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.

Efficient strategies to enhance plasmid stability for fermentation of recombinant Escherichia coli harboring tyrosine phenol lyase.

OBJECTIVE: To solve the bottleneck of plasmid instability during microbial fermentation of L-DOPA with recombinant Escherichia coli expressing heterologous tyrosine phenol lyase. RESULTS: The tyrosine phenol lyase from Fusobacterium nucleatum was constitutively expressed in E. coli and a fed-batch fermentation process with temperature down-shift cultivation was performed. Efficient strategies including replacing the original ampicillin resistance gene, as well as inserting cer site that is active for resolving plasmid multimers were applied. As a result, the plasmid stability was increased. The co-use of cer site on plasmid and kanamycin in culture medium resulted in proportion of plasmid containing cells maintained at 100% after fermentation for 35 h. The specific activity of tyrosine phenol lyase reached 1493 U/g dcw, while the volumetric activity increased from 2943 to 14,408 U/L for L-DOPA biosynthesis. CONCLUSIONS: The established strategies for plasmid stability is not only promoted the applicability of the recombinant cells for L-DOPA production, but also provides important guidance for industrial fermentation with improved microbial productivity.

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 insights into the catalytic mechanism of cysteine (hydroxyl) lyase from the hydrogen sulfide-producing oral pathogen, Fusobacterium nucleatum.

Hydrogen sulfide (H2S) plays important roles in the pathogenesis of periodontitis. Oral pathogens typically produce H2S from l-cysteine in addition to pyruvate and [Formula: see text] However, fn1055 from Fusobacterium nucleatum subsp. nucleatum ATCC 25586 encodes a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the production of H2S and l-serine from l-cysteine and H2O, an unusual cysteine (hydroxyl) lyase reaction (ß-replacement reaction). To reveal the reaction mechanism, the crystal structure of substrate-free Fn1055 was determined. Based on this structure, a model of the l-cysteine-PLP Schiff base suggested that the thiol group forms hydrogen bonds with Asp232 and Ser74, and the substrate α-carboxylate interacts with Thr73 and Gln147 Asp232 is a unique residue to Fn1055 and its substitution to asparagine (D232N) resulted in almost complete loss of ß-replacement activity. The D232N structure obtained in the presence of l-cysteine contained the α-aminoacrylate-PLP Schiff base in the active site, indicating that Asp232 is essential for the addition of water to the α-aminoacrylate to produce the l-serine-PLP Schiff base. Rapid-scan stopped-flow kinetic analyses showed an accumulation of the α-aminoacrylate intermediate during the reaction cycle, suggesting that water addition mediated by Asp232 is the rate-limiting step. In contrast, mutants containing substitutions of other active-site residues (Ser74, Thr73, and Gln147) exhibited reduced ß-replacement activity by more than 100-fold. Finally, based on the structural and biochemical analyses, we propose a mechanism of the cysteine (hydroxyl) lyase reaction by Fn1055. The present study leads to elucidation of the H2S-producing mechanism in F. nucleatum.

Correlation between relative bacterial activity and lactate dehydrogenase gene expression of co-cultures in vitro.

OBJECTIVES: The present study aims at correlating the relative bacterial activity with the H+ concentration and the ldh expression of caries-associated bacteria in co-cultures. MATERIALS AND METHODS: Well plates were prepared with BHI medium and cultures of Lactobacillus paracasei and Fusobacterium nucleatum. Bacterial growth at 37 °C was measured using a microplate-photometer before and after adding sucrose to the samples. Samples of co-cultures (n = 12) and single-species cultures (n = 3) were taken and pH was assessed. Real-time quantitative PCRs were applied targeting the 16S-gene, the 16S-rRNA, the ldh-gene, and the ldh-mRNA. RESULTS: For L. paracasei with sucrose, an increase in relative bacterial activity (62.8% ± 23.5% [mean, SE]) was observed, while F. nucleatum showed a clear decrease in relative bacterial activity (- 35.0% ± 9.6%). Simultaneously, the H+ concentration increased (1.15E-05 mol*l-1 ± 4.61E-07 mol*l-1). Consequently, a significant positive correlation was found between L. paracasei's relative bacterial activity and H+ concentration (Spearman rank correlation, r = 0.638; p = 0.002), while F. nucleatum exhibited a negative correlation (r = - 0.741; p ≤ 0.001). Furthermore L. paracasei with sucrose showed a moderate, but significant positive correlation between relative bacterial activity and ldh-expression (r = 0.307; p = 0.024). CONCLUSIONS AND CLINICAL RELEVANCE: The relative bacterial activity after sucrose pulse showed a significant correlation not only to the acid production (H+ concentration) but also to ldh expression of L. paracasei. However, further research is required to confirm these findings in a mature biofilm in vivo.

Structural Basis of Sequential Allosteric Transitions in Tetrameric d-Lactate Dehydrogenases from Three Gram-Negative Bacteria.

d-Lactate dehydrogenases (d-LDHs) from Fusobacterium nucleatum (FnLDH) and Escherichia coli (EcLDH) exhibit positive cooperativity in substrate binding, and the Pseudomonas aeruginosa enzyme (PaLDH) shows negatively cooperative substrate binding. The apo and ternary complex structures of FnLDH and PaLDH have been determined together with the apo-EcLDH structure. The three enzymes consistently form homotetrameric structures with three symmetric axes, the P-, Q-, and R-axes, unlike Lactobacillus d-LDHs, P-axis-related dimeric enzymes, although apo-FnLDH and EcLDH form asymmetric and distorted quaternary structures. The tetrameric structure allows apo-FnLDH and EcLDH to form wide intersubunit contact surfaces between the opened catalytic domains of the two Q-axis-related subunits in coordination with their asymmetric and distorted quaternary structures. These contact surfaces comprise intersubunit hydrogen bonds and hydrophobic interactions and likely prevent the domain closure motion during initial substrate binding. In contrast, apo-PaLDH possesses a highly symmetrical quaternary structure and partially closed catalytic domains that are favorable for initial substrate binding and forms virtually no intersubunit contact surface between the catalytic domains, which present their negatively charged surfaces to each other at the subunit interface. Complex FnLDH and PaLDH possess highly symmetrical quaternary structures with closed forms of the catalytic domains, which are separate from each other at the subunit interface. Structure-based mutations successfully converted the three enzymes to their dimeric forms, which exhibited no significant cooperativity in substrate binding. These observations indicate that the three enzymes undergo typical sequential allosteric transitions to exhibit their distinctive allosteric functions through the tetrameric structures.

A chemical and biological toolbox for Type Vd secretion: Characterization of the phospholipase A1 autotransporter FplA from Fusobacterium nucleatum.

Fusobacterium nucleatum is an oral pathogen that is linked to multiple human infections and colorectal cancer. Strikingly, F. nucleatum achieves virulence in the absence of large, multiprotein secretion systems (Types I, II, III, IV, and VI), which are widely used by Gram-negative bacteria for pathogenesis. By contrast, F. nucleatum strains contain genomic expansions of Type V secreted effectors (autotransporters) that are critical for host cell adherence, invasion, and biofilm formation. Here, we present the first characterization of an F. nucleatum Type Vd phospholipase class A1 autotransporter (strain ATCC 25586, gene FN1704) that we hereby rename Fusobacterium phospholipase autotransporter (FplA). Biochemical analysis of multiple Fusobacterium strains revealed that FplA is expressed as a full-length 85-kDa outer membrane-embedded protein or as a truncated phospholipase domain that remains associated with the outer membrane. Whereas the role of Type Vd secretion in bacteria remains unidentified, we show that FplA binds with high affinity to host phosphoinositide-signaling lipids, revealing a potential role for this enzyme in establishing an F. nucleatum intracellular niche. To further analyze the role of FplA, we developed an fplA gene knock-out strain, which will guide future in vivo studies to determine its potential role in F. nucleatum pathogenesis. In summary, using recombinant FplA constructs, we have identified a biochemical toolbox that includes lipid substrates for enzymatic assays, potent inhibitors, and chemical probes to detect, track, and characterize the role of Type Vd secreted phospholipases in Gram-negative bacteria.

An efficient colorimetric high-throughput screening method for synthetic activity of tyrosine phenol-lyase.

Tyrosine phenol-lyase (TPL) naturally catalyzes the reversible ß-elimination of l-tyrosine to phenol, pyruvate and ammonium. With its reverse reaction (synthetic activity), l-tyrosine and its derivatives could be synthesized with high atom economy, which are widely used in pharmaceutical industries. In this study, a high-throughput screening method for synthetic activity of TPL was developed. One of the substrate, sodium pyruvate was found to react with salicylaldehyde under alkali condition, forming a yellow color compound. The concentration of sodium pyruvate can be quantified according to the absorbance of the colorimetric compound at wavelength of 465 nm and the activity of TPL could be screened according to the decrease of the absorbance. After optimization of the colorimetric reaction conditions, the established high-throughput screening method was successfully used for screening of TPL with enhanced activity for l-DOPA synthesis. The confirmed sensitivity and accuracy demonstrated the feasibility and application potential of this screening method.

Identification of a highly active tannase enzyme from the oral pathogen Fusobacterium nucleatum subsp. polymorphum.

BACKGROUND: Tannases are tannin-degrading enzymes that have been described in fungi and bacteria as an adaptative mechanism to overcome the stress conditions associated with the presence of these phenolic compounds. RESULTS: We have identified and expressed in E. coli a tannase from the oral microbiota member Fusobacterium nucleatum subs. polymorphum (TanBFnp). TanBFnp is the first tannase identified in an oral pathogen. Sequence analyses revealed that it is closely related to other bacterial tannases. The enzyme exhibits biochemical properties that make it an interesting target for industrial use. TanBFnp has one of the highest specific activities of all bacterial tannases described to date and shows optimal biochemical properties such as a high thermal stability: the enzyme keeps 100% of its activity after prolonged incubations at different temperatures up to 45 °C. TanBFnp also shows a wide temperature range of activity, maintaining above 80% of its maximum activity between 22 and 55 °C. The use of a panel of 27 esters of phenolic acids demonstrated activity of TanBFnp only against esters of gallic and protocatechuic acid, including tannic acid, gallocatechin gallate and epigallocatechin gallate. Overall, TanBFnp possesses biochemical properties that make the enzyme potentially useful in biotechnological applications. CONCLUSIONS: We have identified and characterized a metabolic enzyme from the oral pathogen Fusobacterium nucleatum subsp. polymorphum. The biochemical properties of TanBFnp suggest that it has a major role in the breakdown of complex food tannins during oral processing. Our results also provide some clues regarding its possible participation on bacterial survival in the oral cavity. Furthermore, the characteristics of this enzyme make it of potential interest for industrial use.

Process development for efficient biosynthesis of L-DOPA with recombinant Escherichia coli harboring tyrosine phenol lyase from Fusobacterium nucleatum.

The tyrosine phenol lyase (TPL) catalyzed synthesis of L-DOPA was regarded as one of the most economic route for L-DOPA synthesis. In our previous study, a novel TPL from Fusobacterium nucleatum (Fn-TPL) was exploited for efficient biosynthesis of L-DOPA. However, the catalytic efficiency decreased when the reaction system expanded from 100 mL to 1 L. As such, the bioprocess for scale-up production of L-DOPA was developed in this study. To increase the stability of substrate and product, as well as decrease the by-product formation, the optimum temperature and pH were determined to be 15 °C and pH 8.0, respectively. The initial concentration of pyrocatechol, pyruvate and ammonium acetate was fixed at 8, 5 and 77 g/L and a fed-batch approach was applied with sodium pyruvate, pyrocatechol and ammonium acetate fed in a concentration of 5, 5 and 3.5 g/L, respectively. In addition, L-DOPA crystals were exogenously added to inhibit cell encapsulation by the precipitated product. The final L-DOPA concentration reached higher than 120 g/L with pyrocatechol conversion more than 96% in a 15-L stirred tank, demonstrating the great potential of Fn-TPL for industrial production of L-DOPA.

A new type of Na(+)-driven ATP synthase membrane rotor with a two-carboxylate ion-coupling motif.

The anaerobic bacterium Fusobacterium nucleatum uses glutamate decarboxylation to generate a transmembrane gradient of Na⁺. Here, we demonstrate that this ion-motive force is directly coupled to ATP synthesis, via an F1F0-ATP synthase with a novel Na⁺ recognition motif, shared by other human pathogens. Molecular modeling and free-energy simulations of the rotary element of the enzyme, the c-ring, indicate Na⁺ specificity in physiological settings. Consistently, activity measurements showed Na⁺ stimulation of the enzyme, either membrane-embedded or isolated, and ATP synthesis was sensitive to the Na⁺ ionophore monensin. Furthermore, Na⁺ has a protective effect against inhibitors targeting the ion-binding sites, both in the complete ATP synthase and the isolated c-ring. Definitive evidence of Na⁺ coupling is provided by two identical crystal structures of the c11 ring, solved by X-ray crystallography at 2.2 and 2.6 Šresolution, at pH 5.3 and 8.7, respectively. Na⁺ ions occupy all binding sites, each coordinated by four amino acids and a water molecule. Intriguingly, two carboxylates instead of one mediate ion binding. Simulations and experiments demonstrate that this motif implies that a proton is concurrently bound to all sites, although Na⁺ alone drives the rotary mechanism. The structure thus reveals a new mode of ion coupling in ATP synthases and provides a basis for drug-design efforts against this opportunistic pathogen.

Evaluation of Fusobacterium nucleatum Enoyl-ACP Reductase (FabK) as a Narrow-Spectrum Drug Target.

Fusobacterium nucleatum, a pathobiont inhabiting the oral cavity, contributes to opportunistic diseases, such as periodontal diseases and gastrointestinal cancers, which involve microbiota imbalance. Broad-spectrum antimicrobial agents, while effective against F. nucleatum infections, can exacerbate dysbiosis. This necessitates the discovery of more targeted narrow-spectrum antimicrobial agents. We therefore investigated the potential for the fusobacterial enoyl-ACP reductase II (ENR II) isoenzyme FnFabK (C4N14_ 04250) as a narrow-spectrum drug target. ENRs catalyze the rate-limiting step in the bacterial fatty acid synthesis pathway. Bioinformatics revealed that of the four distinct bacterial ENR isoforms, F. nucleatum specifically encodes FnFabK. Genetic studies revealed that fabK was indispensable for F. nucleatum growth, as the gene could not be deleted, and silencing of its mRNA inhibited growth under the test conditions. Remarkably, exogenous fatty acids failed to rescue growth inhibition caused by the silencing of fabK. Screening of synthetic phenylimidazole analogues of a known FabK inhibitor identified an inhibitor (i.e., 681) of FnFabK enzymatic activity and F. nucleatum growth, with an IC50 of 2.1 µM (1.0 µg/mL) and a MIC of 0.4 µg/mL, respectively. Exogenous fatty acids did not attenuate the activity of 681 against F. nucleatum. Furthermore, FnFabK was confirmed as the intracellular target of 681 based on the overexpression of FnFabK shifting MICs and 681-resistant mutants having amino acid substitutions in FnFabK or mutations in other genetic loci affecting fatty acid biosynthesis. 681 had minimal activity against a range of commensal flora, and it was less active against streptococci in physiologic fatty acids. Taken together, FnFabK is an essential enzyme that is amenable to drug targeting for the discovery and development of narrow-spectrum antimicrobial agents.

Prevalence of ß-lactamase-producing bacteria in human periodontitis.

BACKGROUND AND OBJECTIVE: Beta-lactam antibiotics prescribed in periodontal therapy are vulnerable to degradation by bacterial ß-lactamases. This study evaluated the occurrence of ß-lactamase-positive subgingival bacteria in chronic periodontitis subjects of USA origin, and assessed their in vitro resistance to metronidazole at a breakpoint concentration of 4 µg/mL. MATERIAL AND METHODS: Subgingival plaque specimens from deep periodontal pockets with bleeding on probing were removed from 564 adults with severe chronic periodontitis before treatment. The samples were transported in VMGA III and then plated onto: (i) nonselective enriched Brucella blood agar (EBBA) and incubated anaerobically for 7 d; and (ii) selective trypticase soy-bacitracin-vancomycin (TSBV) and incubated for 3 d in air + 5% CO2 . At the end of the incubation periods, the bacterial test species were identified and quantified. Specimen dilutions were also plated onto EBBA plates supplemented with 2 µg/mL of amoxicillin, a combination of 2 µg/mL of amoxicillin plus 2 µg/mL of the ß-lactamase inhibitor clavulanic acid, or 4 µg/mL of metronidazole, followed by anaerobic incubation for 7 d. Bacterial test species presumptively positive for ß-lactamase production were identified by growth on EBBA primary isolation plates supplemented with amoxicillin alone and no growth on EBBA primary isolation plates containing both amoxicillin plus clavulanic acid. A subset of such isolates was subjected to nitrocefin-based chromogenic disk testing to confirm the presence of ß-lactamase activity. In vitro resistance to 4 µg/mL of metronidazole was noted when growth of test species occurred on metronidazole-supplemented EBBA culture plates. RESULTS: Two-hundred and ninety-four (52.1%) of the study subjects yielded ß-lactamase-producing subgingival bacterial test species, with Prevotella intermedia/nigrescens, Fusobacterium nucleatum and other Prevotella species most frequently identified as ß-lactamase-producing organisms. Of the ß-lactamase-producing bacterial test species strains recovered, 98.9% were susceptible in vitro to metronidazole at 4 µg/mL. CONCLUSION: The occurrence of ß-lactamase-positive subgingival bacterial species in more than half of the subjects with severe chronic periodontitis raises questions about the therapeutic potential of single-drug regimens with ß-lactam antibiotics in periodontal therapy. The in vitro effectiveness of metronidazole against nearly all recovered ß-lactamase-producing subgingival bacterial species further supports clinical periodontitis treatment strategies involving the combination of systemic amoxicillin plus metronidazole.

Purification, crystallization and preliminary X-ray analysis of two hydrogen sulfide-producing enzymes from Fusobacterium nucleatum.

Hydrogen sulfide produced by oral bacteria is responsible for oral malodour. Two homologous hydrogen sulfide-producing enzymes, Fn1220 and Cdl, from Fusobacterium nucleatum (which actively produces hydrogen sulfide) were overproduced, purified and crystallized. X-ray diffraction data were collected from the crystals using a synchrotron-radiation source. The Fn1220 crystal belonged to tetragonal space group P4(1)2(1)2 or P4(3)2(1)2 (unit-cell parameters a=b=116.8, c=99.2 Å) and the Cdl crystal belonged to monoclinic space group P2(1) (unit-cell parameters a=84.9, b=70.9, c=87.6 Å, ß=90.3°).

Identification of an L-methionine γ-lyase involved in the production of hydrogen sulfide from L-cysteine in Fusobacterium nucleatum subsp. nucleatum ATCC 25586.

Fusobacterium nucleatum produces an abundance of hydrogen sulfide (H(2)S) in the oral cavity that is mediated by several enzymes. The identification and characterization of three distinct enzymes (Fn0625, Fn1055 and Fn1220) in F. nucleatum that catalyse the production of H(2)S from l-cysteine have been reported. In the current study, a novel enzyme involved in the production of H(2)S in F. nucleatum ATCC 25586, whose molecular mass had been estimated to be approximately 130 kDa, was identified by two-dimensional electrophoresis combined with MALDI-TOF MS. The enzyme, Fn1419, has previously been characterized as an l-methionine γ-lyase. SDS-PAGE and gel-filtration chromatography indicated that Fn1419 has a molecular mass of 43 kDa and forms tetramers in solution. Unlike other enzymes associated with H(2)S production in F. nucleatum, the quaternary structure of Fn1419 was not completely disrupted by exposure to SDS. The purified recombinant enzyme exhibited a K(m) of 0.32±0.02 mM and a k(cat) of 0.69±0.01 s(-1). Based on current and published data, the enzymic activity for H(2)S production from l-cysteine in F. nucleatum is ranked as follows: Fn1220>Fn1055>Fn1419>Fn0625. Based on kinetic values and relative mRNA levels of the respective genes, as determined by real-time quantitative PCR, the amount of H(2)S produced by Fn1419 was estimated to be 1.9 % of the total H(2)S produced from l-cysteine in F. nucleatum ATCC 25586. In comparison, Fn1220 appeared to contribute significantly to H(2)S production (87.6 %).

Identification and enzymic analysis of a novel protein associated with production of hydrogen sulfide and L-serine from L-cysteine in Fusobacterium nucleatum subsp. nucleatum ATCC 25586.

A third enzyme that produces hydrogen sulfide from L-cysteine was identified in Fusobacterium nucleatum subsp. nucleatum. The fn1055 gene was cloned from a cosmid library constructed with genomic DNA of F. nucleatum ATCC 25586. Despite the database annotation that the product of fn1055 is a cysteine synthase, reverse-phase HPLC revealed that no L-cysteine was produced in vitro by the purified Fn1055 protein; however, the enzyme did produce L-serine. In addition, a cysteine auxotroph, Escherichia coli NK3, transformed with a plasmid containing the fn1055 gene did not grow without cysteine, which further suggests that Fn1055 does not function as a cysteine synthase. The Michaelis-Menten kinetics (K(m) =0.09 ± 0.001 mM and k(cat) =5.43 ± 0.64 s(-1)) of the purified enzyme showed that the capacity of Fn1055 to produce hydrogen sulfide was between that of two other enzymes, Fn0625 and Fn1220. Incubation of Fn1055 with L-cysteine resulted in the production of hydrogen sulfide, but not of pyruvate, ammonia or lanthionine, which are all byproducts produced in addition to hydrogen sulfide when Fn0625 or Fn1220 is incubated with L-cysteine. Instead, Fn1055 produced L-serine in its reaction with L-cysteine. Fn1055 produces hydrogen sulfide from l-cysteine by a mechanism that is different from that of Fn0625 or Fn1220.

Structural and biochemical analyses of the tetrameric carboxypeptidase S9Cfn from Fusobacterium nucleatum.

As one of the most abundant bacteria in the human oral cavity, Fusobacterium nucleatum is closely involved in various oral diseases and is also a risk factor for other diseases. The peptidases of F. nucleatum can digest exogenous peptides into amino acids to satisfy its nutrient requirements. Here, a putative F. nucleatum peptidase, termed S9Cfn, which belongs to the S9C peptidase family was identified. Enzymatic activity assays combined with mass-spectrometric analysis revealed that S9Cfn is a carboxypeptidase, but not an aminopeptidase as previously annotated. The crystal structure of the S9Cfn tetramer was solved at 2.6 Šresolution and was found to contain a pair of oligomeric pores in the center. Structural analysis, together with site-directed mutagenesis and enzymatic activity assays, revealed a substrate-entrance tunnel that extends from each oligomeric pore to the catalytic triad, adjacent to which three conserved arginine residues are responsible for substrate binding. Moreover, comparison with other S9 peptidase structures indicated drastic conformational changes of the oligomeric pores during the catalytic cycle. Together, these findings increase the knowledge of this unique type of tetrameric carboxypeptidase and provide insight into the homeostatic control of microbiota in the human oral cavity.

Inhibitory effect of black cumin (Nigella sativa) seed essential oil on Fusobacterium nucleatum L-methionine-γ-lyase (L-methioninase) activity.

The enzyme L-methionine-γ-lyase is commonly found in a wide range of bacteria and catalyzes the α-elimination and γ-elimination of L-methionine to produce methyl mercaptan, α-ketobutyrate and ammonia. Black cumin seed essential oil (BC oil) reportedly exhibits deodorizing activity against methyl mercaptan. Therefore, we hypothesized that BC oil may also suppress methyl mercaptan production. In this study, we aimed to evaluate the inhibitory effect of BC oil on L-methionine-γ-lyase activity in Fusobacterium nucleatum. Recombinant L-methionine-γ-lyase was incubated under appropriate conditions with BC oil and its constituent thymoquinone. To analyze L-methionine-γ-lyase activity, α-ketobutyric acid and ammonia concentrations were determined. The concentrations of α-ketobutyric acid and ammonia were significantly decreased by 10 µg mL-1 of BC oil (P < 0.01) and 16.4 µg/mL of thymoquinone (P < 0.05). An enzyme kinetic assay showed a mixed inhibition pattern between L-methionine-γ-lyase and thymoquinone. In conclusion, BC oil not only had a deodorizing effect against methyl mercaptan but also an inhibitory effect on methyl mercaptan production through the suppression of L-methionine-γ-lyase activity. Thymoquinone may be mainly responsible for these effects of BC oil. Thus, application of natural BC oil may be adapted not only for medical use but also in other areas of life.