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Literature summary for 6.3.2.B16 extracted from

  • Kino, K.; Arai, T.; Tateiwa, D.
    A novel L-amino acid ligase from Bacillus subtilis NBRC3134 catalyzed oligopeptide synthesis (2010), Biosci. Biotechnol. Biochem., 74, 129-134.
    View publication on PubMed
Search for more literature for 6.3.2.B16

Cloned(Commentary)

Cloned (Comment) Organism
expression in Escherichia coli as His-tagged protein Bacillus subtilis
gene rizB, two splicing variants, rizB-P1 and rizB-P2, transcriptional analysis and DNA and amino acid sequence determination and analysis, sequence comparison, recombinant overexpression of the His-tagged enzyme in Escherichia coli strain BL21(DE3) Bacillus subtilis

Metals/Ions

Metals/Ions Comment Organism Structure
Co2+ required. Mg2+ can be replaced with Mn2+ and Co2+. The use of Mn2+ or Co2+ decreases activity and the maximum length of peptide synthesized becomes shorter Bacillus subtilis
Co2+ can replace Mg2+, but with lower activity Bacillus subtilis
Mg2+ required. Mg2+ can be replaced with Mn2+ and Co2+. The use of Mn2+ or Co2+ decreases activity and the maximum length of peptide synthesized becomes shorter Bacillus subtilis
Mg2+ required, best metal cofactor, can be replaced by Mn2+ or Co2+ Bacillus subtilis
Mn2+ required. Mg2+ can be replaced with Mn2+ and Co2+. The use of Mn2+ or Co2+ decreases activity and the maximum length of peptide synthesized becomes shorter Bacillus subtilis
Mn2+ can replace Mg2+, but with lower activity Bacillus subtilis
additional information in the reaction with Val, tetramers are the longest products synthesized, trimer are formed with Co2+ Bacillus subtilis

Organism

Organism UniProt Comment Textmining
Bacillus subtilis C0STU2
-
-
Bacillus subtilis C0STU2 gene rizB, two variants P1 and P2
-
Bacillus subtilis NBRC3134 C0STU2
-
-
Bacillus subtilis NBRC3134 C0STU2 gene rizB, two variants P1 and P2
-

Purification (Commentary)

Purification (Comment) Organism
-
 Bacillus subtilis
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration Bacillus subtilis

Reaction

Reaction Comment Organism Reaction ID
2 ATP + L-valine + 2 L-Baa = 2 ADP + 2 phosphate + L-Val-(L-Baa)2 Baa: Leu, Ile, Met Bacillus subtilis
a
3 ATP + 3 L-valine + L-Zaa = 3 ADP + 3 phosphate + L-Val-L-Val-L-Val-L-amino-acid-L-Zaa Zaa: Arg, His, Gln, Asn or Phe Bacillus subtilis
a
ATP + 2 L-valine + L-Yaa = 2 ADP + 2 phosphate + L-Val-L-Val-L-Yaa Yaa: Arg, Lys, His, Gln, Asn, Ala, Ser, Thr, Gly, Leu, Ile, Met, Phe or Trp Bacillus subtilis
a
n-1 ATP + n L-Xaa = n-1 ADP + n-1 phosphate + (Xaa)n Xaa: Val, Leu, or Met. n = 2-5 Bacillus subtilis
a
n-1 ATP + n L-Xaa = n-1 ADP + n-1 phosphate + (Xaa)n the enzyme catalyzes the synthesis of oligopeptides from unprotected L-amino acids in an ATP-dependent manner, showing synthesis of various oligopeptides consisting of two to five amino acids, e.g. synthesis of Val-Val-Val tripeptide by the enzyme RizB. The enzyme shows highest activity with Val, Ile, Leu, and Met as substrates. RizB shows specificity toward N-terminal substrates, but is relaxed in the specificity for the C-terminus Bacillus subtilis
a

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2 ATP + 2 L-Val + Gly
-
 Bacillus subtilis 2 ADP + 2 phosphate + L-Val-L-Val-Gly
-
 ?
2 ATP + 2 L-Val + L-Ala
-
 Bacillus subtilis 2 ADP + 2 phosphate + L-Val-L-Val-L-Ala
-
 ?
2 ATP + 2 L-Val + L-Ala
-
 Bacillus subtilis NBRC3134 2 ADP + 2 phosphate + L-Val-L-Val-L-Ala
-
 ?
2 ATP + 2 L-Val + L-Arg
-
 Bacillus subtilis 2 ADP + 2 phosphate + L-Val-L-Val-L-Arg
-
 ?
2 ATP + 2 L-Val + L-Arg
-
 Bacillus subtilis NBRC3134 2 ADP + 2 phosphate + L-Val-L-Val-L-Arg
-
 ?
2 ATP + 2 L-Val + L-Asn
-
 Bacillus subtilis 2 ADP + 2 phosphate + L-Val-L-Val-L-Asn
-
 ?
2 ATP + 2 L-Val + L-Gln
-
 Bacillus subtilis 2 ADP + 2 phosphate + L-Val-L-Val-L-Gln
-
 ?
2 ATP + 2 L-Val + L-His
-
 Bacillus subtilis 2 ADP + 2 phosphate + L-Val-L-Val-L-His
-
 ?
2 ATP + 2 L-Val + L-Ile
-
 Bacillus subtilis 2 ADP + 2 phosphate + L-Val-L-Val-L-Ile
-
 ?
2 ATP + 2 L-Val + L-Leu
-
 Bacillus subtilis 2 ADP + 2 phosphate + L-Val-L-Val-L-Leu
-
 ?
2 ATP + 2 L-Val + L-Lys
-
 Bacillus subtilis 2 ADP + 2 phosphate + L-Val-L-Val-L-Lys
-
 ?
2 ATP + 2 L-Val + L-Met
-
 Bacillus subtilis 2 ADP + 2 phosphate + L-Val-L-Val-L-Met
-
 ?
2 ATP + 2 L-Val + L-Phe
-
 Bacillus subtilis 2 ADP + 2 phosphate + L-Val-L-Val-L-Phe
-
 ?
2 ATP + 2 L-Val + L-Ser
-
 Bacillus subtilis 2 ADP + 2 phosphate + L-Val-L-Val-L-Ser
-
 ?
2 ATP + 2 L-Val + L-Thr
-
 Bacillus subtilis 2 ADP + 2 phosphate + L-Val-L-Val-L-Thr
-
 ?
2 ATP + 2 L-Val + L-Trp
-
 Bacillus subtilis 2 ADP + 2 phosphate + L-Val-L-Val-L-Trp
-
 ?
2 ATP + 3 L-leucine the enzyme synthesizes homo-oligomers of two to five amino acids. ATP cannot by replaced with GTP, CTP or dTTP Bacillus subtilis 2 ADP + 2 phosphate + L-Leu-L-Leu-L-Leu
-
 ?
2 ATP + 3 L-methionine the enzyme synthesizes homo-oligomers of two to five amino acids. ATP cannot by replaced with GTP, CTP or dTTP Bacillus subtilis 2 ADP + 2 phosphate + L-Met-L-Met-L-Met
-
 ?
2 ATP + 3 L-valine the enzyme synthesizes homo-oligomers of two to five amino acids. ATP cannot by replaced with GTP, CTP or dTTP Bacillus subtilis 2 ADP + 2 phosphate + L-Val-L-Val-L-Val
-
 ?
2 ATP + L-Val + 2 L-Leu
-
 Bacillus subtilis 2 ADP + 2 phosphate + L-Val-L-Leu-L-Leu
-
 ?
2 ATP + L-Val + 2 L-Met
-
 Bacillus subtilis 2 ADP + 2 phosphate + L-Met-L-Met-L-Met
-
 ?
2 ATP + L-Val + L-Arg-L-Ser no homopeptides of Val-Val and Val-Val-Val are detected. This suggests that the enzyme preferentially uses dipeptides as the C-terminal substrate Bacillus subtilis 2 ADP + phosphate + L-Val-L-Arg-L-Ser
-
 ?
2 ATP + L-Val + L-Val + L-Arg-L-Ser no homopeptides of Val-Val and Val-Val-Val are detected. This suggests that the enzyme preferentially uses dipeptides as the C-terminal substrate Bacillus subtilis 2 ADP + 2 phosphate + L-Val-L-Val-L-Arg-L-Ser
-
 ?
2 ATP + Val + 2 L-Ile
-
 Bacillus subtilis 2 ADP + 2 phosphate + L-Val-L-Ile-L-Ile
-
 ?
3 ATP + 3 L-Val + L-Arg
-
 Bacillus subtilis 3 ADP + 3 phosphate + L-Val-L-Val-L-Val-L-Arg
-
 ?
3 ATP + 3 L-Val + L-Gln
-
 Bacillus subtilis 3 ADP + 3 phosphate + L-Val-L-Val-L-Val-L-Gln
-
 ?
3 ATP + 3 L-Val + L-His
-
 Bacillus subtilis 3 ADP + 3 phosphate + L-Val-L-Val-L-Val-L-His
-
 ?
3 ATP + 3 L-Val + L-Phe
-
 Bacillus subtilis 3 ADP + 3 phosphate + L-Val-L-Val-L-Val-L-Phe
-
 ?
3 ATP + 4 L-leucine the enzyme synthesizes homo-oligomers of two to five amino acids. ATP cannot by replaced with GTP, CTP or dTTP Bacillus subtilis 3 ADP + 3 phosphate + L-Leu-L-Leu-L-Leu-L-Leu
-
 ?
3 ATP + 4 L-methionine the enzyme synthesizes homo-oligomers of two to five amino acids. ATP cannot by replaced with GTP, CTP or dTTP Bacillus subtilis 3 ADP + 3 phosphate + L-Met-L-Met-L-Met-L-Met
-
 ?
3 ATP + 4 L-valine the enzyme synthesizes homo-oligomers of two to five amino acids. ATP cannot by replaced with GTP, CTP or dTTP Bacillus subtilis 3 ADP + 3 phosphate + L-Val-L-Val-L-Val-L-Val
-
 ?
4 ATP + 5 L-leucine the enzyme synthesizes homo-oligomers of two to five amino acids. ATP cannot by replaced with GTP, CTP or dTTP Bacillus subtilis 4 ADP + 4 phosphate + L-Leu-L-Leu-L-Leu-L-Leu-L-Leu
-
 ?
4 ATP + 5 L-methionine the enzyme synthesizes homo-oligomers of two to five amino acids. ATP cannot by replaced with GTP, CTP or dTTP Bacillus subtilis 4 ADP + 4 phosphate + L-Met-L-Met-L-Met-L-Met-L-Met
-
 ?
4 ATP + 5 L-valine the enzyme synthesizes homo-oligomers of two to five amino acids. ATP cannot by replaced with GTP, CTP or dTTP Bacillus subtilis 4 ADP + 4 phosphate + L-Val-L-Val-L-Val-L-Val-L-Val
-
 ?
ATP + 2 L-leucine the enzyme synthesizes homo-oligomers of two to five amino acids. ATP cannot by replaced with GTP, CTP or dTTP Bacillus subtilis ADP + phosphate + L-Leu-L-Leu
-
 ?
ATP + 2 L-methionine the enzyme synthesizes homo-oligomers of two to five amino acids. ATP cannot by replaced with GTP, CTP or dTTP Bacillus subtilis ADP + phosphate + L-Met-L-Met
-
 ?
ATP + 2 L-valine the enzyme synthesizes homo-oligomers of two to five amino acids. ATP cannot by replaced with GTP, CTP or dTTP Bacillus subtilis ADP + phosphate + L-Val-L-Val
-
 ?
ATP + L-Val + L-Arg
-
 Bacillus subtilis ADP + phosphate + L-Val-L-Arg
-
 ?
ATP + L-Val + L-Arg-L-Ser no homopeptides of Val-Val and Val-Val-Val are detected. The enzyme preferentially uses dipeptides as the C-terminal substrates Bacillus subtilis ADP + phosphate + L-Val-L-Arg-L-Ser + L-Val-L-Val-L-Arg-L-Ser
-
 ?
ATP + L-Val + L-Arg-NHOH
-
 Bacillus subtilis ADP + phosphate + L-Val-L-Arg-NHOH + L-Val-L-Val-L-Arg-NHOH
-
 ?
ATP + L-Val + L-Phe-NHOH
-
 Bacillus subtilis ADP + phosphate + L-Val-L-Phe-NHOH + L-Val-L-Val-L-Phe-NHOH
-
 ?
additional information dipeptides, tripeptides, tetrapeptides and pentapeptides are synthesized in the reaction mixtures containing Val, Leu, or Met as substrate. When Ile is used, the longest peptides are tetrapeptides. No peptides are detected when the other 15 amino acids are used as substrates. When Leu, Ile, or Met is used as substrate with Val, heteropeptides containing two residues of Leu, Ile, and Met are detected. No heteropeptides containing Tyr. Pro, Cys, Asp or Glu are detected. When other amino acids that are not usable for homopeptide synthesis are used as substrate, heteropeptides containing only one amino acid residue of these amino acids are detected. Substrate recognition at the N-terminus influences the resulting heteropeptide sequence. The enzyme does not participate in peptide-to-peptide ligation. Very low activity with D-amino acids Bacillus subtilis ?
-
 ?
additional information the enzyme catalyzes the synthesis of oligopeptides from unprotected L-amino acids in an ATP-dependent manner. Synthesis of various oligopeptides consisting of two to five amino acids, e.g. synthesis of Val-Val-Val tripeptide by the enzyme RizB. The enzyme shows highest activity with Val, Ile, Leu, and Met as substrates. In the reaction with Val, tetramers are the longest products synthesized, trimer are formed with Co2+. RizB shows specificity toward N-terminal substrates, but is relaxed in the specificity for the C-terminus, mass spectrometric reaction analysis, overview Bacillus subtilis ?
-
 ?
additional information dipeptides, tripeptides, tetrapeptides and pentapeptides are synthesized in the reaction mixtures containing Val, Leu, or Met as substrate. When Ile is used, the longest peptides are tetrapeptides. No peptides are detected when the other 15 amino acids are used as substrates. When Leu, Ile, or Met is used as substrate with Val, heteropeptides containing two residues of Leu, Ile, and Met are detected. No heteropeptides containing Tyr. Pro, Cys, Asp or Glu are detected. When other amino acids that are not usable for homopeptide synthesis are used as substrate, heteropeptides containing only one amino acid residue of these amino acids are detected. Substrate recognition at the N-terminus influences the resulting heteropeptide sequence. The enzyme does not participate in peptide-to-peptide ligation. Very low activity with D-amino acids Bacillus subtilis NBRC3134 ?
-
 ?
additional information the enzyme catalyzes the synthesis of oligopeptides from unprotected L-amino acids in an ATP-dependent manner. Synthesis of various oligopeptides consisting of two to five amino acids, e.g. synthesis of Val-Val-Val tripeptide by the enzyme RizB. The enzyme shows highest activity with Val, Ile, Leu, and Met as substrates. In the reaction with Val, tetramers are the longest products synthesized, trimer are formed with Co2+. RizB shows specificity toward N-terminal substrates, but is relaxed in the specificity for the C-terminus, mass spectrometric reaction analysis, overview Bacillus subtilis NBRC3134 ?
-
 ?

Synonyms

Synonyms Comment Organism
oligomer L-amino acid ligase
-
 Bacillus subtilis
oligopeptide ligase
-
 Bacillus subtilis
rizB
-
 Bacillus subtilis

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
 assay at Bacillus subtilis
40
-
-
 Bacillus subtilis
40
-
 recombinant enzyme Bacillus subtilis

Temperature Range [°C]

Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
25 45 activity range, recombinant enzyme, profile overview Bacillus subtilis
30 45 30°C: about 35% of maximal activity, 45°C: about 80% of maximal activity Bacillus subtilis

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
 assay at Bacillus subtilis
9
-
 about, recombinant enzyme Bacillus subtilis
9 9.5
-
 Bacillus subtilis

pH Range

pH Minimum pH Maximum Comment Organism
7.5 9.5 activity range, recombinant enzyme, profile overview Bacillus subtilis
8 10 pH 8.0: about 50% of maximal activity, significant decrease in activity above pH 10.0 Bacillus subtilis

Cofactor

Cofactor Comment Organism Structure
ATP dependent on, cannot be replaced by GTP, CTP, or TTP Bacillus subtilis

General Information

General Information Comment Organism
evolution the enzyme RizB shows high sequence homology to protein BL02410, UniProt ID Q65P25, from Bacillus licheniformis. In contrast to the other L-amino acid ligases, e.g. RizA and YwfE, RizB catalyzes the formation of oligomers from L-amino acids Bacillus subtilis
metabolism the enzyme might be involved in the rhizocticin biosynthesis Bacillus subtilis
physiological function the enzyme is involved in rhizocticin biosynthesis Bacillus subtilis