2S2] cluster. Although the Fe–S cluster is crucial for DHAD function, little is known about the cluster-coordination environment or the mechanism of catalysis and cluster biogenesis.High-resolution structure of a lytic polysaccharide monooxygenase from Hypocrea jecorina reveals a predicted linker as an integral part of the catalytic domain
For decades, the enzymes of the fungus Hypocrea jecorina have served as a model system for the breakdown of cellulose. Three-dimensional structures for almost all H. jecorina cellulose-degrading enzymes are available, except for HjLPMO9A, belonging to the AA9 family of lytic polysaccharide monooxygenases (LPMOs). These enzymes enhance the hydrolytic activity of cellulases and are essential for cost-efficient conversion of lignocellulosic biomass. Here, using structural and spectroscopic analyses, we found that native HjLPMO9A contains a catalytic domain and a family-1 carbohydrate-binding module (CBM1) connected via a linker sequence.Mutual synergy between catalase and peroxidase activities of the bifunctional enzyme KatG is facilitated by electron hole-hopping within the enzyme
KatG is a bifunctional, heme-dependent enzyme in the front-line defense of numerous bacterial and fungal pathogens against H2O2-induced oxidative damage from host immune responses. Contrary to the expectation that catalase and peroxidase activities should be mutually antagonistic, peroxidatic electron donors (PxEDs) enhance KatG catalase activity. Here, we establish the mechanism of synergistic cooperation between these activities. We show that at low pH values KatG can fully convert H2O2 to O2 and H2O only if a PxED is present in the reaction mixture.Glutamate 52-β at the α/β subunit interface of Escherichia coli class Ia ribonucleotide reductase is essential for conformational gating of radical transfer
Ribonucleotide reductases (RNRs) catalyze the conversion of nucleoside diphosphate substrates (S) to deoxynucleotides with allosteric effectors (e) controlling their relative ratios and amounts, crucial for fidelity of DNA replication and repair. Escherichia coli class Ia RNR is composed of α and β subunits that form a transient, active α2β2 complex. The E. coli RNR is rate-limited by S/e-dependent conformational change(s) that trigger the radical initiation step through a pathway of 35 Å across the subunit (α/β) interface.Oxidative Maturation and Structural Characterization of Prenylated FMN Binding by UbiD, a Decarboxylase Involved in Bacterial Ubiquinone Biosynthesis
The activity of the reversible decarboxylase enzyme Fdc1 is dependent on prenylated FMN (prFMN), a recently discovered cofactor. The oxidized prFMN supports a 1,3-dipolar cycloaddition mechanism that underpins reversible decarboxylation. Fdc1 is a distinct member of the UbiD family of enzymes, with the canonical UbiD catalyzing the (de)carboxylation of para-hydroxybenzoic acid-type substrates. Here we show that the Escherichia coli UbiD enzyme, which is implicated in ubiquinone biosynthesis, cannot be isolated in an active holoenzyme form despite the fact active holoFdc1 is readily obtained.Structural Characterization and Ligand/Inhibitor Identification Provide Functional Insights into the Mycobacterium tuberculosis Cytochrome P450 CYP126A1
The Mycobacterium tuberculosis H37Rv genome encodes 20 cytochromes P450, including P450s crucial to infection and bacterial viability. Many M. tuberculosis P450s remain uncharacterized, suggesting that their further analysis may provide new insights into M. tuberculosis metabolic processes and new targets for drug discovery. CYP126A1 is representative of a P450 family widely distributed in mycobacteria and other bacteria. Here we explore the biochemical and structural properties of CYP126A1, including its interactions with new chemical ligands.Characterization of Anammox Hydrazine Dehydrogenase, a Key N2-producing Enzyme in the Global Nitrogen Cycle
Anaerobic ammonium-oxidizing (anammox) bacteria derive their energy for growth from the oxidation of ammonium with nitrite as the electron acceptor. N2, the end product of this metabolism, is produced from the oxidation of the intermediate, hydrazine (N2H4). Previously, we identified N2-producing hydrazine dehydrogenase (KsHDH) from the anammox organism Kuenenia stuttgartiensis as the gene product of kustc0694 and determined some of its catalytic properties. In the genome of K. stuttgartiensis, kustc0694 is one of 10 paralogs related to octaheme hydroxylamine (NH2OH) oxidoreductase (HAO).Substrate-selective Inhibition of Cyclooxygeanse-2 by Fenamic Acid Derivatives Is Dependent on Peroxide Tone
Cyclooxygenase-2 (COX-2) catalyzes the oxygenation of arachidonic acid (AA) and endocannabinoid substrates, placing the enzyme at a unique junction between the eicosanoid and endocannabinoid signaling pathways. COX-2 is a sequence homodimer, but the enzyme displays half-of-site reactivity, such that only one monomer of the dimer is active at a given time. Certain rapid reversible, competitive nonsteroidal anti-inflammatory drugs (NSAIDs) have been shown to inhibit COX-2 in a substrate-selective manner, with the binding of inhibitor to a single monomer sufficient to inhibit the oxygenation of endocannabinoids but not arachidonic acid.Structural and Functional Analysis of a Lytic Polysaccharide Monooxygenase Important for Efficient Utilization of Chitin in Cellvibrio japonicus
Cellvibrio japonicus is a Gram-negative soil bacterium that is primarily known for its ability to degrade plant cell wall polysaccharides through utilization of an extensive repertoire of carbohydrate-active enzymes. Several putative chitin-degrading enzymes are also found among these carbohydrate-active enzymes, such as chitinases, chitobiases, and lytic polysaccharide monooxygenases (LPMOs). In this study, we have characterized the chitin-active LPMO, CjLPMO10A, a tri-modular enzyme containing a catalytic family AA10 LPMO module, a family 5 chitin-binding module, and a C-terminal unclassified module of unknown function.Spectroscopic and Kinetic Properties of the Molybdenum-containing, NAD+-dependent Formate Dehydrogenase from Ralstonia eutropha
We have examined the rapid reaction kinetics and spectroscopic properties of the molybdenum-containing, NAD+-dependent FdsABG formate dehydrogenase from Ralstonia eutropha. We confirm previous steady-state studies of the enzyme and extend its characterization to a rapid kinetic study of the reductive half-reaction (the reaction of formate with oxidized enzyme). We have also characterized the electron paramagnetic resonance signal of the molybdenum center in its MoV state and demonstrated the direct transfer of the substrate Cα hydrogen to the molybdenum center in the course of the reaction.Epoxyqueuosine Reductase Structure Suggests a Mechanism for Cobalamin-dependent tRNA Modification
Background: Little is known about epoxyqueuosine reductase (QueG), which catalyzes the final step in the biosynthesis of queuosine. Results: We report solution and structural characterization of Streptococcus thermophilus QueG. Conclusion: The QueG similarity to reductive dehalogenases is largely limited to cofactor binding. Significance: Our study establishes the link between cobalamin-metabolism and tRNA modification and suggests a mechanism for cobalamin-dependent epoxide reduction.Pyranopterin Coordination Controls Molybdenum Electrochemistry in Escherichia coli Nitrate Reductase
Background: The role of the pyranopterin component of the mononuclear molybdenum cofactor is largely unknown.Results: Variants of pyranopterin-coordinating amino acid residues were generated, and their effects on electrochemistry/catalysis investigated.Conclusion: The pyranopterin environment modulates molybdenum electrochemistry.Significance: The pyranopterin coordination environment enables redox-tuning of the molybdenum atom, and facilitates molybdoenzyme reactivity toward a broad range of substrates.Structural and Functional Characterization of a Lytic Polysaccharide Monooxygenase with Broad Substrate Specificity
Background: The recently discovered lytic polysaccharide monooxygenases (LPMOs) are important in enzymatic conversion of lignocellulosic biomass.Results: We describe structural and functional studies of NcLPMO9C, which cleaves both cellulose and certain hemicelluloses.Conclusion: NcLPMO9C has structural and functional features that correlate with the enzyme's catalytic capabilities.Significance: This study shows how LPMO active sites are tailored to varying functionalities and adds to a growing LPMO knowledge base.Demonstration of Lignin-to-Peroxidase Direct Electron Transfer: A TRANSIENT-STATE KINETICS, DIRECTED MUTAGENESIS, EPR, AND NMR STUDY
Background: Ligninolytic peroxidases are often studied using simple model substrates.Results: Reaction of versatile peroxidase with the lignin polymer is shown by a combination of directed mutagenesis, chromatographic, and spectroscopic techniques.Conclusion: The main reaction implies direct electron transfer to a surface tryptophanyl radical responsible for lignin oxidation.Significance: This is the first time that this central mechanistic aspect is shown for a ligninolytic peroxidase.
