Combined Structural and Functional Investigation of a C-3′′-Ketoreductase Involved in the Biosynthesis of dTDP-l-Digitoxose

Abstract

l-Digitoxose is an unusual dideoxysugar found attached to various pharmacologically active natural products, including the antitumor antibiotic tetrocarcin A and the antibiotics kijanimicin and jadomycin B. Six enzymes are required for its production starting from glucose 1-phosphate. Here we describe a combined structural and functional investigation of KijD10, an NADPH-dependent C-3''-ketoreductase that catalyzes the third step of l-digitoxose biosynthesis in the African soil-dwelling bacterium Actinomadura kijaniata. KijD10 belongs to the glucose-fructose oxidoreductase superfamily. For this investigation, both binary and ternary complexes of KijD10 were crystallized, and their structures were determined to 2.0 Å resolution or better. On the basis of these high-resolution structures, two potential active site acids were identified, Lys 102 and Tyr 186. These residues were individually mutated and the resultant proteins investigated both kinetically and structurally. The Y186F mutant protein demonstrated significant catalytic activity, and its structure was virtually identical to that of the wild-type enzyme except for the positioning of the nicotinamide ring. All lysine mutations, on the other hand, resulted in proteins with either abolished or drastically reduced catalytic activities. Structures for the K102A and K102E mutant proteins were determined and showed that the abrogation of catalytic activity was not a result of large conformational changes. Taken together, these data suggest that Lys 102 donates a proton to the C-3'' keto group during the reaction and that Tyr 186 serves only an auxiliary role. This is in contrast to that proposed for glucose-fructose oxidoreductase and other family members in which the tyrosines, or in some cases similarly positioned histidines, are thought to play major catalytic roles.

Holo Structure and Steady State Kinetics of the Thiazolinyl Imine Reductases for Siderophore Biosynthesis

“…35—38 In the sugar oxidoreductases, the active site residue involved in proton donation has been hypothesized to be either the middle residue of the EKP 35,36 consensus sequence or the final residue of the GGX 3 DX 3 (Y/H) consensus sequence. 33,34,37,38 In the thiazolinyl imine reductases, the EKP consensus sequence is 100 EHP 102 , which may still able to act as a proton donor.…”

“…This residue is conserved in PchG (tyrosine 124). The structure of KijD10, 35 a ketoreductase from Actinomadura kijaniata in the biosynthetic pathway for l -digitoxose, contains a substrate analogue, TDP-benzene, which aligns in three dimensions with HPTT-COOH in Irp3. In KijD10, the proposed lysine general acid (K102) derived from the 101 EKP 103 consensus sequence is 5.7 Å from the double bond being reduced, within range of proton donation (Figure 3).…”

Two Structures of a Thiazolinyl Imine Reductase from Yersinia enterocolitica Provide Insight into Catalysis and Binding to the Nonribosomal Peptide Synthetase Module of HMWP1

“…This sequence is found at the C-terminus of a 28-residue loop and includes the N-terminal two turns of an active site α-helix (Figure 4 lower panels). In GFOR (37), KijD10 (33), and WlbA from Bordetella pertussis (31), which also have the GGX 3 DX 3 (Y/H) substrate binding consensus sequence (grey in Figure 5 for KijD10), the aspartic acid is hypothesized to be important for substrate binding. The final amino acid of the sequence has been hypothesized to serve as the general acid/general base for catalysis.…”

Bacterial Sugar 3,4-Ketoisomerases: Structural Insight into Product Stereochemistry

“…Previous studies from the laboratory have verified that KijD10 from Actinomadura kijaniata functions as an NADPH-dependent C-3′ ketoreductase. 12 The reaction mixtures contained 50 mM HEPES (pH 7.5), 0.2 mM NADPH, 1 mg/mL KijD10, and dTDP-4-keto-6-deoxyglucose varying from 0.005 to 5.0 mM, depending on the enzyme/variant being evaluated. The reactions were initiated by the addition of the 3,4-ketoisomerases to the reaction mixtures in the following concentrations: 0.33 μ M (FdtA wild-type), 0.19 μ M (FdtA Y35F), 2.2 μ M (FdtA R15Y), 0.11 μ M (FdtA H95R) 6.0 μ M (FdtA R15Y/H95R), 0.26 μ M (QdtA wild-type), 21 μ M (QdtA Y37F), 1.2 μ M (QdtA Y17R), 0.79 μ M (QdtA R97H), 8.8 μ M (QdtA Y17R/R97H), and 1.2 μ M (3,4-ketoisomerase domain of FdtD).…”