PduL Is an Evolutionarily Distinct Phosphotransacylase Involved in B ₁₂ -Dependent 1,2-Propanediol Degradation by Salmonella enterica Serovar Typhimurium LT2

Abstract

Salmonella enterica degrades 1,2-propanediol (1,2-PD) in a coenzyme B 12 -dependent manner. Previous enzymatic assays of crude cell extracts indicated that a phosphotransacylase (PTAC) was needed for this process, but the enzyme involved was not identified. Here, we show that the pduL gene encodes an evolutionarily distinct PTAC used for 1,2-PD degradation. Growth tests showed that pduL mutants were unable to ferment 1,2-PD and were also impaired for aerobic growth on this compound. Enzyme assays showed that cell extracts from a pduL mutant lacked measurable PTAC activity in a background that also carried a pta mutation (the pta gene was previously shown to encode a PTAC enzyme). Ectopic expression of pduL corrected the growth defects of a pta mutant. PduL fused to eight C-terminal histidine residues (PduL-His 8 ) was purified, and its kinetic constants were determined: the V max was 51.7 ؎ 7.6 mol min ؊1 mg ؊1 , and the K m values for propionyl-PO 4 2؊ and acetyl-PO 4 2؊ were 0.61 and 0.97 mM, respectively. Sequence analyses showed that PduL is unrelated in amino acid sequence to known PTAC enzymes and that PduL homologues are distributed among at least 49 bacterial species but are absent from the Archaea and Eukarya.

Homolactic Acid Fermentation by the Genetically Engineered Thermophilic Homoacetogen Moorella thermoacetica ATCC 39073

“…It was demonstrated in this study that recombinant proteins translated from pduL1 and pduL2 genes in M. thermoacetica possessed PTA activity. Especially, the specific PTA activity (17,100 U/mg) of PduL2 was 340-fold higher than that of S. enterica serovar Typhimurium LT2 (20) and around 2-fold higher than those of Methanosarcina thermophila (9,006 U/mg) (22) and Clostridium kluyveri (9,100 U/mg) (23), which are the highest specific activities among the previously reported PTAs. The specific activity of PduL1 was 18-fold lower than that of PduL2.…”

“…However, M. thermoacetica lacks any gene that is homologous to pta (18), although activity similar to that of pta has been characterized in M. thermoacetica (19). Instead, Moth_0864 (pduL1) and Moth_1181 (pduL2), two pduL homologs (18), were thought to be alternatives, because propanediol utilization protein (PduL) in Salmonella enterica serovar Typhimurium LT2 was characterized as an evolutionarily distinct PTA (EC 2.3.1.222) (20). Because pduL1 and pduL2 are the only genes thought to encode a PTA in M. thermoacetica (18), they could be expected to be involved in acetate production.…”

In Vivo and in Vitro Analyses of Single-amino Acid Variants of the Salmonella enterica Phosphotransacetylase Enzyme Provide Insights into the Function of Its N-terminal Domain

“…The size of each fragment and their location within the wild-type protein was established by MALDI-TOF mass spectrometry. One fragment was the result of tryptic cleavage after residues Arg 33 and Arg 415 (42 kDa), and the second fragment resulted from cleavage after Lys 70 and Arg 415 (38 kDa) (data not shown). It should be noted that the 38-kDa peptide ran abnormally during SDS-PAGE analysis (supplemental Fig.…”

“…Class I enzymes (Pta I ) are ϳ350 residues in length, whereas class II enzymes (Pta II ) are twice as long as Pta I (ϳ700 residues) (26,32). The pduL gene of S. enterica was recently shown to encode an additional, evolutionarily distinct class of phosphotransacetylase enzymes (33). Pta I enzymes share end-to-end homology with the C-terminal domain of Pta II enzymes; hence, it is inferred that the active site of Pta II enzymes is located within their C-terminal domain.…”

Short N-terminal sequences package proteins into bacterial microcompartments

“…The encapsulated enzymes include coenzyme B 12 -dependent diol dehydratase and CoA-dependent propionaldehyde dehydrogenase (PduP), which convert 1,2-PD to propionyl-CoA via a propionaldehyde intermediate (8,27). Propionyl-CoA exits the MCP into the cytoplasm of the cell, where it is converted to 1-propanol and propionate or enters central metabolism via the methylcitrate pathway (19,(27)(28)(29)(30). The function of the Pdu MCP is to sequester propionaldehyde formed by the first step of 1,2-PD degradation primarily to protect cells from cytotoxicity and DNA damage (31).…”