Preparation of Lipase-Electrospun SiO2 Nanofiber Membrane Bioreactors and Their Targeted Catalytic Ability at the Macroscopic Oil-Water Interface.

Lipase is one of the most widely used enzymes in biocatalysis. Because of the special structure of the catalytic active center, lipases show high catalytic activity at oil-water interfaces. Hence, the interface plays a key role in activating and modulating lipase biocatalysis. Compared with traditional catalytic systems that offer interfaces, such as emulsions, a lipase-membrane bioreactor exhibits many obvious advantages when at the macroscopic oil-water system. In our current research, a series of new Burkholderia cepacia lipase (BCL)-SiO2 nanofiber membrane (NFM) bioreactors prepared via combined electrospinning and immobilization strategies were reported. These SiO2 NFMs assisted BCL in reaching the oil-water interface for efficient catalysis. The enzyme loading capacity and catalytic efficiency of BCL-SiO2 NFMs varied with the surface hydrophobicity of the electrospun NFMs. As the hydrophobicity increased, the activity decreased from 2.43-fold to 0.74-fold that of free BCL. However, the lipase-loading capacity increased obviously when the hydrophobicity of the SiO2 NFMs increased from 0 to 143°, and no significant change was observed when the hydrophobicity of the SiO2 NFMs increased from 143 to 153°. The gel trapping technique proved that the hydrolytic activity of the different BCL-SiO2 NFM bioreactors depends on the contact area of the membrane at the oil-water interface. BCL-SiO2 NFM, BCL-SiO2 NFM-C12, and BCL-SiO2 NFM-C18 retained 32, 83, and 42% of activity, respectively, after five cycles of reuse. The current work was a useful exploration of the construction and modification of lipase-membrane reactors based on electrospun inorganic silicon.

Tailoring a stable and recyclable nanobiocatalyst by immobilization of surfactant treated Burkholderia cepacia lipase on polyaniline nanofibers for biocatalytic application.

Polyaniline nanofibers were synthesized by the oxidative polymerization of aniline. Surfactant treated lipase from Burkholdaria cepacia was immobilized on these polyaniline nanofibers by adsorption. The activity of immobilized preparation was six times higher than that of free lipase with an enhanced dispersion in organic solvents. Five-level-four-factor central composite design was applied for the optimization of immobilization parameters (viz. reaction time, pH, stirring rate and enzyme-support ratio) which were evaluated on the basis of lipase loading and activity. The thermal stability of the lipase in the nanobioconjugate, demonstrated in terms of the half-life at 80 °C was almost sixteen-fold higher than in the free form. The reusability data revealed the utility of the nanoconjugate for seven consecutive cycles with a slow and gradual decline in the activity. However, the nanoconjugate retained almost 30% of their initial activity after seven cycles of reuse revealing its utility of in industrial applications. The nanoconjugate was used in the kinetic resolution of (RS)-1-(7-(3-chloro-2-hydroxypropoxy)benzofuran-2-yl) ethanone, racemic intermediate of an important ß-blocker (Befunolol), with a high conversion rate of 48.2%, 98% ee-value and enantioselectivity (E) of 188, which signify its importance as a nanobiocatalyst.

Surfactant Charge Modulates Structure and Stability of Lipase-Embedded Monolayers at Marine-Relevant Aerosol Surfaces.

Lipases, as well as other enzymes, are present and active within the sea surface microlayer (SSML). Upon bubble bursting, lipases partition into sea spray aerosol (SSA) along with surface-active molecules such as lipids. Lipases are likely to be embedded in the lipid monolayer at the SSA surface and thus have the potential to influence SSA interfacial structure and chemistry. Elucidating the structure of the lipid monolayer at SSA interfaces and how this structure is altered upon interaction with a protein system like lipase is of interest, given the importance of how aerosols interact with sunlight, influence cloud formation, and provide surfaces for chemical reactions. Herein, we report an integrated experimental and computational study of Burkholderia cepacia lipase (BCL) embedded in a lipid monolayer and highlight the important role of electrostatic, rather than hydrophobic, interactions as a driver for monolayer stability. Specifically, we combine Langmuir film experiments and molecular dynamics (MD) simulations to examine the detailed interactions between the zwitterionic dipalmitoylphosphatidylcholine (DPPC) monolayer and BCL. Upon insertion of BCL from the underlying subphase into the lipid monolayer, it is shown that BCL permeates and largely disorders the monolayer while strongly interacting with zwitterionic DPPC molecules, as experimentally observed by Langmuir adsorption curves and infrared reflectance absorbance spectroscopy. Explicitly solvated, all-atom MD is then used to provide insights into inter- and intramolecular interactions that drive these observations, with specific attention to the formation of salt bridges or ionic-bonding interactions. We show that after insertion into the DPPC monolayer, lipase is maintained at high surface pressures and in large BCL concentrations by forming a salt-bridge-stabilized lipase-DPPC complex. In comparison, when embedded in an anionic monolayer at low surface pressures, BCL preferentially forms intramolecular salt bridges, reducing its total favorable interactions with the surfactant and partitioning out of the monolayer shortly after injection. Overall, this study shows that the structure and dynamics of lipase-embedded SSA surfaces vary based on surface charge and pressure and that these variations have the potential to differentially modulate the properties of marine aerosols.

Use of bacterial biosurfactants as natural collectors in the dissolved air flotation process for the treatment of oily industrial effluent.

The aim of the present study was to investigate the separation of oil from water using a bench-scale DAF prototype with the addition of biosurfactants isolated from Pseudomonas cepacia CCT6659 and Bacillus cereus UCP1615. The best operating conditions for the DAF prototype were determined using a central composite rotatable design. The results demonstrated that the biosurfactants from P. cepacia and B. cereus increased the oil separation efficiency from 53.74% (using only microbubbles) to 94.11 and 80.01%, respectively. The prediction models for both DAF-biosurfactant systems were validated, showing an increase in the efficiency of the DAF process from 53.74% to 98.55 and 70.87% using the formulated biosurfactants from P. cepacia and B. cereus, respectively. The biosurfactant from P. cepacia was selected as the more promising product and used for the treatment of oily effluent from a thermoelectric plant, achieving removal rates ranging between 75.74 (isolated biosurfactant) and 95.70% (formulated biosurfactant), respectively.

Impacts of Lipase Enzyme on the Surface Properties of Marine Aerosols.

Triacylglycerol lipases have recently been shown to be transferred from the ocean to the atmosphere in atmospheric sea spray aerosol (SSA). Lipases have the potential to alter the composition of SSA; however, the structure and properties of enzymes in the high salt, high ionic strength, and low pH conditions found in SSA have never been explored. Here, we study the dynamics of Burkholderia cepacia triacylglycerol lipase (BCL) at SSA model surfaces comprised of palmitic acid and dipalmitoylphosphatidic acid (DPPA), two commonly found lipids at SSA surfaces. Surface adsorption Langmuir isotherm experiments and all-atom explicit solvent molecular dynamics simulations together illuminate how and why BCL expands the ordering of lipids at palmitic acid surfaces the most at pH < 4 and the least in DPPA surfaces at pH 6. Taken together, these results represent a first glimpse into the complex interplay between lipid surface structure and protein dynamics within enzyme-containing aerosols.

Lipase Immobilization on Silica Xerogel Treated with Protic Ionic Liquid and its Application in Biodiesel Production from Different Oils.

Treated silica xerogel with protic ionic liquid (PIL) and bifunctional agents (glutaraldehyde and epichlorohydrin) is a novel support strategy used in the effective immobilization of lipase from Burkholderia cepacia (LBC) by covalent binding. As biocatalysts with the highest activity recovery yields, LBC immobilized by covalent binding with epichlorohydrin without (203%) and with PIL (250%), was assessed by the following the hydrolysis reaction of olive oil and characterized biochemically (Michaelis⁻Menten constant, optimum pH and temperature, and operational stability). Further, the potential transesterification activity for three substrates: sunflower, soybean, and colza oils, was also determined, achieving a conversion of ethyl esters between 70 and 98%. The supports and the immobilized lipase systems were characterized using Fourier transform infrared spectra (FTIR), scanning electron microscopy (SEM), elemental analysis, and thermogravimetric (TG) analysis.

Pore Environment Control and Enhanced Performance of Enzymes Infiltrated in Covalent Organic Frameworks.

In the drive toward green and sustainable methodologies for chemicals manufacturing, biocatalysts are predicted to have much to offer in the years to come. That being said, their practical applications are often hampered by a lack of long-term operational stability, limited operating range, and a low recyclability for the enzymes utilized. Herein, we show how covalent organic frameworks (COFs) possess all the necessary requirements needed to serve as ideal host materials for enzymes. The resultant biocomposites of this study have shown the ability boost the stability and robustness of the enzyme in question, namely lipase PS, while also displaying activities far outperforming the free enzyme and biocomposites made from other types of porous materials, such as mesoporous silica and metal-organic frameworks, exemplified in the kinetic resolution of the alcohol assays performed. The ability to easily tune the pore environment of a COF using monomers bearing specific functional groups can improve its compatibility with a given enzyme. As a result, the orientation of the enzyme active site can be modulated through designed interactions between both components, thus improving the enzymatic activity of the biocomposites. Moreover, in comparison with their amorphous analogues, the well-defined COF pore channels not only make the accommodated enzymes more accessible to the reagents but also serve as stronger shields to safeguard the enzymes from deactivation, as evidenced by superior activities and tolerance to harsh environments. The amenability of COFs, along with our increasing understanding of the design rules for stabilizing enzymes in an accessible fashion, gives great promise for providing "off the shelf" biocatalysts for synthetic transformations.

Real-Time PCR Detection of Burkholderia cepacia in Pharmaceutical Products Contaminated with Low Levels of Bacterial Contamination.

A real-time polymerase chain reaction (RT-PCR) assay was developed to detect Burkholderia cepacia in pharmaceutical products contaminated with low levels of bacteria. Different pharmaceutical suspensions were artificially contaminated with B. cepacia, Escherichia coli, Staphylococcus aureus, and Bacillus megaterium After a 24 h incubation in trypticase soy broth with Tween 20, samples were streaked on mannitol salt, phenyl ethyl alcohol, eosin methylene blue, MacConkey, and pseudomonas isolation agar. Microbial DNA was extracted from each sample by using a Tris-EDTA, proteinase K, Tween 20 buffer. Regular PCR targeting the 1.5 kilobases 16S rRNA eubacterial gene and cloning showed the predominant DNA in the extracted mix belonged to E. coli Selective media isolation of bacterial contamination showed B. cepacia only detected on pseudomonas isolation while eosin methylene blue and MacConkey detected only E. coli RT-PCR using primers PSL1 and PSR1 amplified a 209 bp 16S rRNA fragment using a Roche LightCycler 96® system with SYBR green I, a common double-stranded binding dye. The cycle at which fluorescence from amplification exceeds the background fluorescence was referred to as quantification cycle. All samples were found to be positive by standard microbiological testing and RT-PCR. B. cepacia was detected within 30 h in all contaminated samples using RT-PCR. Based upon standard curve analysis of B. cepacia DNA, the minimum DNA concentration that could be detected was 10 fg/uL with a correlation value of 0.98. RT-PCR detection of B. cepacia allowed faster quality control analysis, corrective actions, and process optimization.LAY ABSTRACT: A real-time polymerase chain reaction (RT-PCR) assay was developed to detect Burkholderia cepacia in pharmaceutical products contaminated with low levels of bacteria. B. cepacia is the number one reason for microbial contamination recalls of non-sterile drug products in the USA. RT-PCR using primers PSL1 and PSR1 amplified a 209 bp 16S rRNA fragment using a Roche LightCycler 96® system with SYBR green I, a common double-stranded binding dye. All samples were found to be positive by standard microbiological testing and RT-PCR. B. cepacia was detected within 30 h in all contaminated samples using RT-PCR. RT-PCR detection of B. cepacia allowed faster quality control analysis, corrective actions, and process optimization.

Biosorption of diethyl phthalate ester by living and nonliving Burkholderia cepacia and the role of its cell surface components.

In this study, the dibutyl phthalate (DBP) binding properties of a DBP-tolerant bacterium (B. cepacia) were characterized in terms of adsorption kinetics and isotherm. Living and nonliving cells both exhibited rapid removal of DBP, achieving more than 80% of maximum sorption within 30 min of contact and reached the equilibrium after 3 h. The adsorption isotherms were well fitted with the Sips model and the nonliving cells have greater biosorption capacity and affinity for DBP than the living cells. Furthermore, the absence of an active mechanism dependent on metabolism implied that the DBP bioaccumulation by living cells was mainly attribute to passive surface binding. The optimum pH for DBP adsorption by living and nonliving cells were both observed to be 6.0. The biosorptive mechanism of DBP binding by B. cepacia was further confirmed by FTIR analysis and various chemical treatments. FTIR results indicated that the phosphate and CH2 groups on B. cepacia were the main bounding sites for DBP. Furthermore, 2.28, 2.15, 1.93 and 0.87 g of pretreated cells were obtained from 2.40 g of native cells via extracellular polymeric substances (EPS), superficial layer-capsule, lipids components and cell membrane removal treatments, respectively. Total binding amount of DBP on the native cells, EPS-removed cells, capsule-removed cells, lipids-extracted cells and membrane-removed cells were 26.69, 24.84, 24.93, 16.11 and 10.80 mg, respectively, suggesting that the cell wall lipids, proteins or peptidoglycan might play important roles in the sorption of DBP by B. cepacia. The information could be applied in understanding on the mobility, transport and ultimate fate of PAEs in soil and related environment.

Modification biological activity of S and R forms of Proteus mirabilis and Burkholderia cepacia lipopolysaccharides by carrageenans.

The modification of biological features of S and R forms of Proteus mirabilis and Burkholderia cepacia LPS by kappa/iota and kappa/beta carrageenans was shown in Limulus activation test, ELISA, human complement activation and apoptotic assay. The role of positively charged substituent Ara4N in lipid A was evaluated as a suspected major domain for interactions with sulphate groups of carrageenans.The experiments obtained by three serological methods indicated that not only lipid A part of LPS but also polysaccharide elements such as core and O-specific chain are involved in interaction with carrageenes. Carrageenans turned out to be non-cytotoxic for A549 cells and were able to inhibit the apoptotic effect caused by lipid A of P. mirabilis and B. cepacia.

The members of M20D peptidase subfamily from Burkholderia cepacia, Deinococcus radiodurans and Staphylococcus aureus (HmrA) are carboxydipeptidases, primarily specific for Met-X dipeptides.

Three members of peptidase family M20D from Burkholderia cepacia (BcepM20D; Uniprot accession no. A0A0F7GQ23), Deinococcus radiodurans R1 (DradM20D; Uniprot accession no. Q9RTP6) and Staphylococcus aureus (HmrA; Uniprot accession no. Q99Q45) were characterized in terms of their preference for various substrates. The results thus reveal that all the enzymes including HmrA lack endopeptidase as well as aminopeptidase activities and possess strong carboxypeptidase activity. Further, the amidohydrolase activity exerted on other substrates like N-Acetyl-Amino acids, N-Carbobenzoxyl-Amino acids and Indole acetic acid (IAA)-Amino acids is due to the ability of these enzymes to accommodate different types of chemical groups other than the amino acid at the S1 pocket. Further, data on peptide hydrolysis strongly suggests that all the three enzymes are primarily carboxydipeptidases exhibiting highest catalytic efficiency (kcat/Km 5-36 × 10(5) M(-1) s(-1)) for Met-X substrates, where -X could be Ala/Gly/Ser/Tyr/Phe/Leu depending on the source organism. The supportive evidence for the substrate specificities was also provided with the molecular docking studies carried out using structure of SACOL0085 and homology modelled structure of BcepM20D. The preference for different substrates, their binding at active site of the enzyme and possible role of these enzymes in recycling of methionine are discussed in this study.

Synthesis of the Tetrasaccharide Repeating Unit of the ß-Kdo-Containing Exopolysaccharide from Burkholderia pseudomallei and B. cepacia Complex.

The synthesis of the repeating unit of the immunogenic ß-Kdo-containing exopolysaccharide produced by Burkholderia pseudomallei and bacteria of the B. cepacia complex is described. The target tetrasaccharide was synthesized via stereoselective 1,2-cis- and 1,2-trans-galactosylations and ß-Kdosylation. A [3 + 1] coupling reaction between a trigalactosyl N-phenyl-2,2,2-trifluoroacetimidate donor and a Kdo acceptor has been successfully achieved for the assembly of the tetrasaccharide skeleton.

Synthesis of the tetrasaccharide outer core fragment of Burkholderia multivorans lipooligosaccharide.

The first synthesis of the outer core fragment of Burkholderia multivorans lipooligosaccharide [ß-D-Glc-(1→3)-α-D-GalNAc-(1→3)-ß-D-GalNAc-(1→3)-L-Rha] as α-allyl tetrasaccharide was accomplished. The glycosylations involving GalNAc units were studied in depth testing them under several conditions. This allowed the building of both the α- and the ß-configured glycosidic bonds by employing the same GalNAc glycosyl donor, thus considerably shortening the total number of synthetic steps. The target tetrasaccharide was synthesized with an allyl aglycone to allow its future conjugation with an immunogenic protein en route to the development of a synthetic neoglycoconjugate vaccine against the Burkholderia cepacia pathogens.

Kinetic and dynamic kinetic resolution of secondary alcohols with ionic-surfactant-coated Burkholderia cepacia lipase: substrate scope and enantioselectivity.

Forty-four different secondary alcohols, which can be classified into several types (II-IX), were tested as the substrates of ionic surfactant-coated Burkholderia cepacia lipase (ISCBCL) to see its substrate scope and enantioselectivity in kinetic and dynamic kinetic resolution (KR and DKR). They include 6 boron-containing alcohols, 24 chiral propargyl alcohols, and 14 diarylmethanols. The results from the studies on KR indicate that ISCBCL accepted most of them with high enantioselectivity at ambient temperature and with useful to high enantioselectivity at elevated temperatures. In particular, ISCBCL displayed high enantioselectivity toward sterically demanding secondary alcohols (types VIII and IX) which have two bulky substituents at the hydroxymethine center. DKR reactions were performed by the combination of ISCBCL with a ruthenium-based racemization catalyst at 25-60 °C. Forty-one secondary alcohols were tested for DKR. About half of them were transformed into their acetates of high enantiopurity (>90% ee) with good yields (>80%). It is concluded that ISCBCL appears to be a superb enzyme for the KR and DKR of secondary alcohols.

Synthesis of the trisaccharide outer core fragment of Burkholderia cepacia pv. vietnamiensis lipooligosaccharide.

The synthesis of ß-Gal-(1→3)-α-GalNAc-(1→3)-ß-GalNAc allyl trisaccharide as the outer core fragment of Burkholderia cepacia pv. vietnamiensis lipooligosaccharide was accomplished through a concise, optimized, multi-step synthesis, having as key steps three glycosylations, that were in-depth studied performing them under several conditions. The target trisaccharide was designed with an allyl aglycone in order to open a future access to the conjugation with an immunogenic protein en route to the development of a synthetic neoglycoconjugate vaccine against this Burkholderia pathogen.

Design of biosolvents through hydroxyl functionalization of compounds with high dielectric constant.

We proposed basic principles for biosolvent design on the viewpoint of ionization. Two classes of biosolvents, based on cyclic carbonate moiety and amide moiety, were designed through hydroxyl functionalization of highly dielectric compound. The newly designed compounds, glycerol carbonate (GC) and N-hydroxymethyl formamide (HOF), were synthesized for the development of soluble enzymatic systems and characterized by (13)C NMR and (1)H NMR. All the characterization data were consistent with the expected structures. Using conductance measurements, the pK (a) values of trichloroacetic acid in GC and HOF were determined as 0.80 and 0.85 at 25.0 °C, which was very close to that in water (pK (a) = 0.70), suggesting that the ionizing and dissociating abilities of GC and HOF are similar to those of water. The effects of various reaction parameters on activity and stability of Candida antarctica lipase B and lipase from Pseudomonas cepacia were investigated using the transesterification of ethyl butyrate with n-butanol as a model reaction. The activities of lipases in GC and HOF were comparable to those in water, indicating that the newly designed compounds were biocompactible. Biosolvent design is a promising and versatile method for developing new biosolvents.

Profiling of Burkholderia cepacia secretome at mid-logarithmic and early-stationary phases of growth.

BACKGROUND: Burkholderia cepacia is a Gram-negative pathogen that causes serious respiratory infections in immunocompromised patients and individuals with cystic fibrosis. This bacterium is known to release extracellular proteins that may be involved in virulence. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, B. cepacia grown to mid-logarithmic and early-stationary phases were investigated on their ability to invade and survive intracellularly in A549 lung epithelial cells in order to discern the fate of these bacteria in the pathogenesis of B. cepacia lung infections in in vitro condition. The early-stationary phase B. cepacia was demonstrated to be more invasive than mid-logarithmic phase. In addition, culture supernatants of B. cepacia obtained from these phases of growth were also demonstrated to cause different cytotoxic potency on the A549 human lung epithelial cells. Profiling of the supernatants using the gel-based proteomics approach identified 43 proteins that were commonly released in both the growth phases and 40 proteins newly-released at the early-stationary phase. The latter proteins may account for the higher cytotoxic activity of the early-stationary culture supernatant compared to that obtained at the mid-logarithmic phase. Among the newly-released proteins in the early-stationary phase supernatant were flagellar hook-associated domain protein (FliD), flagellar hook-associated protein (FlgK), TonB-dependent siderophore (Fiu), Elongation factor G (FusA), phosphoglycerate kinase (Pgk) and sulfatase (AslA) which are known for their virulence. CONCLUSION/SIGNIFICANCE: Differences in the ability of B. cepacia to invade and survive intracellularly inside the epithelial cells at different phases of growth may improve our understanding of the varied disease progressions associated with B. cepacia infections. In addition, the identified culture supernatant proteins may be used as targets for the development of new strategies to control B. cepacia infection using agents that can block their release.

O-Acetyl location on cepacian, the principal exopolysaccharide of Burkholderia cepacia complex bacteria.

Cepacian is an exopolysaccharide produced by the majority of the isolates belonging to the Burkholderia cepacia complex bacteria, a group of 17 species, some of which infect cystic fibrosis patients, sometime with fatal outcome. The repeating unit of cepacian consists of a backbone having a trisaccharidic repeating unit with three side chains, as reported in the formula below. The exopolysaccharide is also acetylated, carrying from one to three acetyl esters per repeating unit, depending on the strain examined. The consequences of O-acetyl substitution in a polysaccharide are important both for its biological functions and for industrial applications, including the preparation of conjugated vaccines, since O-acetyl groups are important immunogenic determinants. The location of acetyl groups was achieved by NMR spectroscopy and ESI mass spectrometry and revealed that these substituents are scattered in non-stoichiometric ratio on many sugar residues in different positions, a feature which adds to the already unique carbohydrate structure of the polysaccharide.

Biological control of Botrytis cinerea using the antagonistic and endophytic Burkholderia cepacia Cs5 for vine plantlet protection.

Antifungal activity of the Burkholderia cepacia Cs5 was tested in vitro and in vivo for the control of Botrytis cinerea . Bacterial biomass was significantly improved by the amendment of ZnSO(4), Mo(7)(NH(4))(6)O(24), and mannitol to the NBY medium; consequently, the amount of the secreted fungicides was increased. The quantification of B. cinerea inhibition, in liquid and solid conditions, showed an important sensitivity of this fungus to the strain Cs5 fungicides. Microscopic monitoring impact of these fungicides on mycelium structure showed an important increase in their diameter and ramifications in the presence of 0.75% supernatant. For the in vivo application of the strain Cs5, Vitis vinifera plantlets were inoculated with a Cs5 bacterial suspension, then with B. cinerea spores. The plantlets protection was total and durable when these two inoculations were made 3 weeks apart, which is the time for the endophytic bacterium to colonize the plantlets up to the top leaves. This protection is due to Cs5 antagonism and the elicitation of the plantlets self-defense via the root overgrowth.

Structure of Burkholderia cepacia UDP-glucose dehydrogenase (UGD) BceC and role of Tyr10 in final hydrolysis of UGD thioester intermediate.

Members of the Burkholderia cepacia complex (BCC) are serious respiratory pathogens in immunocompromised individuals and in patients with cystic fibrosis (CF). They are exceptionally resistant to many antimicrobial agents and have the capacity to spread between patients, leading to a decline in lung function and necrotizing pneumonia. BCC members often express a mucoid phenotype associated with the secretion of the exopolysaccharide (EPS) cepacian. There is much evidence supporting the fact that cepacian is a major virulence factor of BCC. UDP-glucose dehydrogenase (UGD) is responsible for the NAD-dependent 2-fold oxidation of UDP-glucose (UDP-Glc) to UDP-glucuronic acid (UDP-GlcA), which is a key step in cepacian biosynthesis. Here, we report the structure of BceC, determined at 1.75-Å resolution. Mutagenic studies were performed on the active sites of UGDs, and together with the crystallographic structures, they elucidate the molecular mechanism of this family of sugar nucleotide-modifying enzymes. Superposition with the structures of human and other bacterial UGDs showed an active site with high structural homology. This family contains a strictly conserved tyrosine residue (Y10 in BceC; shown in italics) within the glycine-rich motif (GXGYXG) of its N-terminal Rossmann-like domain. We constructed several BceC Y10 mutants, revealing only residual dehydrogenase activity and thus highlighting the importance of this conserved residue in the catalytic activity of BceC. Based on the literature of the UGD/GMD nucleotide sugar 6-dehydrogenase family and the kinetic and structural data we obtained for BceC, we determined Y10 as a key catalytic residue in a UGD rate-determining step, the final hydrolysis of the enzymatic thioester intermediate.