HutZ from Inhibits HutW-Mediated Anaerobilin Formation by Sequestering Heme.

Anaerobilin synthase catalyzes the decyclization of the heme protoporphyrin ring, an O-independent reaction that liberates iron and produces the linear tetrapyrrole, anaerobilin. The marine bacterium , the enteric pathogen O157:H7, and the opportunistic oral pathogen encode anaerobilin synthase as part of their heme uptake/utilization operons, designated ( O157:H7), (), and (). and O157:H7 contain accessory proteins (ChuS, ChuY, and HmuF) encoded in their respective operons that mitigate against the cytotoxicity of labile heme and anaerobilin by functioning in heme trafficking and anaerobilin reduction.

New Electron-Transfer Chain to a Flavodiiron Protein in Couples Butyryl-CoA Oxidation to O Reduction.

, a Gram-negative obligate anaerobe, is common to the oral microbiota, but the species is known to infect other sites of the body where it is associated with a range of pathologies. At present, little is known about the mechanisms by which mitigates against oxidative and nitrosative stress. Inspection of the subsp.

Expanding the β-substitution reactions of serine synthase through mutagenesis of aromatic active site residues.

The Gram-negative bacterium, Fusobacterium nucleatum, possesses a fold II type pyridoxal 5'-phosphate-dependent enzyme that catalyzes the reversible β-replacement of l-cysteine and l-serine, generating HS and HO, respectively. This enzyme, termed serine synthase (FN1055), contains an active site Asp that serves as a general base in the activation of a water molecule for nucleophilic attack of the ⍺-aminoacrylate intermediate. A network of hydrophobic residues surrounding Asp are key to catalysis as they increase the basicity of the side chain.

Biosynthesis of meso-lanthionine in Fusobacterium nucleatum.

The opportunistic oral pathogen, Fusobacterium nucleatum contains meso-lanthionine as the diaminodicarboxylic acid in the pentapeptide crosslink of the peptidoglycan layer. The diastereomer, l,l-lanthionine is formed by lanthionine synthase, a PLP-dependent enzyme that catalyzes the β-replacement of l-cysteine with a second equivalent of l-cysteine. In this study, we explored possible enzymatic mechanisms for the formation of meso-lanthionine.

A new member of the flavodoxin superfamily from Fusobacterium nucleatum that functions in heme trafficking and reduction of anaerobilin.

Fusobacterium nucleatum is an opportunistic oral pathogen that is associated with various cancers. To fulfill its essential need for iron, this anaerobe will express heme uptake machinery encoded at a single genetic locus. The heme uptake operon includes HmuW, a class C radical SAM-dependent methyltransferase that degrades heme anaerobically to release Fe and a linear tetrapyrrole called anaerobilin.

Sequence Divergence in the Arginase Domain of Ornithine Decarboxylase/Arginase in Leads to Loss of Function in Oral Associated Species.

A number of species within the family of Gram-negative bacteria uniquely encode for an ornithine decarboxylase/arginase (ODA) that ostensibly channels l-ornithine generated by hydrolysis of l-arginine to putrescine formation. However, two aspartate residues required for coordination to a catalytically obligatory manganese cluster of arginases are substituted for a serine and an asparagine. Curiously, these natural substitutions occur only in a clade of Fusobacterium species that inhabit the oral cavity.

Steady-state and pre-steady state kinetic analysis of ornithine 4,5-aminomutase.

Ornithine 4,5-aminomutase (4,5-OAM) is a pyridoxal 5'-phosphate and adenosylcobalamin-dependent enzyme that catalyzes a 1,2-rearrangement of the terminal amine of d-ornithine to form (2R, 4S)-diaminopentanoate. The gene encoding ornithine 4,5-aminomutase is clustered with other genes that function in the oxidative l-ornithine metabolic pathway present in a number of anaerobic bacteria. This chapter discusses the methodology for measuring 4,5-OAM activity using NAD-dependent diaminopentanoate dehydrogenase, which functions downstream of 4,5-OAM in the l-ornithine metabolic pathway.

A Fold Type II PLP-Dependent Enzyme from Functions as a Serine Synthase and Cysteine Synthase.

Serine synthase (SS) from is a fold type II pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the β-replacement of l-cysteine with water to form l-serine and HS. Herein, we show that SS can also function as a cysteine synthase, catalyzing the β-replacement of l-serine with bisulfide to produce l-cysteine and HO. The forward (serine synthase) and reverse (cysteine synthase) reactions occur with comparable turnover numbers and catalytic efficiencies for the amino acid substrate.

S224 Presents a Catalytic Trade-off in PLP-Dependent l-Lanthionine Synthase from .

Lanthionine synthase from the oral bacterium is a fold type II pyridoxal-5'-phosphate (PLP)-dependent enzyme that catalyzes the β-replacement of l-cysteine by a second molecule of l-cysteine to form HS and l-lanthionine. The -isomer of the latter product is incorporated into the peptidoglycan layer. Herein, we investigated the catalytic role of S224, which engages in hydrogen-bond contact with the terminal carboxylate of l-lanthionine in the closed conformation of the enzyme.

Structural and Kinetic Insight into the Biosynthesis of HS and l-Lanthionine from l-Cysteine by a Pyridoxal l-Phosphate-Dependent Enzyme from .

is a common oral bacterium and a major producer of HS, a toxic gas linked to the pathogenesis of periodontal disease. The bacterium encodes a fold type II pyridoxal l-phosphate (PLP)-dependent enzyme, Fn1220 or lanthionine synthase (LS), that generates HS and l-lanthionine (a component of the peptidoglycan layer) through β-replacement of l-cysteine by a second molecule of l-cysteine. Herein, we show through detailed kinetic analysis that LS elicits catalytic promiscuity as demonstrated for other fold type II PLP-dependent homologues, namely, -acetylserine sulfhydrylase (OASS) and cystathionine β-synthase (CBS).

Structural insight into the high reduction potentials observed for Fusobacterium nucleatum flavodoxin.

Flavodoxins are small flavin mononucleotide (FMN)-containing proteins that mediate a variety of electron transfer processes. The primary sequence of flavodoxin from Fusobacterium nucleatum, a pathogenic oral bacterium, is marked with a number of distinct features including a glycine to lysine (K13) substitution in the highly conserved phosphate-binding loop (T/S-X-T-G-X-T), variation in the aromatic residues that sandwich the FMN cofactor, and a more even distribution of acidic and basic residues. The E (oxidized/semiquinone; -43 mV) and E (semiquinone/hydroquinone; -256 mV) are the highest recorded reduction potentials of known long-chain flavodoxins.

Kinetic characterization of acetone monooxygenase from Gordonia sp. strain TY-5.

Acetone monooxygenase (ACMO) is a unique member of the Baeyer-Villiger monooxygenase (BVMO) family based on its ability to act on small ketones, such as acetone. Herein, we performed a kinetic analysis of ACMO from the propane-utilizing bacterium Gordonia sp. strain TY-5 to assess its preference for smaller ketone substrates.

Active site arginine controls the stereochemistry of hydride transfer in cyclohexanone monooxygenase.

Cyclohexanone monooxygenase (CHMO) uses NADPH and O to insert oxygen into an array of (a)cyclic ketones to form esters or lactones. Herein, the role of two conserved active site residues (R327 and D57) in controlling the binding mode of NADP(H) was investigated. Wild type CHMO elicits a kinetic isotope effect (KIE) of 4.

Active site variants provide insight into the nature of conformational changes that accompany the cyclohexanone monooxygenase catalytic cycle.

Baeyer-Villiger monooxygenases are flavoenzymes that use NADPH and O to convert ketones to esters or lactones. A diagnostic feature of BVMO catalysis is the dual role of the pyridine nucleotide: NADPH functions as a reductant of the FAD cofactor and the resulting NADP acts to stabilize the ensuing C4a-peroxyflavin intermediate. Using cyclohexanone monooxygenase from Acinetobacter sp.

Optimal electrostatic interactions between substrate and protein are essential for radical chemistry in ornithine 4,5-aminomutase.

Ornithine 4,5-aminomutase (OAM) from Clostridium sticklandii is an adenosylcobalamin (AdoCbl) and pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes a 1,2-amino shift, interconverting d-ornithine and 2S, 4R-diaminopentanoate. The reaction occurs via a radical-based mechanism whereby a PLP-bound substrate radical undergoes intramolecular isomerization via an azacyclopropylcarbinyl radical intermediate. Herein, we investigated the catalytic role of active site residues that form non-covalent interactions with PLP and/or substrate, d-ornithine.

Role of active site loop in coenzyme binding and flavin reduction in cytochrome P450 reductase.

Cytochrome P450 reductase (CPR) contains a loop within the active site (comprising Asp(634), Ala(635), Arg(636) and Asn(637); human CPR numbering) that relocates upon NADPH binding. Repositioning of the loop triggers the reorientation of an FAD-shielding tryptophan (Trp(679)) to a partially stacked conformer, reducing the energy barrier for displacement of the residue by the NADPH nicotinamide ring: an essential step for hydride transfer. We used site-directed mutagenesis and kinetic analysis to investigate if the amino acid composition of the loop influences the catalytic properties of CPR.

Kinetic analysis of electron flux in cytochrome P450 reductases reveals differences in rate-determining steps in plant and mammalian enzymes.

Herein, we compare the kinetic properties of CPR from Arabidopsis thaliana (ATR2), with CPR from Artemisia annua (aaCPR) and human CPR (hCPR). While all three CPR forms elicit comparable rates for cytochrome c(3+) turnover, NADPH reduction of the FAD cofactor is ∼50-fold faster in aaCPR and ATR2 compared to hCPR, with a kobs of ∼500 s(-1) (6 °C). Stopped-flow analysis of the isolated FAD-domains reveals that NADP(+)-FADH2 charge-transfer complex formation is also significantly faster in the plant enzymes, but the rate of its decay is comparable for all three proteins.

Isotope effects for deuterium transfer and mutagenesis of Tyr187 provide insight into controlled radical chemistry in adenosylcobalamin-dependent ornithine 4,5-aminomutase.

Adenosylcobalamin-dependent ornithine 4,5-aminomutase (OAM) from Clostridium sticklandii utilizes pyridoxal 5'-phosphate (PLP) to interconvert d-ornithine to 2,4-diaminopentanoate via a multistep mechanism that involves two hydrogen transfer steps. Herein, we uncover features of the OAM catalytic mechanism that differentiate it from its homologue, the more catalytically promiscuous lysine 5,6-aminomutase. Kinetic isotope effects (KIEs) with dl-ornithine-3,3,4,4,5,5-d6 revealed a diminished (D)kcat/Km of 2.

Mitochondrial transcription factor A (Tfam) is a pro-inflammatory extracellular signaling molecule recognized by brain microglia.

Microglia represent mononuclear phagocytes in the brain and perform immune surveillance, recognizing a number of signaling molecules released from surrounding cells in both healthy and pathological situations. The microglia interact with several damage-associated molecular pattern molecules (DAMPs) and recent data indicate that mitochondrial transcription factor A (Tfam) could act as a specific DAMP in peripheral tissues. This study tested the hypothesis that extracellular Tfam induces pro-inflammatory and cytotoxic responses of the microglia.

Proximal FAD histidine residue influences interflavin electron transfer in cytochrome P450 reductase and methionine synthase reductase.

Cytochrome P450 reductase (CPR) and methionine synthase reductase (MSR) transfer reducing equivalents from NADPH to FAD to FMN. In CPR, hydride transfer and interflavin electron transfer are kinetically coupled steps, but in MSR the two catalytic steps are represented by two distinct kinetic phases leading to transient formation of the FAD hydroquinone. In human CPR, His(322) forms a hydrogen-bond with the highly conserved Asp(677), a member of the catalytic triad.

Kinetic analysis of cytochrome P450 reductase from Artemisia annua reveals accelerated rates of NADH-dependent flavin reduction.

Cytochrome P450 reductase from Artemisia annua (aaCPR) is a diflavin enzyme that has been employed for the microbial synthesis of artemisinic acid (a semi-synthetic precursor of the anti-malarial drug, artemisinin) based on its ability to transfer electrons to the cytochrome P450 monooxygenase, CYP71AV1. We have isolated recombinant aaCPR (with the N-terminal transmembrane motif removed) from Escherichia coli and compared its kinetic and thermodynamic properties with other CPR orthologues, most notably human CPR. The FAD and FMN redox potentials and the macroscopic kinetic constants associated with cytochrome c(3+) reduction for aaCPR are comparable to that of other CPR orthologues, with the exception that the apparent binding affinity for the oxidized coenzyme is ~ 30-fold weaker compared to human CPR.

Aromatic substitution of the FAD-shielding tryptophan reveals its differential role in regulating electron flux in methionine synthase reductase and cytochrome P450 reductase.

Methionine synthase reductase (MSR) and cytochrome P450 reductase (CPR) transfer reducing equivalents from NADPH via an FAD and FMN cofactor to a redox partner protein. In both enzymes, hydride transfer from NADPH to FAD requires displacement of a conserved tryptophan that lies coplanar to the FAD isoalloxazine ring. Swapping the tryptophan for a smaller aromatic side chain revealed a distinct role for the residue in regulating MSR and CPR catalysis.

Mutagenesis of a conserved glutamate reveals the contribution of electrostatic energy to adenosylcobalamin co-C bond homolysis in ornithine 4,5-aminomutase and methylmalonyl-CoA mutase.

Binding of substrate to ornithine 4,5-aminomutase (OAM) and methylmalonyl-CoA mutase (MCM) leads to the formation of an electrostatic interaction between a conserved glutamate side chain and the adenosyl ribose of the adenosylcobalamin (AdoCbl) cofactor. The contribution of this residue (Glu338 in OAM from Clostridium sticklandii and Glu392 in human MCM) to AdoCbl Co-C bond labilization and catalysis was evaluated by substituting the residue with a glutamine, aspartate, or alanine. The OAM variants, E338Q, E338D, and E338A, showed 90-, 380-, and 670-fold reductions in catalytic turnover and 20-, 60-, and 220-fold reductions in k(cat)/K(m), respectively.

Tryptophan 697 modulates hydride and interflavin electron transfer in human methionine synthase reductase.

Human methionine synthase reductase (MSR), a diflavin oxidoreductase, plays a vital role in methionine and folate metabolism by sustaining methionine synthase (MS) activity. MSR catalyzes the oxidation of NADPH and shuttles electrons via its FAD and FMN cofactors to inactive MS-cob(II)alamin. A conserved aromatic residue (Trp697) positioned next to the FAD isoalloxazine ring controls nicotinamide binding and catalysis in related flavoproteins.