Family of phenylacetyl-CoA monooxygenases differs in subunit organization from other monooxygenases.
The phenylacetate degradation pathway is present in a wide range of microbes. A key component of this pathway is the four-subunit phenylacetyl-coenzyme A monooxygenase complex (PA-CoA MO, PaaACBE) that catalyzes the insertion of an oxygen in the aromatic ring of PA. This multicomponent enzyme represents a new family of monooxygenases. We have previously determined the structure of the PaaAC subcomplex of catalytic (A) and structural (C) subunits and shown that PaaACB form a stable complex. The PaaB subunit is unrelated to the small subunits of homologous monooxygenases and its role and organization of the PaaACB complex is unknown. From low-resolution crystal structure, electron microscopy and small angle X-ray scattering we show that the PaaACB complex forms heterohexamers, with a homodimer of PaaB bridging two PaaAC heterodimers. Modeling the interactions of reductase subunit PaaE with PaaACB suggested that a unique and conserved 'lysine bridge' constellation near the Fe-binding site in the PaaA subunit (Lys68, Glu49, Glu72 and Asp126) may form part of the electron transfer path from PaaE to the iron center. The crystal structure of the PaaA(K68Q/E49Q)-PaaC is very similar to the wild-type enzyme structure, but when combined with the PaaE subunit the mutant showed 20-50 times reduced activity, supporting the functional importance of the 'lysine bridge'.
Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada.
Andrey M
AM
Grishin
Eunice
E
Ajamian
Limei
L
Tao
Mihnea
M
Bostina
Linhua
L
Zhang
Jean-Francois
JF
Trempe
Robert
R
Menard
Isabelle
I
Rouiller
Miroslaw
M
Cygler
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D020285:Cryoelectron Microscopy; D018360:Crystallography, X-Ray; D007711:Klebsiella pneumoniae / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry; D013869:Thiolester Hydrolases
D001426:Bacterial Proteins; D021122:Protein Subunits; D006899:Mixed Function Oxygenases; D013869:Thiolester Hydrolases; C022339:phenylacetyl Coenzyme A hydrolase
Aromatic compounds degradation; Monooxygenase; Phenylacetate degradation pathway; Quaternary structure