Assignment of biochemical functions to glycosyl transferase genes which are essential for biosynthesis of exopolysaccharides in Sphingomonas strain S88 and Rhizobium leguminosarum.

Glycosyl transferases which recognize identical substrates (nucleotide-sugars and lipid-linked carbohydrates) can substitute for one another in bacterial polysaccharide biosynthesis, even if the enzymes originate in different genera of bacteria. This substitution can be used to identify the substrate specificities of uncharacterized transferase genes. The spsK gene of Sphingomonas strain S88 and the pssDE genes of Rhizobium leguminosarum were identified as encoding glucuronosyl-(B1-->4)-glucosyl transferases based on reciprocal genetic complementation of mutations in the spsK gene and the pssDE genes by segments of cloned DNA and by the SpsK-dependent incorporation of radioactive glucose (Glc) and glucuronic acid (GlcA) into lipid-linked disaccharides in EDTA-permeabilized cells. By contrast, glycosyl transferases which form alternative sugar linkages to the same substrate caused inhibition of polysaccharide synthesis or were deleterious or lethal in a foreign host. The negative effects also suggested specific substrate requirements: we propose that spsL codes for a glucosyl-(beta1-->4)-glucuronosyl transferase in Sphingomonas and that pssC codes for a glucuronosyl-(beta1-->4)-glucuronosyl transferase in R. leguminosarum. Finally, the complementation results indicate the order of attachment of sphingan main-chain sugars to the C55-isoprenylphosphate carrier as -Glc-GlcA-Glc-isoprenylpyrophosphate.

Cloning and characterization of four genes of Rhizobium leguminosarum bv. trifolii involved in exopolysaccharide production and nodulation.

Four different genes of Rhizobium leguminosarum bv. trifolii strain RBL5599 involved in exopolysaccharide (EPS) production were identified by complementation of Tn5-induced EPS-deficient mutants (Exo mutants) with a cosmid bank. On one cosmid pssA was located, which was found to be almost identical to the pss4 gene from R. leguminosarum bv. viciae VF39 and highly homologous to a family of glycosyl transferases. Two pssA mutants, exo2 and exo4, were characterized and found to produce 19 and 1% of the wild-type amount of EPS, respectively. The three other genes were found to be closely linked on a different complementing cosmid. pssC revealed similarity to exoM and exoW of R. meliloti, both encoding glucosyl transferases involved in the synthesis of succinoglycan. A mutation in this gene (mutant exo50) did reduce EPS synthesis to 27% of the wild-type amount. We found an operon closely linked to pssC, consisting of two overlapping genes, pssD and pssE, that is essential for EPS production. Homology of pssD and pssE was found with cps14F and cps14G of Streptococcus pneumoniae, respectively: two genes responsible for the second step in capsule polysaccharide synthesis. Furthermore, pssD and pssE were homologous to the 5' and 3' parts, respectively, of spsK of Sphingomonas S88, which encodes a putative glycosyl transferase. Structural analysis of EPS produced by Exo mutants exo2, exo4, and exo50 showed it to be identical to that of the parental strain RBL5599, with the exception of acetyl groups esterified to one of the glucose residues being absent.

19F-NMR study on the pH-dependent regioselectivity and rate of the ortho-hydroxylation of 3-fluorophenol by phenol hydroxylase from Trichosporon cutaneum. Implications for the reaction mechanism.

The regioselectivity and rate of the ortho-hydroxylation of 3-fluorophenol by phenol hydroxylase from Trichosporon cutaneum (EC 1.14.13.7) was studied using 19F-NMR. The regioselective hydroxylation as well as the rate of ortho-hydroxylation are pH dependent with a pKa of 6.5. At pH values below 6.5, 3-fluorophenol preferentially becomes hydroxylated at the C6 ortho position, resulting in a maximum C6/C2 hydroxylation ratio of 6.7. Upon increasing the pH, the total rate of conversion increases. Also, the C2 ortho-hydroxylation increases relatively to the C6 ortho-hydroxylation and yields a minimum C6/C2 hydroxylation ratio of 2.2 at pH values above 7.5. Based on data from 19F-NMR binding studies and molecular orbital calculations, a hypothesis is put forward which explains the pH-dependent effects observed. A mechanism is proposed involving an active-site amino acid residue acting as a base in the reduced form of the protein. Deprotonation of this residue results in hydrogen bond formation with the hydroxyl moiety of the phenolic substrate, leading to (partial) deprotonation of the substrate. Molecular orbital calculations demonstrate that such a (partial) deprotonation increases (a) the overall reactivity of 3-fluorophenol for an electrophilic attack and (b) the reactivity of C2 relative to the C6 position. The hypothesis may explain the decrease in the C6/C2 hydroxylation ratio. Furthermore the increased amount of ortho-hydroxylated products formed with increasing pH can also be explained by this hypothesis.