Kubiak R.L.,University of Wisconsin - Madison |
Phillips R.K.,University of Wisconsin - Madison |
Zmudka M.W.,University of Wisconsin - Madison |
Ahn M.R.,Edgewood Campus Middle School |
And 4 more authors.
Biochemistry | Year: 2012
Unusual deoxy sugars are often attached to natural products such as antibiotics, antifungals, and chemotherapeutic agents. One such sugar is mycinose, which has been found on the antibiotics chalcomycin and tylosin. An intermediate in the biosynthesis of mycinose is dTDP-6-deoxy-d-allose. Four enzymes are required for the production of dTDP-6-deoxy-d-allose in Streptomyces bikiniensis, a soil-dwelling microbe first isolated from the Bikini and Rongelap atolls. Here we describe a combined structural and functional study of the enzyme ChmJ, which reportedly catalyzes the third step in the pathway leading to dTDP-6-deoxy-d-allose formation. Specifically, it has been proposed that ChmJ is a 3′-epimerase that converts dTDP-4-keto-6-deoxyglucose to dTDP-4-keto-6-deoxyallose. This activity, however, has never been verified in vitro. As reported here, we demonstrate using 1H nuclear magnetic resonance that ChmJ, indeed, functions as a 3′-epimerase. In addition, we determined the structure of ChmJ complexed with dTDP-quinovose to 2.0 Å resolution. The structure of ChmJ shows that it belongs to the well-characterized "cupin" superfamily. Two active site residues, His 60 and Tyr 130, were subsequently targeted for study via site-directed mutagenesis and kinetic analyses, and the three-dimensional architecture of the H60N/Y130F mutant protein was determined to 1.6 Å resolution. Finally, the structure of the apoenzyme was determined to 2.2 Å resolution. It has been previously suggested that the position of a conserved tyrosine, Tyr 130 in the case of ChmJ, determines whether an enzyme in this superfamily functions as a mono-or diepimerase. Our results indicate that the orientation of the tyrosine residue in ChmJ is a function of the ligand occupying the active site cleft. © 2012 American Chemical Society.
Bruender N.A.,University of Wisconsin - Madison |
Thoden J.B.,University of Wisconsin - Madison |
Kaur M.,Edgewood Campus Middle School |
Avey M.K.,Edgewood Campus Middle School |
Holden H.M.,University of Wisconsin - Madison
Biochemistry | Year: 2010
S-Adenosylmethionine (SAM)-dependent methyltransferases are involved in a myriad of biological processes, including signal transduction, chromatin repair, metabolism, and biosyntheses, among others. Here we report the high-resolution structure of a novel C-3'-methyltransferase involved in the production of d-tetronitrose, an unusual sugar found attached to the antitumor agent tetrocarcin A or the antibiotic kijanimicin. Specifically, this enzyme, referred to as TcaB9 and cloned from Micromonospora chalcea, catalyzes the conversion of dTDP-3-amino-2,3,6-trideoxy-4-keto-d-glucose to dTDP-3-amino-2,3,6-trideoxy-4- keto-3-methyl-d-glucose. For this analysis, two structures were determined to 1.5 Å resolution: one in which the enzyme was crystallized in the presence of SAM and dTMP and the other with the protein complexed to S-adenosylhomocysteine and its dTDP-linked sugar product. The overall fold of the monomeric enzyme can be described in terms of three domains. The N-terminal domain harbors the binding site for a zinc ion that is ligated by four cysteines. The middle domain adopts the canonical "SAM-binding" fold with a seven-stranded mixed β-sheet flanked on either side by three α-helices. This domain is responsible for anchoring the SAM cofactor to the protein. Strikingly, the C-terminal domain also contains a seven-stranded β-sheet, and it appears to be related to the middle domain by an approximate 2-fold rotational axis, thus suggesting TcaB9 arose via gene duplication. Key residues involved in sugar binding include His 181, Glu 224, His 225, and Tyr 222. Their possible roles in catalysis are discussed. © 2010 American Chemical Society.