TcUGM activity with UDP-Gal<i>f</i>.

<p>TcUGM was reduced with either 20 mM dithionite (▪), 0.5 mM NADPH(•), or 2.5 mM NADH(▴). Reactions were performed with 200 nM TcUGM incubated with varying concentrations of substrate for 1 min at 37°C. The data were fit to the Michaelis-Menten equation. Summary of data is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032918#pone-0032918-t003" target="_blank">Table 3</a>.</p

Rapid reaction kinetics with reduced TcUGM mixed with substrate and substrate analogs.

<p>The initial spectrum (1.2 ms) is shown as a solid line, the final spectrum (14 ms for UDP-Gal<i>f</i> and 1.5 s for all other analogs) as a dashed line, and intermediate time points as gray lines (spectra every 1.26 ms for UDP-Gal<i>f</i> and every tenth spectrum collected of 400 points on logarithmic time scale over 2 s for all other analogs). Reduced TcUGM mixed with 0.25 mM UDP (A), 0.25 mM UDP-Gal<i>p</i> (B), 0.15 mM UDP-Gal<i>f</i> (C), and formation of the iminium ion monitored at 455 nm (D). The data was fit to equation 3.</p

Anaerobic reduction of TcUGM with NAD(P)H.

<p>Reduction was monitored using the stopped flow spectrophotometer at 15°C. The data was collected from 2 ms to 100 s on a logarithmic timescale. A) Changes in the spectra of oxidized TcUGM after mixing with 0.5 mM NADPH over 23 s. B) Traces of the flavin reduction at various concentrations of NADPH. The data were fit to a single exponential decay equation. C) The k<i>_obs</i> values were plotted as a function NADPH (•) and NADH (▴) concentrations and fitted using the equation 2.</p

Chemical mechanism of TcUGM.

<p>The reaction requires the oxidized flavin cofactor (<b>a</b>) to be reduced for activity. First, NADPH binds to the oxidized enzyme (<b>b</b>), and only after the flavin is reduced (<b>c</b>) will UDP-Gal<i>p</i> bind (<b>d</b>). The flavin then acts as a nucleophile attacking the C1 of galactose and forming a flavin sugar adduct (<b>e</b>), which occurs rapidly (<b>f</b>). This is followed by ring opening and recyclization (<b>g</b>). The rate limiting step in the reaction corresponds to either galactose isomerization or reattachment of the UDP (<b>f</b> to <b>g</b>). We postulate that the rate limiting step is the isomerization step. The final step is release of UDP-Gal<i>f</i>, which occurs rapidly. The enzyme can proceed to the next reaction cycle or be slowly oxidized by molecular oxygen (<b>h</b> to <b>a</b>).</p

Trapping of a covalent flavin intermediate.

<p>A) HPLC traces of the flavin sugar adduct from free FAD. The peak eluding at 22.5 min is the adduct, while the second peak at 23.6 min is FAD. B) Spectrum of the C4a- hydroxyflavin-galactose adduct. C) High resolution mass spectrometry results of the peak containing the flavin adduct. The inset shows the structure of the adduct with a hydroxyl group at the flavin C4a-position.</p

Fluorescence anisotropy assay to measure the affinity of UDP-Gal<i>p</i> to TcUGM.

<p>A) UDP-rhodamine chromophore used in the fluorescence anisotropy experiments. B) Fluorescence polarization binding assay. The binding of UDP-Gal<i>p</i> to chemically reduced TcUGM was monitored by measuring the changes in anisotropy as it displaces UDP-rhodamine from the active site. The K<i>_d</i> values were obtained using equation 3.</p

TcUGM Reduction by NAD(P)H^a.

a<p>Reactions were measured under anaerobic conditions at 15°C in 50 mM phosphate buffer pH 7.0.</p

Viscosity effect on k_cat.

<p>The effect of viscosity was determined by measuring the activity of TcUGM as a function of increasing concentrations of glycerol. The data was fit to a linear equation; the dashed line depicts the results of a diffusion controlled reaction. This line has a slope of 1.</p

The two proposed chemical mechanisms for UGMs.

<p>In one mechanism the reduced FAD (<b>1</b>) is depicted to act as a nucleophile forming a flavin-galactose adduct (either via S_N1 or S_N2) (<b>2</b>) and a subsequent iminium ion (<b>3</b>). These steps are followed by ring contraction forming the galactofuranose (<b>4</b>). An alternative mechanism predicts an electron transfer step, in which one electron is transferred from FADH⁻ to an oxocarbenium ion intermediate (<b>6</b>) creating a flavin and a sugar radical, which react and form the galactose-FAD adduct (<b>2</b>), followed by ring contraction.</p

Oxidase activity of TcUGM with NAD(P)H.

<p>A) Scheme representing the oxidase activity of TcUGM measured in this assay. B) Activity of TcUGM measured in the presence and absence of saturating concentration of UDP-Gal<i>p</i> (0.5 mM) under air saturating conditions at room temperature (NADPH (•), NADH (▴), NADPH+UDP-Gal<i>p</i> (▪), and with NADH+UDP-Gal<i>p</i> (▾)).</p