Purification and characterization of clavaminate synthase from Streptomyces antibioticus. A multifunctional enzyme of clavam biosynthesis.
Clavaminate synthase (CS), a key enzyme in the clavulanic acid biosynthetic pathway, has been purified to electrophoretic homogeneity from Streptomyces antibioticus (Tü 1718), a species that does not produce clavulanic acid. A comparison of the physical and kinetic properties of clavaminate synthase from S. antibioticus (CS3) and the two isozymes from Streptomyces clavuligerus (CS1 and CS2) has been conducted. In oxidative reactions requiring the co-substrates O2, alpha-ketoglutaric acid, and catalytic Fe2+, both CS1 and CS2 catalyze three distinct transformations, the hydroxylation of deoxyguanidinoproclavaminic acid to guanidinoproclavaminic acid, and the cyclization and desaturation of proclavaminic acid to clavaminic acid. We have demonstrated that CS3 from S. antibioticus also catalyzes these three oxidations. The apparent molecular mass of CS3 from matrix-assisted laser desorption mass spectrometry is 35,839 +/- 36 Da. The enzyme is a monomer in solution as determined by gel filtration chromatography. Analysis of the four possible proclavaminic acid diastereomers confirmed the absolute configuration of the substrate to be 2S,3R. Based upon N-terminal sequence comparisons among the three proteins, CS3 possesses the higher degree of homology with the CS1 isozyme from S. clavuligerus. Although previously associated solely with clavulanic acid biosynthesis, we propose these findings and recent precursor incorporation data support the view that clavaminate synthase plays a critical role in the biosynthesis of the clavam metabolites.
Janc
J W
JW
Department of Chemistry, Johns Hopkins University, Baltimore, Maryland 21218.
Egan
L A
LA
Townsend
C A
CA
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000096:biosynthesis; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D019818:Clavulanic Acid; D002969:Clavulanic Acids / Q000096:biosynthesis / Q000737:chemistry; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000201:enzymology; D013303:Streptomyces antibioticus / Q000201:enzymology*; D013379:Substrate Specificity
D000900:Anti-Bacterial Agents; D002969:Clavulanic Acids; D007527:Isoenzymes; D019818:Clavulanic Acid; D006899:Mixed Function Oxygenases; C066046:clavaminate synthase