Regulation of Acetate Kinase Isozymes and Its Importance for MixedAcid Fermentation in Lactococcus lactis

P. Puri, A. Goel, A. Bochynska, B. Poolman

Research output: Contribution to journalArticleAcademicpeer-review

10 Citations (Scopus)

Abstract

Acetate kinase (ACK) converts acetyl phosphate to acetate along with the generation of ATP in the pathway for mixed-acid fermentation in Lactococcus lactis. The reverse reaction yields acetyl phosphate for assimilation purposes. Remarkably, L. lactis has two ACK isozymes, and the corresponding genes are present in an operon. We purified both enzymes (AckA1 and AckA2) from L. lactis MG1363 and determined their oligomeric state, specific activities, and allosteric regulation. Both proteins form homodimeric complexes, as shown by size exclusion chromatography and static light-scattering measurements. The turnover number of AckA1 is about an order of magnitude higher than that of AckA2 for the reaction in either direction. The K-m values for acetyl phosphate, ATP, and ADP are similar for both enzymes. However, AckA2 has a higher affinity for acetate than does AckA1, suggesting an important role under acetate-limiting conditions despite the lower activity. Fructose-1,6-bisphosphate, glyceraldehyde- 3-phosphate, and phospho-enol-pyruvate inhibit the activities of AckA1 and AckA2 to different extents. The allosteric regulation of AckA1 and AckA2 and the pool sizes of the glycolytic intermediates are consistent with a switch from homolactic to mixed-acid fermentation upon slowing of the growth rate.
Original languageEnglish
Pages (from-to)1386-1393
JournalJournal of Bacteriology
Volume196
Issue number7
DOIs
Publication statusPublished - 2014

Fingerprint

Acetate Kinase
Lactococcus lactis
Allosteric Regulation
Isoenzymes
Fermentation
Acetates
Glyceraldehyde 3-Phosphate
Acids
Enzymes
Operon
Pyruvic Acid
Adenosine Diphosphate
Gel Chromatography
Adenosine Triphosphate
Light
Growth
Genes
acetyl phosphate
Proteins

Keywords

  • streptococcus-lactis
  • escherichia-coli
  • methanosarcina-thermophila
  • lysine acetylation
  • membrane-proteins
  • product formation
  • light-scattering
  • in-vivo
  • metabolism
  • phosphate

Cite this

Puri, P. ; Goel, A. ; Bochynska, A. ; Poolman, B. / Regulation of Acetate Kinase Isozymes and Its Importance for MixedAcid Fermentation in Lactococcus lactis. In: Journal of Bacteriology. 2014 ; Vol. 196, No. 7. pp. 1386-1393.
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Regulation of Acetate Kinase Isozymes and Its Importance for MixedAcid Fermentation in Lactococcus lactis. / Puri, P.; Goel, A.; Bochynska, A.; Poolman, B.

In: Journal of Bacteriology, Vol. 196, No. 7, 2014, p. 1386-1393.

Research output: Contribution to journalArticleAcademicpeer-review

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T1 - Regulation of Acetate Kinase Isozymes and Its Importance for MixedAcid Fermentation in Lactococcus lactis

AU - Puri, P.

AU - Goel, A.

AU - Bochynska, A.

AU - Poolman, B.

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N2 - Acetate kinase (ACK) converts acetyl phosphate to acetate along with the generation of ATP in the pathway for mixed-acid fermentation in Lactococcus lactis. The reverse reaction yields acetyl phosphate for assimilation purposes. Remarkably, L. lactis has two ACK isozymes, and the corresponding genes are present in an operon. We purified both enzymes (AckA1 and AckA2) from L. lactis MG1363 and determined their oligomeric state, specific activities, and allosteric regulation. Both proteins form homodimeric complexes, as shown by size exclusion chromatography and static light-scattering measurements. The turnover number of AckA1 is about an order of magnitude higher than that of AckA2 for the reaction in either direction. The K-m values for acetyl phosphate, ATP, and ADP are similar for both enzymes. However, AckA2 has a higher affinity for acetate than does AckA1, suggesting an important role under acetate-limiting conditions despite the lower activity. Fructose-1,6-bisphosphate, glyceraldehyde- 3-phosphate, and phospho-enol-pyruvate inhibit the activities of AckA1 and AckA2 to different extents. The allosteric regulation of AckA1 and AckA2 and the pool sizes of the glycolytic intermediates are consistent with a switch from homolactic to mixed-acid fermentation upon slowing of the growth rate.

AB - Acetate kinase (ACK) converts acetyl phosphate to acetate along with the generation of ATP in the pathway for mixed-acid fermentation in Lactococcus lactis. The reverse reaction yields acetyl phosphate for assimilation purposes. Remarkably, L. lactis has two ACK isozymes, and the corresponding genes are present in an operon. We purified both enzymes (AckA1 and AckA2) from L. lactis MG1363 and determined their oligomeric state, specific activities, and allosteric regulation. Both proteins form homodimeric complexes, as shown by size exclusion chromatography and static light-scattering measurements. The turnover number of AckA1 is about an order of magnitude higher than that of AckA2 for the reaction in either direction. The K-m values for acetyl phosphate, ATP, and ADP are similar for both enzymes. However, AckA2 has a higher affinity for acetate than does AckA1, suggesting an important role under acetate-limiting conditions despite the lower activity. Fructose-1,6-bisphosphate, glyceraldehyde- 3-phosphate, and phospho-enol-pyruvate inhibit the activities of AckA1 and AckA2 to different extents. The allosteric regulation of AckA1 and AckA2 and the pool sizes of the glycolytic intermediates are consistent with a switch from homolactic to mixed-acid fermentation upon slowing of the growth rate.

KW - streptococcus-lactis

KW - escherichia-coli

KW - methanosarcina-thermophila

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KW - product formation

KW - light-scattering

KW - in-vivo

KW - metabolism

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