4ICY

Tracing the Evolution of Angucyclinone Monooxygenases: Structural Determinants for C-12b Hydroxylation and Substrate Inhibition in PgaE

Summary for 4ICY

• Entry DOI: 10.2210/pdb4icy/pdb
• Related: 2QA1
• Descriptor: Polyketide oxygenase PgaE, FLAVIN-ADENINE DINUCLEOTIDE, 1,2-ETHANEDIOL, ... (7 entities in total)
• Functional Keywords: fad-dependent aromatic hydroxylation, monooxygenase, fad, oxidoreductase
• Biological source: Streptomyces sp. PGA64
• Total number of polymer chains: 1
• Total formula weight: 55104.60

Authors

Kallio, P.,Patrikainen, P.,Belogurov, G.,Mantsala, P.,Yang, K.,Niemi, J.,Metsa-Ketela, M. (deposition date: 2012-12-11, release date: 2013-06-12, Last modification date: 2024-04-03)

Primary citation

Kallio, P.,Patrikainen, P.,Belogurov, G.A.,Mantsala, P.,Yang, K.,Niemi, J.,Metsa-Ketela, M.
Tracing the Evolution of Angucyclinone Monooxygenases: Structural Determinants for C-12b Hydroxylation and Substrate Inhibition in PgaE.
Biochemistry, 52:4507-4516, 2013

PubMed Abstract: Two functionally distinct homologous flavoprotein hydroxylases, PgaE and JadH, have been identified as branching points in the biosynthesis of the polyketide antibiotics gaudimycin C and jadomycin A, respectively. These evolutionarily related enzymes are both bifunctional and able to catalyze the same initial reaction, C-12 hydroxylation of the common angucyclinone intermediate prejadomycin. The enzymes diverge in their secondary activities, which include hydroxylation at C-12b by PgaE and dehydration at C-4a/C-12b by JadH. A further difference is that the C-12 hydroxylation is subject to substrate inhibition only in PgaE. Here we have identified regions associated with the C-12b hydroxylation in PgaE by extensive chimeragenesis, focusing on regions surrounding the active site. The results highlight the importance of a hairpin-β motif near the dimer interface, with two nonconserved residues, P78 and I79 (corresponding to Q89 and F90, respectively, in JadH), and invariant residue H73 playing key roles. Kinetic characterization of PgaE variants demonstrates that the secondary C-12b hydroxylation and substrate inhibition by prejadomycin are likely to be interlinked. The crystal structure of the PgaE P78Q/I79F variant at 2.4 Å resolution confirms that the changes do not alter the conformation of the β-strand secondary structure and that the side chains of these residues in effect point away from the active site toward the dimer interface. The results support a catalytic model for PgaE containing two binding modes for C-12 and C-12b hydroxylations, where binding of prejadomycin in the orientation for C-12b hydroxylation leads to substrate inhibition. The presence of an allosteric network is evident based on enzyme kinetics.

PubMed: 23731237
DOI: 10.1021/bi400381s

Experimental method

X-RAY DIFFRACTION (2.4 Å)