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The primary pathway for lactate oxidation in Desulfovibrio vulgaris.

Vita N, Valette O, Brasseur G, Lignon S, Denis Y, Ansaldi M, Dolla A, Pieulle L - Front Microbiol (2015)

Bottom Line: Lactate oxidation by these incomplete oxidizers generates reductants through lactate dehydrogenase (LDH) and pyruvate-ferredoxin oxidoreductase (PFOR), with the latter catalyzing pyruvate conversion into acetyl-CoA.The growth of mutant Δ26-28 was highly disrupted on D-lactate, whereas the growth of mutant Δ32-33 was slower on L-lactate, which could be related to a decrease in the activity of D-lactate or L-lactate oxidase in the corresponding mutants.This result could be related to the identification of several operon enzymes, including LDHs, in the PFOR activity bands, suggesting the occurrence of a lactate-oxidizing supermolecular structure that can optimize the performance of lactate utilization in Desulfovibrio species.

View Article: PubMed Central - PubMed

Affiliation: CNRS, LCB-UMR7283, Aix-Marseille Université Marseille, France.

ABSTRACT
The ability to respire sulfate linked to lactate oxidation is a key metabolic signature of the Desulfovibrio genus. Lactate oxidation by these incomplete oxidizers generates reductants through lactate dehydrogenase (LDH) and pyruvate-ferredoxin oxidoreductase (PFOR), with the latter catalyzing pyruvate conversion into acetyl-CoA. Acetyl-CoA is the source of substrate-level phosphorylation through the production of ATP. Here, we show that these crucial steps are performed by enzymes encoded by a nonacistronic transcriptional unit named now as operon luo (for lactate utilization operon). Using a combination of genetic and biochemical techniques, we assigned a physiological role to the operon genes DVU3027-28 and DVU3032-33. The growth of mutant Δ26-28 was highly disrupted on D-lactate, whereas the growth of mutant Δ32-33 was slower on L-lactate, which could be related to a decrease in the activity of D-lactate or L-lactate oxidase in the corresponding mutants. The DVU3027-28 and DVU3032-33 genes thus encode functional D-LDH and L-LDH enzymes, respectively. Scanning of the genome for lactate utilization revealed several lactate permease and dehydrogenase homologs. However, transcriptional compensation was not observed in any of the mutants except for lactate permease. Although there is a high degree of redundancy for lactate oxidase, it is not functionally efficient in LDH mutants. This result could be related to the identification of several operon enzymes, including LDHs, in the PFOR activity bands, suggesting the occurrence of a lactate-oxidizing supermolecular structure that can optimize the performance of lactate utilization in Desulfovibrio species.

No MeSH data available.


Domains and motifs found in the Dld-II and LldEFG orthologs in the DvH genome (according to the Pfam database). The FAD-binding and FAD-linked oxidase domains (Pfam accession nos. PFO1565 and PFO2913, respectively; blue). H highlighted in gray corresponds to the essential histidine conserved in enzymes that binds to lactate (Griffin et al., 1992). The 4Fe-4S dicluster domain (PF13183; yellow). Two CCG domains (PFO2754; orange). Two DUF162 domains (PFO2589) were identified in all of the LldEFG orthologs (green). So, Shewanella oneidensis.
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Figure 2: Domains and motifs found in the Dld-II and LldEFG orthologs in the DvH genome (according to the Pfam database). The FAD-binding and FAD-linked oxidase domains (Pfam accession nos. PFO1565 and PFO2913, respectively; blue). H highlighted in gray corresponds to the essential histidine conserved in enzymes that binds to lactate (Griffin et al., 1992). The 4Fe-4S dicluster domain (PF13183; yellow). Two CCG domains (PFO2754; orange). Two DUF162 domains (PFO2589) were identified in all of the LldEFG orthologs (green). So, Shewanella oneidensis.

Mentions: A schematic representation of the DvH organic acid oxidation region consisting of nine open reading frames (ORFs) is represented in Figure 1, and the corresponding annotations1 are reported in Table 1. Certain ORFs are annotated as the encoding enzymes that are most likely involved in the phosphoroclastic reaction, including DVU3025 (also called por), DVU3029 and DVU3030, which encode PFOR, Pta and Ack, respectively. A sequence analysis of the Pta (DVU3029) revealed the presence of three conserved domains: a catalytic PTA_PTB protein domain, which is found in all Pta, and AAA and DRTGG domains, which are only found in class II enzymes (Campos-Bermudez et al., 2010). DVU3026 encoded a putative lactate permease; however, annotation of the remaining ORFs was unclear: DVU3031 encoded a conserved hypothetical protein consisting of the AAA and DRTGG domains but without a PTA_PTB protein domain, thus excluding putative Pta activity for this protein. DVU3027 and 3028 were annotated as a glycolate oxidase subunit and iron-sulfur cluster-binding protein encoding gene, respectively. However, their amino acid sequences suggested that they corresponded to two subunits of a flavin- and iron sulfur-containing oxidoreductase homolog of the monomeric D-iLDH (Dld-II), which is characterized in Shewanella oneidensis (Pinchuk et al., 2009) as already proposed in Pereira et al. (2011). Despite the low pairwise sequence identity (17% sequence identity, Supplementary Figure S1), DVU3027-28 consisted of the same protein domains and motifs, including the FAD-binding domain (Pfam accession no. PF01565), FAD-linked oxidase domain (PF02913), the 4Fe-4S dicluster domain (PF13183) and CCG domain (PF02754), (Figure 2). The C-terminal FAD-linked oxidase domain of DVU3027 contained a sequence close to the motif GEHGD and an essential histidine conserved in enzymes that bind lactate (Griffin et al., 1992). DVU3032 and DVU3033 were annotated as a conserved hypothetical protein and iron sulfur cluster-binding protein, respectively. However, their amino-acid sequences shared 26% amino acid sequence identity with the three subunits of the non-flavin iron-sulfur containing oxidoreductase (LldEFG, Supplementary Figure S2) of S. oneidensis (Pinchuk et al., 2009). Moreover, both proteins shared the same multi-domain composition, which is shown in Figure 2. In addition to the iron sulfur-containing domains, DVU3033 contained an N-terminal domain of unknown function (DUF162), and this protein domain was also detected in DVU3032. Therefore, this analysis suggests that DVU3027-3028-3032-3033 genes are candidate genes for lactate utilization enzymes belonging to the Shewanella LDHs family, although with a different subunit organization resulting from either genes fusion or splitting.


The primary pathway for lactate oxidation in Desulfovibrio vulgaris.

Vita N, Valette O, Brasseur G, Lignon S, Denis Y, Ansaldi M, Dolla A, Pieulle L - Front Microbiol (2015)

Domains and motifs found in the Dld-II and LldEFG orthologs in the DvH genome (according to the Pfam database). The FAD-binding and FAD-linked oxidase domains (Pfam accession nos. PFO1565 and PFO2913, respectively; blue). H highlighted in gray corresponds to the essential histidine conserved in enzymes that binds to lactate (Griffin et al., 1992). The 4Fe-4S dicluster domain (PF13183; yellow). Two CCG domains (PFO2754; orange). Two DUF162 domains (PFO2589) were identified in all of the LldEFG orthologs (green). So, Shewanella oneidensis.
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Related In: Results  -  Collection

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Show All Figures
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Figure 2: Domains and motifs found in the Dld-II and LldEFG orthologs in the DvH genome (according to the Pfam database). The FAD-binding and FAD-linked oxidase domains (Pfam accession nos. PFO1565 and PFO2913, respectively; blue). H highlighted in gray corresponds to the essential histidine conserved in enzymes that binds to lactate (Griffin et al., 1992). The 4Fe-4S dicluster domain (PF13183; yellow). Two CCG domains (PFO2754; orange). Two DUF162 domains (PFO2589) were identified in all of the LldEFG orthologs (green). So, Shewanella oneidensis.
Mentions: A schematic representation of the DvH organic acid oxidation region consisting of nine open reading frames (ORFs) is represented in Figure 1, and the corresponding annotations1 are reported in Table 1. Certain ORFs are annotated as the encoding enzymes that are most likely involved in the phosphoroclastic reaction, including DVU3025 (also called por), DVU3029 and DVU3030, which encode PFOR, Pta and Ack, respectively. A sequence analysis of the Pta (DVU3029) revealed the presence of three conserved domains: a catalytic PTA_PTB protein domain, which is found in all Pta, and AAA and DRTGG domains, which are only found in class II enzymes (Campos-Bermudez et al., 2010). DVU3026 encoded a putative lactate permease; however, annotation of the remaining ORFs was unclear: DVU3031 encoded a conserved hypothetical protein consisting of the AAA and DRTGG domains but without a PTA_PTB protein domain, thus excluding putative Pta activity for this protein. DVU3027 and 3028 were annotated as a glycolate oxidase subunit and iron-sulfur cluster-binding protein encoding gene, respectively. However, their amino acid sequences suggested that they corresponded to two subunits of a flavin- and iron sulfur-containing oxidoreductase homolog of the monomeric D-iLDH (Dld-II), which is characterized in Shewanella oneidensis (Pinchuk et al., 2009) as already proposed in Pereira et al. (2011). Despite the low pairwise sequence identity (17% sequence identity, Supplementary Figure S1), DVU3027-28 consisted of the same protein domains and motifs, including the FAD-binding domain (Pfam accession no. PF01565), FAD-linked oxidase domain (PF02913), the 4Fe-4S dicluster domain (PF13183) and CCG domain (PF02754), (Figure 2). The C-terminal FAD-linked oxidase domain of DVU3027 contained a sequence close to the motif GEHGD and an essential histidine conserved in enzymes that bind lactate (Griffin et al., 1992). DVU3032 and DVU3033 were annotated as a conserved hypothetical protein and iron sulfur cluster-binding protein, respectively. However, their amino-acid sequences shared 26% amino acid sequence identity with the three subunits of the non-flavin iron-sulfur containing oxidoreductase (LldEFG, Supplementary Figure S2) of S. oneidensis (Pinchuk et al., 2009). Moreover, both proteins shared the same multi-domain composition, which is shown in Figure 2. In addition to the iron sulfur-containing domains, DVU3033 contained an N-terminal domain of unknown function (DUF162), and this protein domain was also detected in DVU3032. Therefore, this analysis suggests that DVU3027-3028-3032-3033 genes are candidate genes for lactate utilization enzymes belonging to the Shewanella LDHs family, although with a different subunit organization resulting from either genes fusion or splitting.

Bottom Line: Lactate oxidation by these incomplete oxidizers generates reductants through lactate dehydrogenase (LDH) and pyruvate-ferredoxin oxidoreductase (PFOR), with the latter catalyzing pyruvate conversion into acetyl-CoA.The growth of mutant Δ26-28 was highly disrupted on D-lactate, whereas the growth of mutant Δ32-33 was slower on L-lactate, which could be related to a decrease in the activity of D-lactate or L-lactate oxidase in the corresponding mutants.This result could be related to the identification of several operon enzymes, including LDHs, in the PFOR activity bands, suggesting the occurrence of a lactate-oxidizing supermolecular structure that can optimize the performance of lactate utilization in Desulfovibrio species.

View Article: PubMed Central - PubMed

Affiliation: CNRS, LCB-UMR7283, Aix-Marseille Université Marseille, France.

ABSTRACT
The ability to respire sulfate linked to lactate oxidation is a key metabolic signature of the Desulfovibrio genus. Lactate oxidation by these incomplete oxidizers generates reductants through lactate dehydrogenase (LDH) and pyruvate-ferredoxin oxidoreductase (PFOR), with the latter catalyzing pyruvate conversion into acetyl-CoA. Acetyl-CoA is the source of substrate-level phosphorylation through the production of ATP. Here, we show that these crucial steps are performed by enzymes encoded by a nonacistronic transcriptional unit named now as operon luo (for lactate utilization operon). Using a combination of genetic and biochemical techniques, we assigned a physiological role to the operon genes DVU3027-28 and DVU3032-33. The growth of mutant Δ26-28 was highly disrupted on D-lactate, whereas the growth of mutant Δ32-33 was slower on L-lactate, which could be related to a decrease in the activity of D-lactate or L-lactate oxidase in the corresponding mutants. The DVU3027-28 and DVU3032-33 genes thus encode functional D-LDH and L-LDH enzymes, respectively. Scanning of the genome for lactate utilization revealed several lactate permease and dehydrogenase homologs. However, transcriptional compensation was not observed in any of the mutants except for lactate permease. Although there is a high degree of redundancy for lactate oxidase, it is not functionally efficient in LDH mutants. This result could be related to the identification of several operon enzymes, including LDHs, in the PFOR activity bands, suggesting the occurrence of a lactate-oxidizing supermolecular structure that can optimize the performance of lactate utilization in Desulfovibrio species.

No MeSH data available.