Literature summary for 4.99.1.9 extracted from

Dali, A.; Gabler, T.; Sebastiani, F.; Destinger, A.; Furtmueller, P.G.; Pfanzagl, V.; Becucci, M.; Smulevich, G.; Hofbauer, S.
Active site architecture of coproporphyrin ferrochelatase with its physiological substrate coproporphyrin III propionate interactions and porphyrin core deformation (2023), Protein Sci., 32, e4534.

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified wild-type and mutant R45L enzymes in apo and in substrate coproporphyrin III-bound forms, apoprotein-LmCpfC is dissolved in 50 mM HEPES, pH 7.4, and mixed with 0.0.05-0.010 mM cpIII in 50 mM HEPES, pH 7.4, the crystallization solution contains 15-25% PEG MME 2000, 0.1 M Bis-Tris, pH 5.8-6.3, and 0.1-0.4 M calcium acetate, mixing of 150 nl each. The LmCpfC R45L mutant in complex with cpIII is crystallized in 18% w/v PEG 8000 and 20% glycerol with a 1:1000 dilution of apo wild-type germination solution containing crushed apo wild-type crystals and 5 mM phosphate buffer, one drop contains 150 nl of 0.35 mM cpIII-R45L protein, 200 nl of mother liquor, and 30 nl of 1:1000 seed solution, X-ray diffraction structure determination and analysis at 1.51 A and 2.64 A resolution, respectively, molecular replacement using the apo-LmCpfC structure (PDB ID 6RWV) and refined to Rfree values of 0.1816 (cpIII-LmCpfC wild-type) and 0.2112 (cpIII-LmCpfC R45L) as templates	Listeria monocytogenes

Crystallization (Comment)

Organism

purified wild-type and mutant R45L enzymes in apo and in substrate coproporphyrin III-bound forms, apoprotein-LmCpfC is dissolved in 50 mM HEPES, pH 7.4, and mixed with 0.0.05-0.010 mM cpIII in 50 mM HEPES, pH 7.4, the crystallization solution contains 15-25% PEG MME 2000, 0.1 M Bis-Tris, pH 5.8-6.3, and 0.1-0.4 M calcium acetate, mixing of 150 nl each. The LmCpfC R45L mutant in complex with cpIII is crystallized in 18% w/v PEG 8000 and 20% glycerol with a 1:1000 dilution of apo wild-type germination solution containing crushed apo wild-type crystals and 5 mM phosphate buffer, one drop contains 150 nl of 0.35 mM cpIII-R45L protein, 200 nl of mother liquor, and 30 nl of 1:1000 seed solution, X-ray diffraction structure determination and analysis at 1.51 A and 2.64 A resolution, respectively, molecular replacement using the apo-LmCpfC structure (PDB ID 6RWV) and refined to Rfree values of 0.1816 (cpIII-LmCpfC wild-type) and 0.2112 (cpIII-LmCpfC R45L) as templates

Listeria monocytogenes

Protein Variants

Protein Variants	Comment	Organism
R45L	site-directed mutagenesis, the mutant lacks the H-bonding with the propionate at position 6 (p6), determination and analysis of the mutant enzyme structure as apo-enzyme and with bound substrate coproporphyrin III, structure comparison with the wild-type enzyme, overview	Listeria monocytogenes

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
coproporphyrin III + Fe2+	Listeria monocytogenes	-	Fe-coproporphyrin III + 2 H+	-	?

Organism

Organism	UniProt	Comment	Textmining
Listeria monocytogenes	A0A5D5VUB4	-	-

Purification (Commentary)

Purification (Comment)	Organism
the recombinant reconstituted cpIII-LmCpfC complex is purified to monodispersity by gel filtration	Listeria monocytogenes

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
coproporphyrin III + Fe2+	-	Listeria monocytogenes	Fe-coproporphyrin III + 2 H+	-	?

Synonyms

Synonyms	Comment	Organism
CpfC	-	Listeria monocytogenes

General Information

General Information	Comment	Organism
additional information	active site architecture of wild-type and R45L mutant coproporphyrin ferrochelatases with bound physiological substrate coproporphyrin III, crystal structure analysis, propionate interactions and porphyrin core deformation, detailed overview. A cleft, build by structural elements of both ferredoxin-like domains, contains several conserved amino acid residues (distal H182 and E263, proximal Y12) and is the porphyrin binding site, where catalysis happens. The monomeric enzyme exhibits two ferredoxin-like domains, each with a four-stranded parallel beta-sheet flanked by alpha-helices. In cpIII-LmCpfC, as well as in the coproheme-bound structure, the porphyrin is oriented in the active site with the propionates 2 and 4 (p2 and p4) pointing to the inner core of the protein, p6 and p7 face towards the protein surface and are much more solvent exposed. In both cpIII and coproheme complexes with LmCpfC the H-bond interactions between the four propionates and six amino acids, Thr14, Arg29, Arg45, Tyr46, Ser53, and Tyr124, are fundamental to the porphyrin stabilization	Listeria monocytogenes