( 2010 )
X-ray structure of kijd3, a key enzyme involved in the biosynthesis of D-kijanose.
PMID : 20334431 : DOI : 10.1021/bi100318v
D-kijanose is an unusual nitrosugar found attached to the antibiotic kijanimicin. Ten enzymes are required for its production in Actinomadura kijaniata, a soil-dwelling actinomycete. The focus of this investigation is on the protein encoded by the kijd3 gene and hereafter referred to as KijD3. On the basis of amino acid sequence analyses, KijD3 has been proposed to be an FAD-dependent oxidoreductase, which catalyzes the sixth step in d-kijanose biosynthesis by converting dTDP-3-amino-2,3,6-trideoxy-4-keto-3-methyl-d-glucose into its C-3' nitro derivative. This putative activity, however, has never been demonstrated in vivo or in vitro. Here we report the first structural study of this enzyme. For our investigation, crystals of KijD3 were grown in the presence of dTDP, and the structure was solved to 2.05-A resolution. The enzyme is a tetramer with each subunit folding into three distinct regions: a five alpha-helical bundle, an eight-stranded beta-sheet, and a second five alpha-helical bundle. The dTDP moiety is anchored to the protein via the side chains of Glu 113, Gln 254, and Arg 330. The overall fold of KijD3 places it into the well-characterized fatty acyl-CoA dehydrogenase superfamily. There is a decided cleft in each subunit with the appropriate dimensions to accommodate a dTDP-linked sugar. Strikingly, the loop defined by Phe 383 to Ala 388, which projects into the active site, contains two adjacent cis-peptide bonds, Pro 386 and Tyr 387. Activity assays demonstrate that KijD3 requires FAD for activity and that it produces a hydroxylamino product. The molecular architecture of KijD3 described in this report serves as a paradigm for a new family of enzymes that function on dTDP-linked sugar substrates.
( 2007 )
Elucidation of the kijanimicin gene cluster: insights into the biosynthesis of spirotetronate antibiotics and nitrosugars.
PMID : 17985890 : DOI : 10.1021/ja0744854 PMC : PMC2515274
The antibiotic kijanimicin produced by the actinomycete Actinomadura kijaniata has a broad spectrum of bioactivities as well as a number of interesting biosynthetic features. To understand the molecular basis for its formation and to develop a combinatorial biosynthetic system for this class of compounds, a 107.6 kb segment of the A. kijaniata chromosome containing the kijanimicin biosynthetic locus was identified, cloned, and sequenced. The complete pathway for the formation of TDP-l-digitoxose, one of the two sugar donors used in construction of kijanimicin, was elucidated through biochemical analysis of four enzymes encoded in the gene cluster. Sequence analysis indicates that the aglycone kijanolide is formed by the combined action of a modular Type-I polyketide synthase, a conserved set of enzymes involved in formation, attachment, and intramolecular cyclization of a glycerate-derived three-carbon unit, which forms the core of the spirotetronate moiety. The genes involved in the biosynthesis of the unusual deoxysugar d-kijanose [2,3,4,6-tetradeoxy-4-(methylcarbamyl)-3-C-methyl-3-nitro-d-xylo-hexopyranose], including one encoding a flavoenzyme predicted to catalyze the formation of the nitro group, have also been identified. This work has implications for the biosynthesis of other spirotetronate antibiotics and nitrosugar-bearing natural products, as well as for future mechanistic and biosynthetic engineering efforts.
( 2016 )
Structural studies on KijD1, a sugar C-3'-methyltransferase.
PMID : 27595766 : DOI : 10.1002/pro.3034 PMC : PMC5119555
Kijanimicin is an antitumor antibiotic isolated from Actinomadura kijaniata. It is composed of three distinct moieties: a pentacyclic core, a monosaccharide referred to as d-kijanose, and a tetrasaccharide chain composed of l-digitoxose units. d-Kijanose is a highly unusual nitro-containing tetradeoxysugar, which requires at least ten enzymes for its production. Here we describe a structural analysis of one of these enzymes, namely KijD1, which functions as a C-3'-methyltransferase using S-adenosylmethionine as its cofactor. For this investigation, two ternary complexes of KijD1, determined in the presence of S-adenosylhomocysteine (SAH) and dTDP or SAH and dTDP-3-amino-2,3,6-trideoxy-4-keto-3-methyl-d-glucose, were solved to 1.7 or 1.6 ? resolution, respectively. Unexpectedly, these structures, as well as additional biochemical analyses, demonstrated that the quaternary structure of KijD1 is a dimer. Indeed, this is in sharp contrast to that previously observed for the sugar C-3'-methyltransferase isolated from Micromonospora chalcea. By the judicious use of site-directed mutagenesis, it was possible to convert the dimeric form of KijD1 into a monomeric version. The quaternary structure of KijD1 could not have been deduced based solely on bioinformatics approaches, and thus this investigation highlights the continuing need for experimental validation.
( 2011 )
Combined structural and functional investigation of a C-3''-ketoreductase involved in the biosynthesis of dTDP-L-digitoxose.
PMID : 21598943 : DOI : 10.1021/bi200514b
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 ? 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.
( 2013 )
Active site architecture of a sugar N-oxygenase.
PMID : 23621882 : DOI : 10.1021/bi400407x
KijD3 is a flavin-dependent N-oxygenase implicated in the formation of the nitro-containing sugar d-kijanose, found attached to the antibiotic kijanimicin. For this investigation, the structure of KijD3 in complex with FMN and its dTDP-sugar substrate was solved to 2.1 ? resolution. In contrast to the apoenzyme structure, the C-terminus of the protein becomes ordered and projects into the active site cleft [Bruender, N. A., Thoden, J. B., and Holden, H. M. (2010) Biochemistry 49, 3517-3524]. The amino group of the dTDP-aminosugar that is oxidized is located 4.9 ? from C4a of the flavin ring. The model provides a molecular basis for understanding the manner in which KijD3 catalyzes its unusual chemical transformation.