Literature summary for 4.1.1.11 extracted from

Arnott, Z.L.P.; Nozaki, S.; Monteiro, D.C.F.; Morgan, H.E.; Pearson, A.R.; Niki, H.; Webb, M.E.
The mechanism of regulation of pantothenate biosynthesis by the PanD-PanZ-AcCoA complex reveals an additional mode of action for the antimetabolite N-pentyl pantothenamide (N5-Pan) (2017), Biochemistry, 56, 4931-4939 .

Activating Compound

Activating Compound	Comment	Organism	Structure
PanZ	the Escherichia coli enzyme requires the regulatroy factor PanZ for proteolytic cleavage of the zymogen to form the mature enzyme, the PanZ mutant N45A is unable to complement a panZ deletion strain. Complex formation of the site-directed mutants PanZ(R73A) and PanD(K119A) leads to a complex that still complements the beta-alanine auxotrophy of the DELTApanZ and DELTApanD strains, indicating that catalytically active PanD is formed, but no growth inhibition is observed as a result of PanZ overexpression	Escherichia coli

Application

Application	Comment	Organism
drug development	the PanDZ complex is a target for antibiotic development	Escherichia coli

Cloned(Commentary)

Cloned (Comment)	Organism
gene panD, recombinant overexpression of wild-type and mutant enzymes in Escherichia coli strain C41(DE3), subcloning in Escherichia coli strain MG1655. Overexpression-linked growth inhibition is dependent upon CoA-dependent interaction of PanZ with PanD	Escherichia coli

Crystallization (Commentary)

Crystallization (Comment)	Organism
co-crystallization of fully activated PanD and PanZ in complex, in a 10:11 PanD:PanZ ratio (protomer to monomer), hanging drop vapor diffusion method, mixing of 0.003 ml of 9 mg/ml protein in 50 mM Tris, 100 mM NaCl, and 0.1 mM DTT, pH 7.5, with 00.001 ml of reservoir solution containing 200 mM KSCN, 100 mM Bis-Tris propane, pH 6.5, and 20% w/v PEG 3350 at 18°C, X-ray diffraction structure determination and analysis at 1.16 A resolution. The same structure is observed using both room-temperature and cryo-cooled crystals, indicating that the hydrate is formed from the pyruvoyl cofactor and is not an intermediate in the activation reaction. This state is stabilized by a hydrogen bond to the amide of Gly24, which is held in place by interactions with PanZ	Escherichia coli

Crystallization (Comment)

Organism

co-crystallization of fully activated PanD and PanZ in complex, in a 10:11 PanD:PanZ ratio (protomer to monomer), hanging drop vapor diffusion method, mixing of 0.003 ml of 9 mg/ml protein in 50 mM Tris, 100 mM NaCl, and 0.1 mM DTT, pH 7.5, with 00.001 ml of reservoir solution containing 200 mM KSCN, 100 mM Bis-Tris propane, pH 6.5, and 20% w/v PEG 3350 at 18°C, X-ray diffraction structure determination and analysis at 1.16 A resolution. The same structure is observed using both room-temperature and cryo-cooled crystals, indicating that the hydrate is formed from the pyruvoyl cofactor and is not an intermediate in the activation reaction. This state is stabilized by a hydrogen bond to the amide of Gly24, which is held in place by interactions with PanZ

Escherichia coli

Protein Variants

Protein Variants	Comment	Organism
K115A	site-directed mutagenesis, the mutation is introduced in vitro by overlap extension PCR	Escherichia coli
K119A	site-directed mutagenesis, the mutation is introduced in vitro by overlap extension PCR. Complex formation of the site-directed mutants PanZ(R73A) and PanD(K119A) leads to a complex that still complements the beta-alanine auxotrophy of the DELTApanZ and DELTApanD strains, indicating that catalytically active PanD is formed, but no growth inhibition is observed as a result of PanZ overexpression	Escherichia coli
K14A	site-directed mutagenesis, the mutation is introduced in vitro by overlap extension PCR	Escherichia coli
K53A	site-directed mutagenesis, the mutation is introduced in vitro by overlap extension PCR	Escherichia coli

Inhibitors

Inhibitors	Comment	Organism	Structure
acetyl-CoA	CoA-dependent interaction of PanZ and PanD. Growth inhibition is due to the CoA-dependent PanD-PanZ interaction and the inhibition occurs at native concentrations of PanD and PanZ in the cell. The production of beta-alanine is feedback-regulated by the PanZ-AcCoA complex	Escherichia coli
pentyl pantothenamide	high-potency growth inhibition by pentyl pantothenamide is dependent upon the PanD-PanZ interaction. Substitution of the Escherichia coli panD for the noninteracting Bacillus panD leads to resistance against the inhibitor	Escherichia coli

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
L-aspartate	Escherichia coli	-	beta-alanine + CO2	-	?

Organism

Organism	UniProt	Comment	Textmining
Escherichia coli	-	-	-

Posttranslational Modification

Posttranslational Modification	Comment	Organism
additional information	the Escherichia coli enzyme requires the regulatroy factor PanZ for proteolytic cleavage of the zymogen to form the mature enzyme	Escherichia coli

Purification (Commentary)

Purification (Comment)	Organism
recombinant wild-type and mutant enzymes from Escherichia coli strain C41(DE3) by sequential immobilized metal affinity chromatography and gel filtration	Escherichia coli

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
L-aspartate	-	Escherichia coli	beta-alanine + CO2	-	?

Synonyms

Synonyms	Comment	Organism
ADC	-	Escherichia coli
PanD	-	Escherichia coli

Cofactor

Cofactor	Comment	Organism	Structure
pyruvoyl cofactor	-	Escherichia coli

General Information

General Information	Comment	Organism
malfunction	both regulatory protein PanZ overexpression-linked beta-alanine auxotrophy and pentyl pantothenamide toxicity are due to formation of the PanDZ complex between enzyme PanD and effector protein PanZ. Formation of such a complex between activated aspartate decarboxylase (PanD) and PanZ leads to sequestration of the pyruvoyl cofactor as a ketone hydrate and demonstrates that both PanZ overexpression-linked beta-alanine auxotrophy and pentyl pantothenamide toxicity are due to formation of this complex. Substitution of the Escherichia coli panD for the noninteracting Bacillus panD suppresses the phenotype	Escherichia coli
metabolism	enzyme PanD is responsible for the production of beta-alanine in the pantothenate biosynthesis pathway. The production of beta-alanine is feedback-regulated by the PanZ-AcCoA complex	Escherichia coli
additional information	PanD-PanZ complex three-dimensional structure analysis, overview	Escherichia coli
physiological function	the PanDZ complex regulates the pantothenate biosynthetic pathway in a cellular context in Escherichia coli by limiting the supply of beta-alanine in response to coenzyme A concentration. Formation of such a complex between activated aspartate decarboxylase (PanD) and regulatory protein PanZ leads to sequestration of the pyruvoyl cofactor as a ketone hydrate. Regulation of PanD is due to CoA-dependent interaction of PanZ and PanD	Escherichia coli