Molecular characterization of the genes encoding pyruvate formate-lyase and its activating enzyme of Clostridium pasteurianum

Weidner, Gerhard; Sawers, R. Gary

doi:10.1128/jb.178.8.2440-2444.1996

Cited by 33 publications

(33 citation statements)

References 37 publications

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“…As observed in other genomes, C. thermocellum pfl is adjacent to a gene encoding a PFL-activating enzyme (act, gene 506; Table 2), which is required for activation of PFL, but is transcribed independently in most organisms studied including C. pasteurianum [20]. This enzyme shares 53 % amino acid similarity with the PFL-AE encoded by Bacillus cereus G9241 (E score = 2e-73).…”

Section: Resultsmentioning

confidence: 92%

Pyruvate catabolism and hydrogen synthesis pathway genes of Clostridium thermocellum ATCC 27405

et al. 2008

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Clostridium thermocellum is a gram-positive, acetogenic, thermophilic, anaerobic bacterium that degrades cellulose and carries out mixed product fermentation, catabolising cellulose to acetate, lactate, and ethanol under various growth conditions, with the concomitant release of H 2 and CO 2 . Very little is known about the factors that determine metabolic fl uxes infl uencing H 2 synthesis in anaerobic, cellulolytic bacteria like C. thermocellum. We have begun to investigate the relationships between genome content, gene expression, and end-product synthesis in C. thermocellum cultured under different conditions. Using bioinformatics tools and the complete C. thermocellum 27405 genome sequence, we identifi ed genes encoding key enzymes in pyruvate catabolism and H 2 -synthesis pathways, and have confi rmed transcription of these genes throughout growth on α-cellulose by reverse transcriptase polymerase chain reaction. Bioinformatic analyses revealed two putative lactate dehydrogenases, one pyruvate formate lyase, four pyruvate:formate lyase activating enzymes, and at least three putative pyruvate:ferredoxin oxidoreductase (POR) or POR-like enzymes. Our data suggests that hydrogen may be generated through the action of either a Ferredoxin (Fd)-dependent NiFe hydrogenase, often referred to as "Energy-converting Hydrogenases", or via NAD(P)Hdependent Fe-only hydrogenases which would permit H 2 production from NADH generated during the glyceraldehyde-3-phosphate dehydrogenase reaction. Furthermore, our fi ndings show the presence of a gene cluster putatively encoding a membrane integral NADH:Fd oxidoreductase, suggesting a possible mechanism in which electrons could be transferred between NADH and ferredoxin. The elucidation of pyruvate catabolism pathways and mechanisms of H 2 synthesis is the fi rst step in developing strategies to increase hydrogen yields from biomass. Our studies have outlined the likely pathways leading to hydrogen synthesis in C. thermocellum strain 27405, but the actual functional roles of these gene products during pyruvate catabolism and in H 2 synthesis remain to be elucidated, and will need to be confi rmed using both expression analysis and protein characterization.

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Section: Resultsmentioning

confidence: 92%

Pyruvate catabolism and hydrogen synthesis pathway genes of Clostridium thermocellum ATCC 27405

et al. 2008

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“…Homology searches in the databases revealed similarity with glycyl radical enzymes like pyruvate formate lyase (PFL) (EC 2.3.1.54) and PFL homologues. The lowest level of identity (around 25%) was found with the well characterized PFLs PflB from E. coli (28) and C. pasteurianum (29). The highest degree of identity was obtained with PflD and f810 (41% and 37%, respectively), two Pfl homologues from E. coli with unknown functions (30), and the recently characterized enzyme involved in anaerobic toluene metabolism, benzylsuccinate synthase from Thauera aromatica (32% identity) (31) and TutD from Thauera sp.…”

Section: Resultsmentioning

confidence: 94%

Molecular characterization of the 1,3-propanediol (1,3-PD) operon of Clostridium butyricum

Raynaud

Sarçabal

Meynial-Salles

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

207

160

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The genes encoding the 1,3-propanediol (1,3-PD) operon of Clostridium butyricum VPI1718 were characterized from a molecular and a biochemical point of view. This operon is composed of three genes, dhaB1, dhaB2, and dhaT. When grown in a vitamin B12-free mineral medium with glycerol as carbon source, Escherichia coli expressing dhaB1, dhaB2, and dhaT produces 1,3-PD and high glycerol dehydratase and 1,3-PD dehydrogenase activities. dhaB1 and dhaB2 encode, respectively, a new type of glycerol dehydratase and its activator protein. The deduced proteins DhaB1 and DhaB2, with calculated molecular masses of 88,074 and 34,149 Da, respectively, showed no homology with the known glycerol dehydratases that are all B12 dependent but significant similarity with the pyruvate formate lyases and pyruvate formate lyases activating enzymes and their homologues. The 1,158-bp dhaT gene codes for a 1,3-PD dehydrogenase with a calculated molecular mass of 41,558 Da, revealing a high level of identity with other DhaT proteins from natural 1,3-PD producers. The expression of the 1,3-PD operon in C. butyricum is regulated at the transcriptional level, and this regulation seems to involve a two-component signal transduction system DhaAS͞DhaA, which may have a similar function to DhaR, a transcriptional regulator found in other natural 1,3-PD producers. The discovery of a glycerol dehydratase, coenzyme B12 independent, should significantly influence the development of an economical vitamin B12-free biological process for the production of 1,3-PD from renewable resources.

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“…Analysis of the promoter region of the C. pasteurianum pfl gene did not allow the identification of putative FNR boxes. In this bacterium, PFL has been suggested to play a role in anabolism and to supply the cells with C 1 units, and consequently, regulation of expression may not include FNR (39). The possible role of FNR-1 and FNR-2 in the regulation of pfl expression in L. lactis is currently being investigated, and the recent isolation of fnr-like genes (13) represents an invaluable tool for achieving this goal.…”

Section: Discussionmentioning

confidence: 99%

“…The enzyme is activated via free-radical formation and is degraded in the presence of oxygen (9,19). pfl homologs have been characterized in only a few bacteria including Clostridium pasteurianum, Haemophilus influenzae, and Streptococcus mutans (8,39,41). Here we describe the cloning, sequence analysis, and characterization of the L. lactis pfl gene.…”

mentioning

confidence: 99%

Cloning, expression, and characterization of the Lactococcus lactis pfl gene, encoding pyruvate formate-lyase

Arnau¹,

Jørgensen²,

Madsen³

et al. 1997

J Bacteriol

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The Lactococcus lactis pfl gene, encoding pyruvate formate-lyase (PFL), has been cloned and characterized. The deduced amino acid sequence of the L. lactis PFL protein showed high similarity to those of other bacterial PFL proteins and included the conserved glycine residue involved in posttranslational activation of PFL. The genetic organization of the chromosomal pfl region in L. lactis showed differences from other characterized pfl loci, with an upstream open reading frame independently transcribed in the same orientation as the pfl gene. The gene coding for PFL-activase (act), normally found downstream of pfl, was not identified in L. lactis. Analysis of pfl expression showed a strong induction under anaerobiosis at the transcriptional level independent of the growth medium used. During growth with galactose, pfl showed the highest levels of expression. Constructed L. lactis pfl strains were unable to produce formate under anaerobic growth. Higher levels of diacetyl and acetoin were produced anaerobically in the constructed Lactococcus lactis subsp. lactis biovar diacetylactis pfl strain.Pyruvate has a key role in the metabolic network of Lactococcus lactis. In this industrially relevant organism, sugars are metabolized predominantly to generate energy and as a carbon source. The catabolism of sugars leads to pyruvate, whose further metabolism determines the nature of the fermentation (see reference 5 for a review). Lactate is the major end product responsible for acidification, while other minor metabolites, mainly diacetyl, are responsible for aroma in fermented milk products. Several genes involved in pyruvate metabolism have been studied for their role in the production of diacetyl (Fig.

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Molecular characterization of the genes encoding pyruvate formate-lyase and its activating enzyme of Clostridium pasteurianum

Cited by 33 publications

References 37 publications

Pyruvate catabolism and hydrogen synthesis pathway genes of Clostridium thermocellum ATCC 27405

Pyruvate catabolism and hydrogen synthesis pathway genes of Clostridium thermocellum ATCC 27405

Molecular characterization of the 1,3-propanediol (1,3-PD) operon of Clostridium butyricum

Cloning, expression, and characterization of the Lactococcus lactis pfl gene, encoding pyruvate formate-lyase

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