Pharmacological inhibition of bacterial ß-glucuronidase prevents irinotecan-induced diarrhea without impairing its antitumor efficacy in vivo.

Irinotecan (CPT-11), a first-line chemotherapy for advanced colorectal cancer, causes serious diarrhea in patients receiving treatment. The underlying mechanism has been shown that the active metabolite of CPT-11, SN-38, is metabolized to the inactive metabolite SN-38 glucuronide (SN-38 G) during hepatic glucuronidation, and subsequently is exported into the intestine, where SN-38 G is hydrolyzed by bacterial ß-glucuronidase (ßG) to be SN-38, thus leading to intestinal toxicity. Thus, inhibition of the intestinal bacterial ßG activity is expected to prevent CPT-11-induced diarrhea. However, the effects of such inhibition on serum pharmacokinetics of SN-38, the key determinant of CPT-11 treatment, are uncertain. Here, we determined the effects of a potent E. coli ßG (eßG)-specific inhibitor pyrazolo[4,3-c]quinoline derivative (TCH-3562) for the potential use in preventing CPT-11-induced diarrhea. TCH-3562 exhibited efficacious inhibitory potency of endogenous ßG activity in two anaerobes, Eubacteriumsp. and Peptostreptococcus anaerobius. Oral administration of TCH-3562 also effectively reduced the bacterial ßG activity in mice intestine. Moreover, pharmacokinetic analysis of TCH-3562 revealed a relatively low amount of TCH-3562 was detected in the plasma whereas the majority of TCH-3562 was found in the feces. Importantly, co-treatment of CPT-11 and TCH-3562 did not decrease active SN-38 level in mice plasma. Finally, we established that TCH-3562 as an adjuvant treatment showed protective effects on CPT-11-induced diarrhea and had no negative effects on the therapeutic efficacy of CPT-11 in tumor-bearing mice. Therefore, inhibition of the intestinal bacterial ßG activity by the specific inhibitor, TCH-3562, is promising to prevent CPT-11-induced diarrhea while maintaining its anti-tumor efficacy that may have clinical potentials for the treatment with CPT-11.

Filifactor alocis collagenase can modulate apoptosis of normal oral keratinocytes.

To successfully colonize host cells, pathogenic bacteria must circumvent the host's structural barrier such as the collagen-rich extracellular matrix (ECM), as a preliminary step to invasion and colonization of the periodontal tissue. Filifactor alocis possesses a putative Peptidase U32 family protein (HMPREF0389_00504) with collagenase activity that may play a significant role in colonization of host tissue during periodontitis by breaking down collagen into peptides and disruption of the host cell. Domain architecture of the HMPREF0389_00504 protein predicted the presence of a characteristic PrtC-like collagenase domain, and a peptidase domain. Our study demonstrated that the recombinant F. alocis peptidase U32 protein (designated PrtFAC) can interact with, and degrade, type I collagen, heat-denatured collagen and gelatin in a calcium-dependent manner. PrtFAC decreased viability and induced apoptosis of normal oral keratinocytes (NOKs) in a time and dose-dependent manner. Transcriptome analysis of NOK cells treated with PrtFAC showed an upregulation of the genes encoding human pro-apoptotic proteins: Apoptotic peptidase activating factor 1 (Apaf1) cytochrome C, as well as caspase 3 and caspase 9, suggesting the involvement of the mitochondrial apoptotic pathway. There was a significant increase in caspase 3/7 activity in NOK cells treated with PrtFAC. Taken together, these findings suggest that F. alocis PrtFAC protein may play a role in the virulence and pathogenesis of F. alocis.

Degradation of serine-containing oligopeptides by Peptostreptococcus micros ATCC 33270.

BACKGROUND/AIMS: Microorganisms of Peptostreptococcus micros are asaccharolytic, anaerobic gram-positive cocci that are frequently isolated from human oral sites such as periodontal pockets. Preliminary study showed that several amino acids, including serine, enhanced slightly the growth of P. micros. Therefore, we investigated the degradation of serine and serine-containing oligopeptides. METHODS: Metabolic end products were determined with high-performance liquid chromatography. The related enzymatic activities in cell-free extract were also assayed. RESULTS: Washed P. micros degraded serine-tripeptides (Ser-Ser-Ser), and produced formate, pyruvate, acetate, and ammonia. They also degraded serinyl-tyrosine (Ser-Tyr) to the same products. Related enzymatic activities, such as serine dehydratase, pyruvate formate-lyase, formate dehydrogenase, pyruvate oxidoreductase, phosphate acetyltransferase, and acetate kinase, were detected in the cell-free extract, indicating that the organisms produced ATP in the serine metabolism. CONCLUSION: P. micros utilized serine-containing oligopeptides as exogenous metabolic substrates rather than serine itself, and degraded Ser-Ser-Ser and Ser-Tyr to formate, pyruvate, acetate, and ammonia with ATP generation.

Cloning and expression of the two genes coding for L-serine dehydratase from Peptostreptococcus asaccharolyticus: relationship of the iron-sulfur protein to both L-serine dehydratases from Escherichia coli.

The structural genes sdhA and sdhB, coding for the alpha- and beta-subunits of the [4Fe-4S] cluster containing L-serine dehydratase from Peptostreptococcus asaccharolyticus, have been cloned and sequenced. Expression of modified sdhB together with sdhA in Escherichia coli led to overproduction of active His6-tagged L-serine dehydratase. E. coli MEW22, deficient in the L-serine dehydratase L-SD1, was complemented by this sdhBA construct. The derived amino acid sequence of SdhBA shares similarities with both monomeric L-serine dehydratases, L-SD1 and L-SD2, from E. coli and with a putative L-serine dehydratase from Haemophilus influenzae, which suggests that these three enzymes are also iron-sulfur proteins.

Bioactivation of cysteine conjugates of 1-nitropyrene oxides by cysteine conjugate beta-lyase purified from Peptostreptococcus magnus.

To determine the role of cysteine conjugate beta-lyase (beta-lyase) in the metabolism of mutagenic nitropolycyclic aromatic hydrocarbons, we determined the effect of beta-lyase on the mutagenicities and DNA binding of cysteine conjugates of 4,5-epoxy-4,5-dihydro-1-nitropyrene (1-NP 4,5-oxide) and 9,10-epoxy-9,10-dihydro-1-nitropyrene (1-NP 9,10-oxide), which are detoxified metabolites of the mutagenic compound 1-nitropyrene. We purified beta-lyase from Peptostreptococcus magnus GAI0663, since P. magnus is one of the constituents of the intestinal microflora and exhibits high levels of degrading activity with cysteine conjugates of 1-nitropyrene oxides (1-NP oxide-Cys). The activity of purified beta-lyase was optimal at pH 7.5 to 8.0, was completely inhibited by aminooxyacetic acid and hydroxylamine, and was eliminated by heating the enzyme at 55 degrees C for 5 min. The molecular weight of beta-lyase was 150,000, as determined by fast protein liquid chromatography. S-Arylcysteine conjugates were good substrates for this enzyme. As determined by the Salmonella mutagenicity test, 5 ng of beta-lyase protein increased the mutagenicity of the cysteine conjugate of 1-NP 9,10-oxide (10 nmol per plate) 4.5-fold in Salmonella typhimurium TA98 and 4.1-fold in strain TA100. However, beta-lyase had little effect on the cysteine conjugate of 1-NP 4,5-oxide (10 nmol per plate). Both conjugates exhibited only low levels of mutagenicity with nitroreductase-deficient strain TA98NR. In vitro binding of 1-NP oxide-Cys to calf thymus DNA was increased by adding purified beta-lyase or xanthine oxidase.(ABSTRACT TRUNCATED AT 250 WORDS)

Iron-sulfur cluster-containing L-serine dehydratase from Peptostreptococcus asaccharolyticus: correlation of the cluster type with enzymatic activity.

Investigations were performed with regard to the function of the iron-sulfur cluster of L-serine dehydratase from Peptostreptococcus asaccharolyticus, an enzyme which is novel in the class of deaminating hydro-lyases in that it lacks pyridoxal-5'-phosphate. Anaerobically purified L-serine dehydratase from P. asaccharolyticus revealed EPR spectra characteristic of a [3Fe-4S]+ cluster constituting 1% of the total enzyme concentration. Upon incubation of the enzyme under air the intensity of the [3Fe-4S]+ signal increased correlating with the loss of enzymatic activity. Addition of L-serine prevented this. Hence, active L-serine dehydratase probably contains a diamagnetic [4Fe-4S]2+ cluster which is converted by oxidation and loss of one iron ion to a paramagnetic [3Fe-4S]+ cluster, resulting in inactivation of the enzyme. In analogy to the mechanism elucidated for aconitase, it is proposed that L-serine is coordinated via its hydroxyl and carboxyl groups to the labile iron atom of the [4Fe-4S]2+ cluster.

Bacterial L-serine dehydratases: a new family of enzymes containing iron-sulfur clusters.

Two families of enzymes are described which catalyse identical chemical reactions but differ in their prosthetic groups and hence in their mechanism of action. One family, the pyridoxal-5'-phosphate (PLP)-dependent L-threonine dehydratases, also use L-serine as substrate. The other, hitherto unrecognized family is the iron-dependent, highly specific bacterial L-serine dehydratases. It has been shown that L-serine dehydratase from the anaerobic bacterium Peptostreptococcus asaccharolyticus contains an iron-sulfur cluster but no PLP. A mechanism for the dehydration of L-serine which is similar, but not identical, to that of the dehydration of citrate catalysed by aconitase is proposed.

The iron-sulfur-cluster-containing L-serine dehydratase from Peptostreptococcus asaccharolyticus. Stereochemistry of the deamination of L-threonine.

The stereochemistry of the deamination of L-threonine to 2-oxobutyrate, catalyzed by purified L-serine dehydratase of Peptostreptococcus asaccharolyticus, was elucidated. For this purpose the enzyme reaction was carried out with unlabelled L-threonine in 2H2O and in 3HOH, as well as with L-[3-3H]threonine in unlabelled water. Isotopically labelled 2-oxobutyrate thus formed was directly reduced in a coupled reaction with L- or D-lactate dehydrogenase and NADH. The (2R)- or (2S)-2-hydroxybutyrate species obtained were then subjected to configurational analyses of their labelled methylene group. The results from 1H-NMR spectroscopy and, after degradation of 2-hydroxybutyrate to propionate, the transcarboxylase assay consistently indicated that the deamination of L-threonine catalyzed by L-serine dehydratase of P. asaccharolyticus proceeds with inversion and retention in a 2:1 ratio. This partial racemization is the first ever to be observed for a reaction catalyzed by serine dehydratase, therefore confirming the distinction of the L-serine dehydratase of P. asaccharolyticus as an iron-sulfur protein from those dehydratases dependent on pyridoxal phosphate. For the latter enzymes exclusively, retention has been reported.

Selection, expression, and nucleotide sequencing of the glutamate dehydrogenase gene of Peptostreptococcus asaccharolyticus.

The gene for the catabolic NAD-linked glutamate dehydrogenase of Peptostreptococcus asaccharolyticus was cloned by selection of Escherichia coli for complementation of a biosynthetic defect. Cloned fragments containing the gene and the P. asaccharolyticus transcription and translation signals are very highly expressed in E. coli. The nucleotide sequence of the cloned gene was determined. It codes for a polypeptide of 421 amino acids, the sequence of which is similar to those of the NADP-accepting glutamate dehydrogenases. The sequence similarity of this protein to the mammalian glutamate dehydrogenases, which accept both NADP and NAD, is greater than its similarity to the bacterial NADP-specific dehydrogenases, suggesting that this NAD-specific bacterial glutamate dehydrogenase and the NADP-specific bacterial dehydrogenases diverged separately from the line leading to the dual-specificity mammalian glutamate dehydrogenases.

Purification and properties of an iron-sulfur-containing and pyridoxal-phosphate-independent L-serine dehydratase from Peptostreptococcus asaccharolyticus.

L-Serine dehydratase with a specific activity of 15 nkat/mg protein was present in the anaerobic eubacterium Peptostreptococcus asaccharolyticus grown either on L-glutamate or L-serine. The enzyme was highly specific for L-serine with the lowest Km = 0.8 mM ever reported for an L-serine dehydratase. L-Threonine (Km = 22 mM) was the only other substrate. V/Km for L-serine was 500 times higher than that for L-threonine. L-Cysteine was the best inhibitor (Ki = 0.3 mM, competitive towards L-serine). The enzyme was purified 400-fold to homogeneity under anaerobic conditions (specific activity 6 mukat/mg). PAGE in the presence of SDS revealed two subunits with similar intensities (alpha, 30 kDa; beta, 25 kDa). The molecular mass of the native enzyme was estimated as 200 +/- 20 kDa (gel filtration) and 180 kDa (gradient PAGE). In the absence of oxygen the enzyme was moderately stable even in the presence of sodium borohydride or phenylhydrazine (5 mM each). However, by exposure to air the activity was lost, especially when the latter agent was added. The enzyme was reactivated by ferrous ion under anaerobic conditions. The inability of several nucleophilic agents to inactivate the enzyme indicated the absence of pyridoxal phosphate. This was confirmed by a microbiological determination of pyridoxal phosphate. However, the enzyme contained 3.8 +/- 0.2 mol Fe and 5.6 +/- 0.3 mol inorganic sulfur/mol heterodimer (55 kDa) indicating the presence of an [Fe-S] center. The enzyme was successfully applied to measure L-serine concentrations in bacterial media and in human sera.

Kinetic parameters of lactate dehydrogenases of some rumen bacterial species, the anaerobic ciliate Isotricha prostoma and mixed rumen microorganisms.

A number of kinetic parameters of the lactate dehydrogenases of three rumen bacterial species (Peptostreptococcus productus, Propionibacterium acnes and Actinomyces viscosus), the rumen ciliate Isotricha prostoma and mixed rumen microorganisms (MRM) with respect to NADH, pyruvate, fructose-1,6-diphosphate (FDP) as well as the effects of several nucleotide phosphates were studied. Partially purified LDH of Peptostr. productus had the same kinetic parameters as in crude cell free extracts. Values for Km, determined by Michaelis-Menten kinetics with pyruvate as the substrate, were in the same range for all lactate dehydrogenases. After feeding a cow, changes in the apparent Km and Vmax values for NADH of the total LDH activity in MRM were followed. It is suggested that of the factors studied the ratio NADH/NAD(H) and ATP are the most important regulatory factors for the lactate dehydrogenases of mixed rumen microorganisms.

Electron-transferring flavoprotein of Peptostreptococcus elsdenii that functions in the reduction of acrylyl-coenzyme A.

In Peptostreptococcus elsdenii, a three-component flavoprotein electron transfer system catalyzes the oxidation of lactate and the reduction of crotonyl-coenzyme A (CoA). Spectral evidence showed that D-lactate dehydrogenase, when reduced by D-lactate, was able to reduce butyryl-CoA dehydrogenase, but only in the presence of the electron-transferring flavoprotein. Reduced nicotinamide adenine dinucleotide could replace reduced D-lactate dehydrogenase. A reconstituted system, containing the three partially purified enzymes, excess D-lactate, and a limiting amount of crotonyl-CoA, reduced the crotonyl-CoA to butyryl-CoA, but only if all components were present. The electron-transferring flavoprotein activity, purified 22-fold, was separated into two major flavoprotein components, A and B, after polyacrylamide gel electrophoresis. Elution of the proteins and subsequent kinetic assays of the eluates showed that component B catalyzes the reduction of butyryl-CoA dehydrogenase by reduced D-lactate dehydrogenase, whereas component A does not. Both A and B catalyzed the reduction of butyryl-CoA dehydrogenase by reduced nicotinamide adenine dinucleotide. The results suggest that the D-lactate dehydrogenase-dependent reduction involves a heretofore unrecognized component of the electron-transferring protein group which may utilize an unusual flavin, 6-hydroxy-7,8-dimethyl-10-(ribityl-5'-adenosine diphosphate)-isoalloxazine.