Designing a metal-binding site in the scaffold of Escherichia coli KDO8PS.

KDO8PS (3-deoxy-d-manno-octulosonate-8-phosphate synthase) and DAH7PS (3-deoxy-d-arabino-heptulosonic acid-7-phosphate synthase) enzymes catalyse analogous condensation reactions between phosphoenolpyruvate and arabinose 5-phosphate or erythrose 4-phosphate, respectively. All known DAH7PS and some of KDO8PS enzymes (Aquifex aeolicus KDO8PS) require a metal ion for activity whereas another class of KDO8PS (including Escherichia coli KDO8PS) does not. Based on sequence alignment of all known KDO8PS and DAH7PS enzymes, we identified a single amino acid residue that might define the metal dependence of KDO8PS activity. One of the four metal-binding residues, a cysteine, is conserved only among metal-binding KDO8PS and DAH7PS enzymes and is replaced by an asparagine residue in other KDO8PS enzymes. We introduced a metal binding site into E.coli KDO8PS by a single N26C and a double M25P N26C mutation, which led to an increased k(cat) of the enzymes in the presence of activating Mn(2+) ions. The M25P N26C mutant of E.coli KDO8PS had a value of k(cat)/K(M) in the presence of Mn(2+) ions four times higher than A.aeolicus KDO8PS. KDO8PS and DAH7PS may have evolved from a common ancestor protein that required a divalent metal ion for activity. A non-metal-binding KDO8PSs may have evolved from an ancestor protein that was able to bind Mn(2+) but no longer required Mn(2+) to function and eventually lost one of metal-binding residues.

Engineering of complex polyketide biosynthesis--insights from sequencing of the monensin biosynthetic gene cluster.

The biosynthesis of complex reduced polyketides is catalysed in actinomycetes by large multifunctional enzymes, the modular Type I polyketide synthases (PKSs). Most of our current knowledge of such systems stems from the study of a restricted number of macrolide-synthesising enzymes. The sequencing of the genes for the biosynthesis of monensin A, a typical polyether ionophore polyketide, provided the first genetic evidence for the mechanism of oxidative cyclisation through which polyethers such as monensin are formed from the uncyclised products of the PKS. Two intriguing genes associated with the monensin PKS cluster code for proteins, which show strong homology with enzymes that trigger double bond migrations in steroid biosynthesis by generation of an extended enolate of an unsaturated ketone residue. A similar mechanism operating at the stage of an enoyl ester intermediate during chain extension on a PKS could allow isomerisation of an E double bond to the Z isomer. This process, together with epoxidations and cyclisations, form the basis of a revised proposal for monensin formation. The monensin PKS has also provided fresh insight into general features of catalysis by modular PKSs, in particular into the mechanism of chain initiation.

Analysis of a kanamycin resistance gene (kmr) from Streptomyces kanamyceticus and a mutant with increased aminoglycoside resistance.

Kanamycin resistance gene (kmr) from the overproducing mutant strain of Streptomyces kanamyceticus ISP1375 (strain 1) and from its gentamicin-resistant mutant were cloned into the high copy number vector pIJ702 and transformed into S. lividans 66. This gene provides resistance to kanamycin, gentamicin, sisomicin and tobramicin. The resistance of the transformed recipient strains was higher than the resistance level of the donor S. kanamyceticus 1. Sequencing of the kmr gene (EMBL Nucleotide Sequence Database accession no. Y15838 revealed 53.9% identity in 274 aa with the kgmB gene product (16S rRNA methylase) of S. tenebrarius. Hybridisation analysis using a 0.85 kb fragment carrying the kmr gene revealed that other gentamicin-resistant mutants of S. kanamyceticus 1 and unstable kanamycin-nonproducing mutant had a high level of kmr amplification. We found no homology between the kmr gene and the total DNA of the neomycin producer S. fradiae IFO3718; the sisomicin producer M. zionensis IFO14116 and the gentamicin producer M. purpurea ATCC15835.