1HJG

Alteration of the co-substrate selectivity of deacetoxycephalosporin C synthase: The role of arginine-258

Summary for 1HJG

• Entry DOI: 10.2210/pdb1hjg/pdb
• Related: 1DCS 1E5H 1E5I 1HJF 1RXF 1RXG
• Descriptor: DEACETOXYCEPHALOSPORIN C SYNTHASE, FE (II) ION, 3-METHYL-2-OXOBUTANOIC ACID, ... (4 entities in total)
• Functional Keywords: oxidoreductase, alternative 2-oxoacids, cephem antibiotic biosynthesis, chemical cosubstrate rescue, co-substrate selectivity, 2- oxoglutarate-dependent oxygenase
• Biological source: STREPTOMYCES CLAVULIGERUS
• Total number of polymer chains: 1
• Total formula weight: 34734.53

Authors

Lee, H.J.,Lloyd, M.D.,Clifton, I.J.,Harlos, K.,Dubus, A.,Baldwin, J.E.,Frere, J.M.,Schofield, C.J. (deposition date: 2001-01-15, release date: 2001-06-01, Last modification date: 2023-12-13)

Primary citation

Lee, H.J.,Lloyd, M.D.,Harlos, K.,Clifton, I.J.,Baldwin, J.E.,Schofield, C.J.
Alteration of the 2-Oxoacid Cosubstrate Selectivity in Deacetoxycephalosporin C Synthase: The Role of Arginine-258
J.Biol.Chem., 276:18290-, 2001

PubMed Abstract: Deacetoxycephalosporin C synthase is an iron(II) 2-oxoglutaratedependent oxygenase that catalyzes the oxidative ring-expansion of penicillin N to deacetoxycephalosporin C. The wild-type enzyme is only able to efficiently utilize 2-oxoglutarate and 2-oxoadipate as a 2-oxoacid co-substrate. Mutation of arginine 258, the side chain of which forms an electrostatic interaction with the 5-carboxylate of the 2-oxoglutarate co-substrate, to a glutamine residue reduced activity to about 5% of the wild-type enzyme with 2-oxoglutarate. However, other aliphatic 2-oxoacids, which were not co-substrates for the wild-type enzyme, were utilized by the R258Q mutant. These 2-oxoacids "rescued" catalytic activity to the level observed for the wild-type enzyme as judged by penicillin N and G conversion. These co-substrates underwent oxidative decarboxylation as observed for 2-oxoglutarate in the normal reaction with the wild-type enzyme. Crystal structures of the iron(II)- 2-oxo-3-methylbutanoate (1.5 A), and iron(II)-2-oxo-4-methylpentanoate (1.6 A) enzyme complexes were obtained, which reveal the molecular basis for this "chemical co-substrate rescue" and help to rationalize the co-substrate selectivity of 2-oxoglutaratedependent oxygenases.

PubMed: 11279000
DOI: 10.1074/JBC.M100085200

Experimental method

X-RAY DIFFRACTION (1.5 Å)