Structural and Functional Studies on a 3′-Epimerase Involved in the Biosynthesis of dTDP-6-deoxy-d-allose

Abstract

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 (1)H 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.

Structural and Biochemical Characterization of a Bifunctional Ketoisomerase/N-Acetyltransferase from Shewanella denitrificans

“…However, the structure of a C-3' epimerase from Streptomyces bikiniensis , another cupin family member, was recently solved in our laboratory in the presence of dTDP-6-deoxyglucose. 23 The only difference between dTDP-6-deoxyglucose and dTDP-4-keto-6-deoxyglucose is the presence of a hydroxyl rather than keto group on the C-4' carbon. The S. bikiniensis C-3' epimerase functions in the biosynthesis of dTDP-6-deoxy- d -allose.…”

Stereospecific Biosynthesis of β‐Methyltryptophan from L‐Tryptophan Features a Stereochemical Switch

“…The only difference between the one‐base and two‐base models is the existence of the second base for reprotonation for the latter (Scheme ). Several typical two‐base epimerases such as the epimerization (E) domain in nonribosomal peptide synthetases20 and dTDP‐4‐keto sugar epimerases21–24 have been confirmed to employ Tyr for reprotonation. Therefore, the only conserved and putative proton donor, Tyr70, was mutated to Ala (see Figure S5 a).…”

Stereospecific Biosynthesis of β‐Methyltryptophan from L‐Tryptophan Features a Stereochemical Switch

“…The only difference between the one-base and two-base models is the existence of the second base for reprotonation for the latter (Scheme 2). Several typical two-base epimerases such as the epimerization (E) domain in nonribosomal peptide synthetases [20] and dTDP-4-keto sugar epimerases [21][22][23][24] have been confirmed to employ Tyr for reprotonation. Therefore, the only conserved and putative proton donor, Tyr70, was mutated to Ala (see Figure S5 a).…”