Characterization of a Structurally Distinct ATP-Dependent Reactivating Factor of Adenosylcobalamin-Dependent Lysine 5,6-Aminomutase.

Several anaerobic bacterial species, including the Gram-negative oral bacterium Fusobacterium nucleatum, ferment lysine to produce butyrate, acetate, and ammonia. The second step of the metabolic pathway─isomerization of ß-l-lysine to erythro-3,5-diaminohexanoate─is catalyzed by the adenosylcobalamin (AdoCbl) and pyridoxal 5'-phosphate (PLP)-dependent enzyme, lysine 5,6-aminomutase (5,6-LAM). Similar to other AdoCbl-dependent enzymes, 5,6-LAM undergoes mechanism-based inactivation due to loss of the AdoCbl 5'-deoxyadenosyl moiety and oxidation of the cob(II)alamin intermediate to hydroxocob(III)alamin. Herein, we identified kamB and kamC, two genes responsible for ATP-dependent reactivation of 5,6-LAM. KamB and KamC, which are encoded upstream of the genes corresponding to α and ß subunits of 5,6-LAM (kamD and kamE), co-purified following coexpression of the genes in Escherichia coli. KamBC exhibited a basal level of ATP-hydrolyzing activity that was increased 35% in a reaction mixture that facilitated 5,6-LAM turnover with ß-l-lysine or d,l-lysine. Ultraviolet-visible (UV-vis) spectroscopic studies performed under anaerobic conditions revealed that KamBC in the presence of ATP/Mg2+ increased the steady-state concentration of the cob(II)alamin intermediate in the presence of excess ß-l-lysine. Using a coupled UV-visible spectroscopic assay, we show that KamBC is able to reactivate 5,6-LAM through exchange of the damaged hydroxocob(III)alamin for AdoCbl. KamBC is also specific for 5,6-LAM as it had no effect on the rate of substrate-induced inactivation of the homologue, ornithine 4,5-aminomutase. Based on sequence homology, KamBC is structurally distinct from previously characterized B12 chaperones and reactivases, and correspondingly adds to the list of proteins that have evolved to maintain the cellular activity of B12 enzymes.

Expanding the ß-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 ß-replacement of l-cysteine and l-serine, generating H2S and H2O, respectively. This enzyme, termed serine synthase (FN1055), contains an active site Asp232 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 Asp232 are key to catalysis as they increase the basicity of the side chain. However, these residues severely restrict the range of nucleophilic substrates that can react with the ⍺-aminoacrylate, making the enzyme an ineffective biocatalyst for noncanonical amino acid biosynthesis. Herein, we systematically substituted four aromatic active residues (Trp99, Phe125, Phe148 and Phe234) to an alanine to determine their catalytic importance in serine/cysteine synthase reactions and if their substitution could broaden the scope of nucleophiles that could react with the ⍺-aminoacrylate intermediate. All four single site mutants W99A, F125A, F148A, and F234A could form the ⍺-aminoacrylate intermediate upon reaction with either l-cysteine or l-serine; however, the rate constant associated with the elimination of the ß-hydroxyl group from l-serine was 150 to 200-fold lower in the F125A and F148A variants. Substitution of Phe125 and Phe148, situated ∼3-4 Å from the general base, also abolished the serine synthase reaction due to their inability to activate a water molecule for nucleophilic attack of the ⍺-aminoacrylate. Overall, the mutational studies indicate that the clustering of aromatic residues disproportionately benefits the serine synthase reaction as they increase the binding affinity for l-cysteine, decrease the binding of the product, l-serine, and promote the activation of a water molecule. Notably, the aminoacrylate species present in F125A and F148A was able to react with thiophenol, signifying that serine synthase has biocatalytic potential in the synthesis of noncanonical amino acids.

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 ß-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. Our inhibition studies with lanthionine synthase, described herein, revealed that meso-diaminopimelate, a bioisostere of meso-lanthionine, is a more potent inhibitor of lanthionine synthase compared to the diastereomer, l,l-diaminopimelate. These results suggested that lanthionine synthase could also form meso-lanthionine by the ß-replacement of l-cysteine with d-cysteine. Through steady-state and pre-steady state kinetic analysis, we confirm that d-cysteine reacts with the ⍺-aminoacylate intermediate with a kon that was 2-3-fold faster and Kd value that was 2-3fold lower compared to l-cysteine. However, given that intracellular levels of d-cysteine levels are assumed to be significantly lower than that of l-cysteine, we also determined if the gene product, FN1732, with low sequence identity to diaminopimelate epimerase could convert l,l-lanthionine to meso-lanthionine. Using diaminopimelate dehydrogenase in a coupled spectrophotometric assay, we show that FN1732 can convert l,l-lanthionine to meso-lanthionine with a kcat of 0.07 ± 0.001 s-1 and a KM of 1.9 ± 0.1 mM. In summary, our results provide two possible enzymatic mechanisms for the biosynthesis of meso-lanthionine in F. nucleatum.

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 Fe2+ and a linear tetrapyrrole called anaerobilin. The last gene in the operon, hmuF encodes a member of the flavodoxin superfamily of proteins. We discovered that HmuF and a paralog, FldH, bind tightly to both FMN and heme. The structure of Fe3+-heme-bound FldH (1.6 Å resolution) reveals a helical cap domain appended to the ⍺/ß core of the flavodoxin fold. The cap creates a hydrophobic binding cleft that positions the heme planar to the si-face of the FMN isoalloxazine ring. The ferric heme iron is hexacoordinated to His134 and a solvent molecule. In contrast to flavodoxins, FldH and HmuF do not stabilize the FMN semiquinone but instead cycle between the FMN oxidized and hydroquinone states. We show that heme-loaded HmuF and heme-loaded FldH traffic heme to HmuW for degradation of the protoporphyrin ring. Both FldH and HmuF then catalyze multiple reductions of anaerobilin through hydride transfer from the FMN hydroquinone. The latter activity eliminates the aromaticity of anaerobilin and the electrophilic methylene group that was installed through HmuW turnover. Hence, HmuF provides a protected path for anaerobic heme catabolism, offering F. nucleatum a competitive advantage in the colonization of anoxic sites of the human body.

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. The use of ornithine racemace, which functions upstream of 4,5-OAM, for the synthesis of d,l-ornithine-3,3,4,4,5,5-d6 is also presented. Finally, this chapter describes the anaerobic stopped-flow spectrophotometric analysis of 4,5-OAM. Information is provided on the integration of a stopped-flow system in the anaerobically-maintained glove, the preparation of anaerobic solutions, and the experimental approach.

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

A number of species within the Fusobacteriaceae 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. Herein, we expressed and isolated full-length ODA from the opportunistic oral pathogen Fusobacterium nucleatum along with the individual arginase and ornithine decarboxylase components. The crystal structure of the arginase domain reveals that it adopts the classical α/ß arginase-fold, but metal ions are absent in the active site. As expected, the ureohydrolase activity with l-arginine was not detected for wild-type ODA or the isolated arginase domain. However, engineering of the complete metal coordination environment through site-directed mutagenesis restored Mn2+ binding capacity and arginase activity, although the catalytic efficiency for l-arginine was low (60-100 M-1 s-1). Full-length ODA and the isolated ODC component were able to decarboxylate both l-ornithine and l-arginine to form putrescine and agmatine, respectively, but kcat/KM of l-ornithine was ∼20-fold higher compared to l-arginine. We discuss environmental conditions that may have led to the natural selection of an inactive arginase in the oral associated species of Fusobacterium.

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

Serine synthase (SS) from Fusobacterium nucleatum is a fold type II pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the ß-replacement of l-cysteine with water to form l-serine and H2S. Herein, we show that SS can also function as a cysteine synthase, catalyzing the ß-replacement of l-serine with bisulfide to produce l-cysteine and H2O. The forward (serine synthase) and reverse (cysteine synthase) reactions occur with comparable turnover numbers and catalytic efficiencies for the amino acid substrate. Reaction of SS with l-cysteine leads to transient formation of a quinonoid species, suggesting that deprotonation of the Cα and ß-elimination of the thiolate group from l-cysteine occur via a stepwise mechanism. In contrast, the quinonoid species was not detected in the formation of the α-aminoacrylate intermediate following reaction of SS with l-serine. A key active site residue, D232, was shown to stabilize the more chemically reactive ketoenamine PLP tautomer and also function as an acid/base catalyst in the forward and reverse reactions. Fluorescence resonance energy transfer between PLP and W99, the enzyme's only tryptophan residue, supports ligand-induced closure of the active site, which shields the PLP cofactor from the solvent and increases the basicity of D232. These results provide new insight into amino acid metabolism in F. nucleatum and highlight the multiple catalytic roles of D232 in a new member of the fold type II family of PLP-dependent enzymes.

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

Lanthionine synthase from the oral bacterium Fusobacterium nucleatum is a fold type II pyridoxal-5'-phosphate (PLP)-dependent enzyme that catalyzes the ß-replacement of l-cysteine by a second molecule of l-cysteine to form H2S and l-lanthionine. The meso-isomer of the latter product is incorporated into the F. nucleatum 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. Unexpectedly, the S224A variant elicited a 7-fold increase in the turnover rate for H2S and lanthionine formation and a 70-fold faster rate constant for the formation of the α-aminoacrylate intermediate compared to the wild-type enzyme. Presteady state kinetic analysis further showed that the reaction between S224A and l-cysteine leads to the formation of the more reactive ketoenamine tautomer of the α-aminoacrylate. The α-aminoacrylate with the protonated Schiff base is not an observable intermediate in the analogous reaction with the wild type, which may account for its attenuated kinetic properties. However, the S224A substitution is detrimental to other aspects of the catalytic cycle; it facilitates the α,ß-elimination of l-lanthionine, and it weakens the enzyme's catalytic preference for the formation of l-lanthionine over that of l-cystathionine.

Structural and Kinetic Insight into the Biosynthesis of H2S and l-Lanthionine from l-Cysteine by a Pyridoxal l-Phosphate-Dependent Enzyme from Fusobacterium nucleatum.

Fusobacterium nucleatum is a common oral bacterium and a major producer of H2S, 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 H2S and l-lanthionine (a component of the peptidoglycan layer) through ß-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, O-acetylserine sulfhydrylase (OASS) and cystathionine ß-synthase (CBS). Like OASS, LS can assimilate H2S by catalyzing the ß-replacement of O-acetyl-l-serine by sulfide to form l-cysteine. However, the turnover for this reaction in LS is slower than that of other studied OASS enzymes due to slower conversion to the α-aminoacrylate intermediate. Similar to yeast and human CBS, LS can generate H2S and l-cystathionine through ß-replacement of l-cysteine by a second molecule of l-homocysteine; however, whereas this is the main H2S-forming reaction in CBS, it is not for LS. LS shows a marked preference for forming H2S and l-lanthionine through the condensation of 2 equiv of l-cysteine. Sequence alignment of LS with other CBS and OASS enzymes and inspection of the LS crystal structure in the external aldimine state with l-lanthionine reveal that LS possesses a unique loop that engages in hydrogen-bond contact with the product, providing a structural rationale for the enzyme's catalytic preference for H2S and l-lanthionine biosynthesis.

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 Eox/sq (oxidized/semiquinone; -43 mV) and Esq/hq (semiquinone/hydroquinone; -256 mV) are the highest recorded reduction potentials of known long-chain flavodoxins. These more electropositive values are a consequence of the apoprotein binding to the FMN hydroquinone anion with ~70-fold greater affinity compared to the oxidized form of the cofactor. Inspection of the FnFld crystal structure revealed the absence of a hydrogen bond between the protein and the oxidized FMN N5 atom, which likely accounts for the more electropositive Eox/sq . The more electropositive Esq/hq is likely attributed to only one negatively charged group positioned within 12 Å of the FMN N1. We show that natural substitutions of highly conserved residues partially account for these more electropositive reduction potentials.

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. Steady state kinetic analysis of ACMO with a range of linear (C3-C7) and cyclic ketones (C4-C6) reveals that the enzyme elicits the highest catalytic efficiency towards butanone and cyclobutanone. Stopped-flow and inhibition studies further revealed that ACMO has a relatively weak binding affinity for the coenzyme with a dissociation constant of 120 µM. We show through mutagenesis that sequence variation in the residue that coordinates to the 2'-phosphate of NADP(H) partially accounts for the weaker binding affinity observed. As for shown for related BVMOs, NADP+ stabilizes the C4a-peroxyflavin intermediate in ACMO; however, the rate of its formation is considerably slower in ACMO. The observed rate constant for NADPH-dependent flavin reduction was 60 s-1 at 25 °C, and experiments performed with 4(R)-[4-2H]NADPH confirm that the C4-pro-R-hydride from the nicotinamide ring is transferred to the FAD. The latter experimental result suggests that the nicotinamide ring rotates within the active site to carry out its two functional roles: reduction of the FAD cofactor and stabilization of the C4a-peroxyflavin adduct.

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

Cyclohexanone monooxygenase (CHMO) uses NADPH and O2 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 4.7 ±â€¯0.1 and 1.1 ±â€¯0.1 with 4(R)-[4-2H]NADPH and 4(S)-[4-2H]NADPH, respectively, consistent with transfer of the proR hydrogen to FAD. Strikingly, the R327K variant appears to lack stereospecificity for hydride transfer as a KIE of 1.5 ±â€¯0.1 and 2.5 ±â€¯0.1 was observed for the proR and proS deuterated forms of NADPH. 1H NMR of the NADP+ products confirmed that the R327K variant abstracts either the proR or proS hydrogen from NADPH. While the D57A variant retained stereospecificity for the proR hydrogen, this substitution resulted in slow decomposition of the C4a-peroxyflavin intermediate in the presence of cyclohexanone. Based on published structures of a related flavin monooxygenase, we suggest that elimination of the hydrogen bond between D57 and R327 in the D57A variant causes R327 to adopt a substrate-induced conformation that slows substrate access to the active site, thereby prolonging the lifetime of the C4a-peroxyflavin intermediate.

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 O2 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. NCIMB 9871 as a model system, we investigated the catalytic role of T187 and W490, which hydrogen bond to the phosphate and ribose of the nicotinamide mononucleotide half of NADP(H), respectively. Eliminating either hydrogen bond through creation of a T187A or a W490F variant leads to a 15-fold reduction in turnover of cyclohexanone. Substitution of either residue does not affect the rate of FAD reduction or the coupling efficiency. Rather, T187A and W490F disrupt distinct steps of the oxidative half-reaction. Kinetic and spectroscopic analysis of T187A reveals that this residue is critical for locking NADP+ in a configuration that dramatically accelerates O2 activation by the reduced flavin. W490 also promotes O2 activation (albeit less so than T187) and accelerates the reaction between the C4a-peroxyflavin and cyclohexanone. The results provide insight into the conformation of CHMO and the coenzyme for optimal catalysis.

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. Kinetic analyses revealed that residues that form salt bridges to the α-carboxylate (R297) or the α-amine (E81) of d-ornithine are most critical for OAM activity as conservative substitutions of these residues results in a 300-600-fold reduction in catalytic turnover and a more pronounced 1000- to 14,000-fold decrease in catalytic efficiency. In contrast, mutating residues that solely interact with the PLP cofactor led to more modest decreases (10-60-fold) in kcat and kcat/Km. All but one variant (S162A) elicited an increase in the kinetic isotope effect on kcat and kcat/Km with d,l-ornithine-3,3,4,4,5,5-d6 as the substrate, which indicates that hydrogen atom abstraction is more rate determining. Electron paramagnetic resonance spectra of the variants reveal that while the substitutions decrease the extent of CoC bond homolysis, they do not affect the structural integrity of the active site. Our experimental results, discussed in context with published computational work, suggests that the protonation state of the PLP cofactor has less of a role in radical-mediated chemistry compared to electrostatic interactions between the substrate and protein.

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. The D634A and D634N variants elicited a modest increase in coenzyme binding affinity coupled with a 36- and 10-fold reduction in cytochrome c(3+) turnover and a 17- and 3-fold decrease in the pre-steady state rate of flavin reduction. These results, in combination with a reduction in the kinetic isotope effect for hydride transfer, suggest that diminished activity is due to destabilization of the partially stacked conformer of Trp(677) and slower release of NADP(+). In contrast, R636A, R636S and an A635G/R636S double mutant led to a modest increase in cytochrome c(3+) reduction, which is linked to weaker coenzyme binding and faster interflavin electron transfer. A potential mechanism by which Arg(636) influences catalysis is discussed.

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. In hCPR, transfer of a hydride ion from NADPH to FAD is tightly coupled to subsequent FAD to FMN electron transfer, indicating that the former catalytic event is slow relative to the latter. In contrast, interflavin electron transfer is slower than NADPH hydride transfer in aaCPR and ATR2, occurring with an observed rate constant of ∼50 s(-1). Finally, the transfer of electrons from FMN to cytochrome c(3+) is rapid (>10(3) s(-1)) in all three enzymes and does not limit catalytic turnover. In combination, the data reveal differences in rate-determining steps between plant CPR and their mammalian equivalent in mediating the flux of reducing equivalents from NADPH to external electron acceptors.

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 2.5 ± 0.4 relative to a (D)kcat of 7.6 ± 0.5, suggesting slow release of the substrate from the active site. In contrast, a KIE was not observed on the rate constant associated with Co-C bond homolysis as this step is likely "gated" by the formation of the external aldimine. The role of tyrosine 187, which lies planar to the PLP pyridine ring, was also investigated via site-directed mutagenesis. The 25- and 1260-fold reduced kcat values for Y187F and Y187A, respectively, are attributed to a slower rate of external aldimine formation and a diminution of adenosylcobalamin Co-C bond homolysis. Notably, electron paramagnetic resonance studies of Y187F suggest that the integrity of the active site is maintained as cob(II)alamin and the PLP organic radical (even at lower concentrations) remain tightly exchange-coupled. Modeling of d-lysine and l-ß-lysine into the 5,6-LAM active site reveals interactions between the substrate and protein are weaker than those in OAM and fewer in number. The combined data suggest that the level of protein-substrate interactions in aminomutases not only influences substrate specificity, but also controls radical chemistry.

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. Three different types of human mononuclear phagocytes were used to model human microglia: human peripheral blood monocytes from healthy donors, human THP-1 monocytic cells, and human primary microglia obtained from autopsy samples. When combined with interferon (IFN)-γ, recombinant human Tfam (rhTfam) induced secretions that were toxic to human SH-SY5Y neuroblastoma cells in all three models. Similar cytotoxic responses were observed when THP-1 cells and human microglia were exposed to human mitochondrial proteins in the presence of IFN-γ. rhTfam alone induced expression of pro-inflammatory cytokines interleukin (IL)-1ß, IL-6 and IL-8 by THP-1 cells. This induction was further enhanced in the presence of IFN-γ. Upregulated secretion of IL-6 in response to rhTfam plus IFN-γ was confirmed in primary human microglia. Use of specific inhibitors showed that the rhTfam-induced cytotoxicity of human THP-1 cells depended partially on activation of c-Jun N-terminal kinase (JNK), but not p38 mitogen-activated protein kinase (MAPK). Overall, our data support the hypothesis that, in the human brain, Tfam could act as an intercellular signaling molecule that is recognized by the microglia to cause pro-inflammatory and cytotoxic responses.

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. The catalytic triad is present in MSR, but Ala(312) replaces the histidine residue. To examine if this structural variation accounts for differences in their kinetic behavior, reciprocal substitutions were created. Substitution of His(322) for Ala in CPR does not affect the rate of NADPH hydride transfer or the FAD redox potentials, but does impede interflavin electron transfer. For MSR, swapping Ala(312) for a histidine residue resulted in the kinetic coupling of hydride and interflavin electron transfer, and eliminated the formation of the FAD hydroquinone intermediate. For both enzymes, placement of the His residue in the active site weakens coenzyme binding affinity. The data suggest that the proximal FAD histidine residue accelerates proton-coupled electron transfer from FADH2 to the higher potential FMN; a mechanism for this catalytic role is discussed.

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. CPR from A. annua shows a 3.5-fold increase in uncoupled NADPH oxidation compared to human CPR and a strong preference (85 100-fold) for NADPH over NADH. Strikingly, reduction of the enzyme by the first and second equivalent of NADPH is much faster in aaCPR, with rates of > 500 and 17 s(-1) at 6 °C. We also optically detect a charge-transfer species that rapidly forms in < 3 ms and then persists during the reductive half reaction. Additional stopped-flow kinetic studies with NADH and (R)-[4-(2) H]NADPH suggest that the accelerated rate of flavin reduction is attributed to the relatively weak binding affinity of aaCPR for NADP(+) .