Molecular insight into substrate recognition and catalysis of Baeyer-Villiger monooxygenase MtmOIV, the key frame-modifying enzyme in the biosynthesis of anticancer agent mithramycin.

Baeyer-Villiger monooxygenases (BVMOs) have been shown to play key roles for the biosynthesis of important natural products. MtmOIV, a homodimeric FAD- and NADPH-dependent BVMO, catalyzes the key frame-modifying steps of the mithramycin biosynthetic pathway, including an oxidative C-C bond cleavage, by converting its natural substrate premithramycin B into mithramycin DK, the immediate precursor of mithramycin. The drastically improved protein structure of MtmOIV along with the high-resolution structure of MtmOIV in complex with its natural substrate premithramycin B are reported here, revealing previously undetected key residues that are important for substrate recognition and catalysis.

The crystal structure and mechanism of an unusual oxidoreductase, GilR, involved in gilvocarcin V biosynthesis.

GilR is a recently identified oxidoreductase that catalyzes the terminal step of gilvocarcin V biosynthesis and is a unique enzyme that establishes the lactone core of the polyketide-derived gilvocarcin chromophore. Gilvocarcin-type compounds form a small distinct family of anticancer agents that are involved in both photo-activated DNA-alkylation and histone H3 cross-linking. High resolution crystal structures of apoGilR and GilR in complex with its substrate pregilvocarcin V reveals that GilR belongs to the small group of a relatively new type of the vanillyl-alcohol oxidase flavoprotein family characterized by bicovalently tethered cofactors.

Characterization of the terminal activation step catalyzed by oxygenase CmmOIV of the chromomycin biosynthetic pathway from Streptomyces griseus.

Inactivation and initial interrogation of key oxygenase CmmOIV of the biosynthetic pathway of chromomycin A(3) in Streptomyces griseus ssp. griseus revealed that a completely methylated and acetylated prechromomycin is the preferred substrate of this enzyme. This suggests that the three sugar decoration reactions, two O-acetylations and an O-methylation, which were previously believed to occur as the final steps of chromomycin A(3) biosynthesis, indeed take place prior to the CmmOIV reaction.

Isolation of Streptomyces species from soil.

This unit describes a general protocol for the isolation of Streptomyces species from soil and fresh water, using a procedure for the selective growth of Streptomyces species. Preparation of the necessary growth medium, recognition of the morphology of the bacteria, and safety considerations are also covered.

Genetic manipulation of Streptomyces species.

This unit includes general protocols for the genetic manipulation of Streptomyces species, including genomic DNA isolation, genomic library preparation, intergeneric conjugation of Streptomyces with E. coli, generation and transformation of Streptomyces protoplasts, electroporation of Streptomyces mycelia, and colony PCR.

Laboratory maintenance of Streptomyces argillaceus and Streptomyces griseus.

This unit describes general protocols for the laboratory maintenance of Streptomyces argillaceus and griseus, including growth on solid and liquid media, as well as specific considerations for the type of medium to be used with these species.

Laboratory maintenance of Streptomyces species.

This unit includes general protocols for the laboratory maintenance of Streptomyces species, including growth in liquid media, growth on solid agar, and short- and long-term storage. Considerations for the handling of Streptomyces species and the morphology of the bacteria are also reviewed.

Crystal structure of Baeyer-Villiger monooxygenase MtmOIV, the key enzyme of the mithramycin biosynthetic pathway .

Baeyer-Villiger monooxygenases (BVMOs), mostly flavoproteins, were shown to be powerful biocatalysts for synthetic organic chemistry applications and were also suggested to play key roles for the biosyntheses of various natural products. Here we present the three-dimensional structure of MtmOIV, a 56 kDa homodimeric FAD- and NADPH-dependent monooxygenase, which catalyzes the key frame-modifying step of the mithramycin biosynthetic pathway and currently the only BVMO proven to react with its natural substrate via a Baeyer-Villiger reaction. MtmOIV's structure was determined by X-ray crystallography using molecular replacement to a resolution of 2.

Loop202-208 in avian sarcoma virus integrase mediates tetramer assembly and processing activity.

Integrase (IN) catalyzes insertion of the retroviral genome into the host via two sequential reactions. The processing activity cleaves the 3'-dinucleotides from the two ends of the viral DNA which are then inserted into the host DNA. Tetramers are required for the joining step.