The glycosyltransferase termed MshA catalyzes the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to 1-Lmyo-inositol-1-phosphate in the first committed step of mycothiol biosynthesis. The structure of MshA from Corynebacterium glutamicum was determined both in the absence of substrates and in a complex with UDP and 1-L-myo-inositol-1-phosphate. MshA belongs to the GT-B structural family whose members have a two-domain structure with both domains exhibiting a Rossman-type fold. Binding of the donor sugar to the C-terminal domain produces a 97°rotational reorientation of the N-terminal domain relative to the C-terminal domain, clamping down on UDP and generating the binding site for 1-Lmyo-inositol-1-phosphate. The structure highlights the residues important in binding of UDP-N-acetylglucosamine and 1-L-myo-inositol-1-phosphate. Molecular models of the ternary complex suggest a mechanism in which the ␤-phosphate of the substrate, UDP-N-acetylglucosamine, promotes the nucleophilic attack of the 3-hydroxyl group of 1-L-myo-inositol-1-phosphate while at the same time promoting the cleavage of the sugar nucleotide bond.

Tyrosine hydroxylase (TyrH 1), the key enzyme in the biosynthesis of catecholamine neurotransmitters, is one of three members of the aromatic amino acid hydroxylase enzyme family. 2,3 The enzyme is found in the brain and adrenal gland where it catalyses the conversion of L-tyrosine to L-DOPA. The other members of the family are phenylalanine hydroxylase, which catabolizes excess phenylalanine to tyrosine, and tryptophan hydroxylase, which catalyzes the rate limiting step in the biosynthesis of the neurotransmitter serotonin. All three enzymes have a mononuclear non-heme iron, coordinated by the common His 2-Glu facial triad motif, 4,5 and use a tetrahydropterin to activate dioxygen for hydroxylation of the aromatic side chains of their corresponding amino acid substrates. 2,3 In the proposed mechanism 6-8 (Scheme 1), oxygen reacts with ferrous iron and tetrahydropterin to produce a Fe(IV)O (ferryl) hydroxylating intermediate and 4a-hydroxypterin (4a-HOPH 3). Then, through an electrophilic aromatic substitution, the ferryl species reacts with the aromatic side chain of the tyrosine substrate (Tyr) to form the product dihydroxyphenylalanine (DOPA). To date there has been no direct evidence for this ferryl species. Here, we report the detection of an Fe(IV) intermediate, which is likely to be the proposed ferryl species, in the TyrH reaction by the use of rapid reaction methods. The anaerobic TyrH•Fe(II) •6-MePH 4 •Tyr complex 9 was reacted with oxygen and quenched by rapid-freeze at time points from 20 ms to 390 ms. 10 Figure 1 (left panel) shows representative Mössbauer spectra of the samples from such a time course. The spectrum of the reactant complex reveals the presence of two broad lines with parameters typical of high-spin Fe(II). The asymmetry suggests the presence of at least two distinct Fe(II) complexes. A new line at ~0.9 mm/s is observed in the spectra of samples in which the reactant complex was exposed to oxygen for either 20 ms or 100 ms, but it is not detected in the spectrum of a sample reacted for 390 ms. Thus, this peak is associated with a reaction intermediate which exhibits a quadrupole doublet in a weak external magnetic field. The low-energy line of this quadrupole doublet overlaps with the low-energy line of the Fe(II). The features of the intermediate are similar to those observed for Fe(IV) intermediates in other mononuclear non-heme enzymes. 11,12

Acetyl-CoA carboxylase catalyzes the first committed step in the biosynthesis of long-chain fatty acids. The Escherichia coli form of the enzyme consists of a biotin carboxylase protein, a biotin carboxyl carrier protein, and a carboxyltransferase protein. In this report a system for site-directed mutagenesis of the biotin carboxylase component is described. The wild-type copy of the enzyme, derived from the chromosomal gene, is separated from the mutant form of the enzyme which is coded on a plasmid. Separation of the two forms is accomplished using a histidine-tag attached to the amino terminus of the mutant form of the enzyme and nickel affinity chromatography. This system was used to mutate four active site residues, E211, E288, N290, and R292, to alanine followed by their characterization with respect to several different reactions catalyzed by biotin carboxylase. In comparison to wild-type biotin carboxylase, all four mutant enzymes gave very similar results in all the different assays, suggesting that the mutated residues have a common function. The mutations did not affect the bicarbonate-dependent ATPase reaction. In contrast, the mutations decreased the maximal velocity of the biotin-dependent ATPase reaction 1000-fold but did not affect the Km for biotin. The activity of the ATP synthesis reaction catalyzed by biotin carboxylase where carbamoyl phosphate reacts with ADP was decreased 100-fold by the mutations. The ATP synthesis reaction required biotin to stimulate the activity in the wild-type; however, biotin did not stimulate the activity of the mutant enzymes. The results showed that the mutations have abolished the ability of biotin to increase the activity of the enzyme. Thus, E211, E288, N290, and R292 were responsible, at least in part, for the substrate-induced synergism by biotin in biotin carboxylase.

Lewis X (Le x )-containing glycans play important roles in numerous cellular processes. However, the absence of robust, facile, and cost-effective methods for the synthesis of Le x and its structurally related analogs has severely hampered the elucidation of the specific functions of these glycan epitopes. Here we demonstrate that chemically defined guanidine 5-diphosphate-␤-L-fucose (GDPfucose), the universal fucosyl donor, the Le x trisaccharide, and their C-5 substituted derivatives can be synthesized on preparative scales, using a chemoenzymatic approach. This method exploits L-fucokinase/GDP-fucose pyrophosphorylase (FKP), a bifunctional enzyme isolated from Bacteroides fragilis 9343, which converts L-fucose into GDP-fucose via a fucose-1-phosphate (Fuc-1-P) intermediate. Combining the activities of FKP and a Helicobacter pylori ␣1,3 fucosyltransferase, we prepared a library of Le x trisaccharide glycans bearing a wide variety of functional groups at the fucose C-5 position. These neoglycoconjugates will be invaluable tools for studying Le x -mediated biological processes.glycobiology ͉ catalysis ͉ enzyme