Crystal Structure Of Enoylpyruvate Transferase In Streptococcus Pneumoniae

Two thirds of the clinically useful antibiotics are naturally produced in actinomycetes, especially in Streptomyces species.These antibiotics vary highly in their chemical structures, some examples being amphenicols (chloramphenicol), polyketides (tetracyclin) or aminoglycosides (streptomycin).Enzymes involved in the synthesis of aminoglycoside antibiotics (AGAs) are organized in large gene clusters containing 24 or more enzymes.The AGA family can be further divided into several subfamilies; the NEO subfamily takes the common precursor paromamine, which is further modified to AGAs such as neomycin, ribostamycin and lividomycin.[1] Lividomycin B and neomycin B -members of the NEO subfamily -are produced by enzymes of the LIV/NEO gene cluster.The aminotransferases that catalyze the terminal transamination reaction (LivB and NeoB respectively) utilise the cofactor pyridoxal-5'phosphate (PLP).LivB catalyzes the transamination reaction of 6'''oxoparomomycin to the antibiotic paromomycin, which is also a precursor of lividomycin B, whereas NeoB performs the transamination of 6'''-oxoneomycin C to neomycin C. [1] LivB and NeoB were expressed in Streptomyces sp., purified via Ni-affinity chromatography and crystallized.The structure of LivB could be solved using the "magic triangle compound" I3C [2] for SAD phasing, and that of NeoB by molecular replacement using the LivB structure as search model.Soaking of LivB crystals with the cofactor PLP, an amino donor and the end product paromomycin yielded crystal structures of the PLP-bound enzyme and the complex structure of LivB with an aldimine paromomycin-PLP intermediate.The latter represents a molecular snapshot of a key intermediate of the enzymatic reaction -the transamination at the 6''' position of the lividomycin B. These structures provide a basis for analyzing substrate specificities of other carbohydrate-modifying aminotransferases.