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Literature summary for 6.2.1.67 extracted from
- Phosphinothricin tripeptide synthetases in Streptomyces viridochromogenes Tü494 (2005), Antimicrob. Agents Chemother., 49, 4598-4607 .
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| gene phsB, DNA and amino acid sequence determination and analysis, promoter analysis, both phsB and phsC appear to be cotranscribed together with two other genes from a single promoter, and they are located at a distance of 20 kb from the gene phsA, encoding PhsA, in the PTT biosynthesis gene cluster, heterologous coexpression of phsB/phsC in Streptomyces lividans strain TK23 using vector pDS209 | Streptomyces viridochromogenes |
| gene phsC, DNA and amino acid sequence determination and analysis, promoter analysis, both phsB and phsC appear to be cotranscribed together with two other genes from a single promoter, and they are located at a distance of 20 kb from the gene phsA, encoding PhsA, in the PTT biosynthesis gene cluster, heterologous coexpression of phsB/phsC in Streptomyces lividans strain TK23 using vector pDS209 | Streptomyces viridochromogenes |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| additional information | gene disruption mutagenesis in phsC using the temperature-sensitive plasmid pDS104 is performed. Mutants PHSB and PHSBC are generated using the nonreplicative plasmids pMS100 and pDS199, respectively, complementation of the mutants PHSB and PHSBC and heterologous expression of phsB/phsC in Streptomyces lividans. Construction of a phsB null mutant (B3-14), which is unable to produce phosphinothricin tripeptide (PTT), no alanine-activating activity attributable to PTT synthetase III is detected. Construction of a triple PTT mutant (phsB, orfM, and phsC), mutant PHSBC has lost the ability to produce PTT. Transformation of PHSBC with either phsB including orfM (pDS207) or phsC (pDS208) does not restore PTT synthesis, indicating that PhsB cannot take on the function of PhsC or vice versa | Streptomyces viridochromogenes |
| additional information | gene disruption mutagenesis in phsC using the temperature-sensitive plasmid pDS104 is performed. Mutants PHSB and PHSBC are generated using the nonreplicative plasmids pMS100 and pDS199, respectively, complementation of the mutants PHSB and PHSBC and heterologous expression of phsB/phsC in Streptomyces lividans. No activity attributable to PTT synthetase II or phosphinothricin tripeptide (PTT) synthesis is detected in the phsC disruption mutant. Construction of a triple PTT mutant (phsB, orfM, and phsC), mutant PHSBC has lost the ability to produce PTT. Transformation of PHSBC with either phsB including orfM (pDS207) or phsC (pDS208) does not restore PTT synthesis, indicating that PhsB cannot take on the function of PhsC or vice versa | Streptomyces viridochromogenes |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Mg2+ | required | Streptomyces viridochromogenes |  |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| (L-alanyl)adenylate + holo-[L-alanyl-carrier protein] | Streptomyces viridochromogenes | - |
AMP + L-alanyl-[L-alanyl-carrier protein] | - |
ir | |
| (L-alanyl)adenylate + holo-[L-alanyl-carrier protein] | Streptomyces viridochromogenes Tü494 | - |
AMP + L-alanyl-[L-alanyl-carrier protein] | - |
ir | |
| ATP + L-glutamate | Streptomyces viridochromogenes | - |
diphosphate + (L-alanyl)adenylate | - |
ir | |
| ATP + L-glutamate | Streptomyces viridochromogenes Tü494 | - |
diphosphate + (L-alanyl)adenylate | - |
ir | |
| ATP + L-glutamate + holo-[L-alanyl-carrier protein] | Streptomyces viridochromogenes | - |
AMP + diphosphate + L-alanyl-[L-alanyl-carrier protein] | - |
ir | |
| ATP + L-glutamate + holo-[L-alanyl-carrier protein] | Streptomyces viridochromogenes Tü494 | - |
AMP + diphosphate + L-alanyl-[L-alanyl-carrier protein] | - |
ir | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Streptomyces viridochromogenes | Q5IW58 | - |
- |
| Streptomyces viridochromogenes | Q5IW60 | - |
- |
| Streptomyces viridochromogenes Tü494 | Q5IW58 | - |
- |
| Streptomyces viridochromogenes Tü494 | Q5IW60 | - |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| native enzyme from mycelium by ammonium sulfate fractionation, gel filtration, and anion exchange chromatography | Streptomyces viridochromogenes |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| (L-alanyl)adenylate + holo-[L-alanyl-carrier protein] | - |
Streptomyces viridochromogenes | AMP + L-alanyl-[L-alanyl-carrier protein] | - |
ir | |
| (L-alanyl)adenylate + holo-[L-alanyl-carrier protein] | - |
Streptomyces viridochromogenes Tü494 | AMP + L-alanyl-[L-alanyl-carrier protein] | - |
ir | |
| ATP + L-glutamate | - |
Streptomyces viridochromogenes | diphosphate + (L-alanyl)adenylate | - |
ir | |
| ATP + L-glutamate | - |
Streptomyces viridochromogenes Tü494 | diphosphate + (L-alanyl)adenylate | - |
ir | |
| ATP + L-glutamate + holo-[L-alanyl-carrier protein] | - |
Streptomyces viridochromogenes | AMP + diphosphate + L-alanyl-[L-alanyl-carrier protein] | - |
ir | |
| ATP + L-glutamate + holo-[L-alanyl-carrier protein] | - |
Streptomyces viridochromogenes Tü494 | AMP + diphosphate + L-alanyl-[L-alanyl-carrier protein] | - |
ir | |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| alanine thioester-forming activity | - |
Streptomyces viridochromogenes |
| peptide synthetase PhsB | UniProt | Streptomyces viridochromogenes |
| peptide synthetase PhsC | UniProt | Streptomyces viridochromogenes |
| phosphinothricin tripeptide synthetase | - |
Streptomyces viridochromogenes |
| phosphinothricin tripeptide synthetase III | UniProt | Streptomyces viridochromogenes |
| phsB | - |
Streptomyces viridochromogenes |
| PhsC | - |
Streptomyces viridochromogenes |
| PTT synthetase II | - |
Streptomyces viridochromogenes |
| PTT synthetase III | - |
Streptomyces viridochromogenes |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 30 | - |
assay at | Streptomyces viridochromogenes |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| 4'-phosphopantetheine | - |
Streptomyces viridochromogenes | |
| ATP | - |
Streptomyces viridochromogenes | |
General Information
| General Information | Comment | Organism |
|---|---|---|
| metabolism | the tripeptide backbone of phosphinothricin (PT) tripeptide (PTT), a compound with herbicidal activity from Streptomyces viridochromogenes, is assembled by three stand-alone peptide synthetase modules. The enzyme PhsA (66 kDa) recruits the PT-precursor N-acetyl-demethylphosphinothricin (N-Ac-DMPT), whereas the two alanine residues of PTT are assembled by the enzymes PhsB and PhsC (129 and 119 kDa, respectively). During or after assembly, the N-Ac-DMPT residue in the peptide is converted to PT by methylation and deacetylation. Both phsB and phsC appear to be cotranscribed together with two other genes from a single promoter and they are located at a distance of 20 kb from the gene phsA, encoding PhsA, in the PTT biosynthesis gene cluster of Streptomyces viridochromogenes | Streptomyces viridochromogenes |
| physiological function | the tripeptide backbone of phosphinothricin (PT) tripeptide (PTT), a compound with herbicidal activity from Streptomyces viridochromogenes, is assembled by three stand-alone peptide synthetase modules. The enzyme PhsA (66 kDa) recruits the PT-precursor N-acetyl-demethylphosphinothricin (N-Ac-DMPT), whereas the two alanine residues of PTT are assembled by the enzymes PhsB and PhsC (129 and 119 kDa, respectively). During or after assembly, the N-Ac-DMPT residue in the peptide is converted to PT by methylation and deacetylation. PhsB and PhsC represent single nonribosomal peptide synthetase elongation modules lacking a thioesterase domain | Streptomyces viridochromogenes |
| physiological function | the tripeptide backbone of phosphinothricin (PT) tripeptide (PTT), a compound with herbicidal activity from Streptomyces viridochromogenes, is assembled by three stand-alone peptide synthetase modules. The enzyme PhsA (66 kDa) recruits the PT-precursor N-acetyl-demethylphosphinothricin (N-Ac-DMPT), whereas the two alanine residues of PTT are assembled by the enzymes PhsB and PhsC (129 and 119 kDa, respectively). During or after assembly, the N-Ac-DMPT residue in the peptide is converted to PT by methylation and deacetylation. PhsB and PhsC represent single nonribosomal peptide synthetase elongation modules lacking a thioesterase domain. Gene inactivations, genetic complementations, determinations of substrate specificity of the heterologously produced proteins, and comparison of PhsC sequence with the N-terminus of the alanine-activating nonribosomal peptide synthetase PTTSII from Streptomyces viridochromogenes confirm the role of the two genes in the bialanylation of Ac-DMPT. The lack of an integral thioesterase domain in the PTT assembly system points to product release possibly involving two type II thioesterase genes (the1 and the2) located in the PTT gene cluster alone or in conjunction with another mechanism of product release | Streptomyces viridochromogenes |
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