Direct and indirect roles of His‐418 in metal binding and in the activity of β‐galactosidase (E. coli)

The active site of ß‐galactosidase (E. coli) contains a Mg²⁺ ion ligated by Glu‐416, His‐418 and Glu‐461 plus three water molecules. A Na⁺ ion binds nearby. To better understand the role of the active site Mg²⁺ and its ligands, His‐418 was substituted with Asn, Glu and Phe. The Asn‐418 and Glu‐418 variants could be crystallized and the structures were shown to be very similar to native enzyme. The Glu‐418 variant showed increased mobility of some residues in the active site, which explains why the substitutions at the Mg²⁺ site also reduce Na⁺ binding affinity. The Phe variant had reduced stability, bound Mg²⁺ weakly and could not be crystallized. All three variants have low catalytic activity due to large decreases in the degalactosylation rate. Large decreases in substrate binding affinity were also observed but transition state analogs bound as well or better than to native. The results indicate that His‐418, together with the Mg²⁺, modulate the central role of Glu‐461 in binding and as a general acid/base catalyst in the overall catalytic mechanism. Glucose binding as an acceptor was also dramatically decreased, indicating that His‐418 is very important for the formation of allolactose (the natural inducer of the lac operon).     

Kinetic characterization of galacto‐oligosaccharide (GOS) synthesis by three commercially important β‐galactosidases

Many β‐galactosidases show large differences in galacto‐oligosaccharide (GOS) production and lactose hydrolysis. In this study, a kinetic model is developed in which the effect of lactose, glucose, galactose, and oligosaccharides on the oNPG converting activity of various β‐galactosidases is quantified. The use of oNPG as a competing substrate to lactose yields more information than can be obtained by examining only the conversion of lactose itself. The reaction rate with lactose or oligosaccharides as substrate relative to that with water as acceptor is much higher for the β‐galactosidase of Bacillus circulans than the β‐galactosidases of Aspergillus oryzae and Kluyveromyces lactis. In addition, the β‐galactosidase of B.circulans has a high reaction rate with galactose as acceptor, in contrast to those of A. oryzae and K. lactis. The latter two are strongly inhibited by galactose. These differences explain why β‐galactosidase of B. circulans gives higher yields in GOS production than other β‐galactosidases. Many of the reaction rate constants for the β‐galactosidase isoforms of B. circulans increase with increasing molecular weight of the isoform. This indicates that the largest isoform β‐gal‐A is most active in GOS production. However, its hydrolysis rate is also much higher than that of the other isoforms, which results in a faster hydrolysis of oligosaccharides as well. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:38–47, 2014     

The structure of DesR from Streptomyces venezuelae,a β‐glucosidase involved in macrolide activation

Antibiotics have, indeed, altered the course of human history as is evidenced by the increase in human life expectancy since the 1940s. Many of these natural compounds are produced by bacteria that, by necessity, must have efficient self‐resistance mechanisms. The methymycin/pikromycin producing species Streptomyces venezuelae, for example, utilizes β‐glucosylation of its macrolide products to neutralize their effects within the confines of the cell. Once released into the environment, these compounds are activated by the removal of the glucose moiety. In S. venezuelae, the enzyme responsible for removal of the sugar from the parent compound is encoded by the desR gene and referred to as DesR. It is a secreted enzyme containing 828 amino acid residues, and it is known to be a retaining glycosidase. Here, we describe the structure of the DesR/D ‐glucose complex determined to 1.4‐Å resolution. The overall architecture of the enzyme can be envisioned in terms of three regions: a catalytic core and two auxiliary domains. The catalytic core harbors the binding platform for the glucose ligand. The first auxiliary domain adopts a “PA14 fold,” whereas the second auxiliary domain contains an immunoglobulin‐like fold. Asp 273 and Glu 578 are in the proper orientation to function as the catalytic base and proton donor, respectively, required for catalysis. The overall fold of the core region places DesR into the GH3 glycoside hydrolase family of enzymes. Comparison of the DesR structure with the β‐glucosidase from Kluyveromyces marxianus shows that their PA14 domains assume remarkably different orientations.     

Inter‐subunit interaction of gastric H+,K+‐ATPase prevents reverse reaction of the transport cycle

The gastric H⁺,K⁺‐ATPase is an ATP‐driven proton pump responsible for generating a million‐fold proton gradient across the gastric membrane. We present the structure of gastric H⁺,K⁺‐ATPase at 6.5 Å resolution as determined by electron crystallography of two‐dimensional crystals. The structure shows the catalytic α‐subunit and the non‐catalytic β‐subunit in a pseudo‐E₂P conformation. Different from Na⁺,K⁺‐ATPase, the N‐terminal tail of the β‐subunit is in direct contact with the phosphorylation domain of the α‐subunit. This interaction may hold the phosphorylation domain in place, thus stabilizing the enzyme conformation and preventing the reverse reaction of the transport cycle. Indeed, truncation of the β‐subunit N‐terminus allowed the reverse reaction to occur. These results suggest that the β‐subunit N‐terminus prevents the reverse reaction from E₂P to E₁P, which is likely to be relevant for the generation of a large H⁺ gradient in vivo situation.     

Structural and functional analysis of tomato β‐galactosidase 4: insight into the substrate specificity of the fruit softening‐related enzyme

Plant β‐galactosidases hydrolyze cell wall β‐(1,4)‐galactans to play important roles in cell wall expansion and degradation, and turnover of signaling molecules, during ripening. Tomato β‐galactosidase 4 (TBG4) is an enzyme responsible for fruit softening through the degradation of β‐(1,4)‐galactan in the pericarp cell wall. TBG4 is the only enzyme among TBGs 1–7 that belongs to the β‐galactosidase/exo‐β‐(1,4)‐galactanase subfamily. The enzyme can hydrolyze a wide range of plant‐derived (1,4)‐ or 4‐linked polysaccharides, and shows a strong ability to attack β‐(1,4)‐galactan. To gain structural insight into its substrate specificity, we determined crystal structures of TBG4 and its complex with β‐d ‐galactose. TBG4 comprises a catalytic TIM barrel domain followed by three β‐sandwich domains. Three aromatic residues in the catalytic site that are thought to be important for substrate specificity are conserved in GH35 β‐galactosidases derived from bacteria, fungi and animals; however, the crystal structures of TBG4 revealed that the enzyme has a valine residue (V548) replacing one of the conserved aromatic residues. The V548W mutant of TBG4 showed a roughly sixfold increase in activity towards β‐(1,6)‐galactobiose, and ~0.6‐fold activity towards β‐(1,4)‐galactobiose, compared with wild‐type TBG4. Amino acid residues corresponding to V548 of TBG4 thus appear to determine the substrate specificities of plant β‐galactosidases towards β‐1,4 and β‐1,6 linkages.     

High‐level expression of Aspergillus niger β‐galactosidase in Ashbya gossypii

Ashbya gossypii has been recently considered as a host for the expression of recombinant proteins. The production levels achieved thus far were similar to those obtained with Saccharomyces cerevisiae for the same proteins. Here, the β‐galactosidase from Aspergillus niger was successfully expressed and secreted by A. gossypii from 2‐µm plasmids carrying the native signal sequence at higher levels than those secreted by S. cerevisiae laboratorial strains. Four different constitutive promoters were used to regulate the expression of β‐galactosidase: A. gossypii AgTEF and AgGPD promoters, and S. cerevisiae ScADH1 and ScPGK1 promoters. The native AgTEF promoter drove the highest expression levels of recombinant β‐galactosidase in A. gossypii, leading to 2‐ and 8‐fold higher extracellular activity than the AgGPD promoter and the heterologous promoters, respectively. In similar production conditions, the levels of active β‐galactosidase secreted by A. gossypii were up to 37 times higher than those secreted by recombinant S. cerevisiae and ～2.5 times higher than those previously reported for the β‐galactosidase‐high producing S. cerevisiae NCYC869‐A3/pVK1.1. The substitution of glucose by glycerol in the production medium led to a 1.5‐fold increase in the secretion of active β‐galactosidase by A. gossypii. Recombinant β‐galactosidase secreted by A. gossypii was extensively glycosylated, as are the native A. niger β‐galactosidase and recombinant β‐galactosidase produced by yeast. These results highlight the potential of A. gossypii as a recombinant protein producer and open new perspectives to further optimize recombinant protein secretion in this fungus. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:261–268, 2014     

Structure of a glycoside hydrolase family 50 enzyme from a subfamily that is enriched in human gut microbiome bacteroidetes

The polysaccharide utilization locus in Bacteroides plebeius that confers the ability to catabolize porphyran contains a putative GH50 β‐agarase (BACPLE_01683, BpGH50). BpGH50 did not show any clear activity on agarose or on the related algal galactans porphyran and carrageenan. However, the 1.4 Å resolution X‐ray crystal structure of BpGH50 confirmed its possession of the core (α/β)₈ barrel fold found in GH50 enzymes as well as the structural conservation of the catalytic residues and some substrate binding residues. Examination of the structure supports assignment of this protein as a β‐galactosidase but suggests that it may utilize a different, possibly hybrid, algal galactan substrate. Proteins 2016; 85:182–187. © 2016 Wiley Periodicals, Inc.     

Re‐examining the role of Lys67 in class C β‐lactamase catalysis

Lys67 is essential for the hydrolysis reaction mediated by class C β‐lactamases. Its exact catalytic role lies at the center of several different proposed reaction mechanisms, particularly for the deacylation step, and has been intensely debated. Whereas a conjugate base hypothesis postulates that a neutral Lys67 and Tyr150 act together to deprotonate the deacylating water, previous experiments on the K67R mutants of class C β‐lactamases suggested that the role of Lys67 in deacylation is mainly electrostatic, with only a 2‐ to 3‐fold decrease in the rate of the mutant vs the wild type enzyme. Using the Class C β‐lactamase AmpC, we have reinvestigated the activity of this K67R mutant enzyme, using biochemical and structural studies. Both the rates of acylation and deacylation were affected in the AmpC K67R mutant, with a 61‐fold decrease in k_cat, the deacylation rate. We have determined the structure of the K67R mutant by X‐ray crystallography both in apo and transition state‐analog complexed forms, and observed only minimal conformational changes in the catalytic residues relative to the wild type. These results suggest that the arginine side chain is unable to play the same catalytic role as Lys67 in either the acylation or deacylation reactions catalyzed by AmpC. Therefore, the activity of this mutant can not be used to discredit the conjugate base hypothesis as previously concluded, although the reaction catalyzed by the K67R mutant itself likely proceeds by an alternative mechanism. Indeed, a manifold of mechanisms may contribute to hydrolysis in class C β‐lactamases, depending on the enzyme (wt or mutant) and the substrate, explaining why different mutants and substrates seem to support different pathways. For the WT enzyme itself, the conjugate base mechanism may be well favored.     

Structural basis for the β‐lactamase activity of EstU1, a family VIII carboxylesterase

EstU1 is a unique family VIII carboxylesterase that displays hydrolytic activity toward the amide bond of clinically used β‐lactam antibiotics as well as the ester bond of p‐nitrophenyl esters. EstU1 assumes a β‐lactamase‐like modular architecture and contains the residues Ser100, Lys103, and Tyr218, which correspond to the three catalytic residues (Ser64, Lys67, and Tyr150, respectively) of class C β‐lactamases. The structure of the EstU1/cephalothin complex demonstrates that the active site of EstU1 is not ideally tailored to perform an efficient deacylation reaction during the hydrolysis of β‐lactam antibiotics. This result explains the weak β‐lactamase activity of EstU1 compared with class C β‐lactamases. Finally, structural and sequential comparison of EstU1 with other family VIII carboxylesterases elucidates an operative molecular strategy used by family VIII carboxylesterases to extend their substrate spectrum. Proteins 2013; 81:2045–2051. © 2013 Wiley Periodicals, Inc.     

Chaperone‐like activities of different molecular forms of β‐casein. Importance of polarity of N‐terminal hydrophilic domain

As a member of intrinsically unstructured protein family, β‐casein (β‐CN) contains relatively high amount of prolyl residues, adopts noncompact and flexible structure and exhibits chaperone‐like activity in vitro. Like many chaperones, native β‐CN does not contain cysteinyl residues and exhibits strong tendencies for self‐association. The chaperone‐like activities of three recombinant β‐CNs wild type (WT) β‐CN, C4 β‐CN (with cysteinyl residue in position 4) and C208 β‐CN (with cysteinyl residue in position 208), expressed and purified from E. coli, which, consequently, lack the phosphorylated residues, were examined and compared with that of native β‐CN using insulin and alcohol dehydrogenase as target/substrate proteins. The dimers (β‐CND) of C4‐β‐CN and C208 β‐CN were also studied and their chaperone‐like activities were compared with those of their monomeric forms. Lacking phosphorylation, WT β‐CN, C208 β‐CN, C4 β‐CN and C4 β‐CND exhibited significantly lower chaperone‐like activities than native β‐CN. Dimerization of C208 β‐CN with two distal hydrophilic domains considerably improved its chaperone‐like activity in comparison with its monomeric form. The obtained results demonstrate the significant role played by the polar contributions of phosphorylated residues and N‐terminal hydrophilic domain as important functional elements in enhancing the chaperone‐like activity of native β‐CN. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 623–632, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

C‐terminal β‐strand swapping in a consensus‐derived fibronectin Type III scaffold

The crystal structures of six different fibronectin Type III consensus‐derived Tencon domains, whose solution properties exhibit no, to various degrees of, aggregation according to SEC, have been determined. The structures of the five variants showing aggregation reveal 3D domain swapped dimers. In all five cases, the swapping involves the C‐terminal β‐strand resulting in the formation of Tencon dimers in which the target‐binding surface is blocked. All of the variants differ in sequence in the FG loop, which is the hinge loop in the β‐strand‐swapped dimers. The six tencon variants have between 0 and 5 residues inserted between positions 77 and 78 in the FG loop. Analysis of the structures suggests that a non‐glycine residue at position 77 and insertions of <4 residues may destabilize the β‐turn in the FG loop promoting β‐strand swapping. Swapped dimers with an odd number of inserted residues may be less stable, particularly if they contain proline residues, because they cannot form perfect β‐bridges in the FG regions that link the swapped dimers. The Tencon β‐swapped variants with the longest FG sequences are observed to form higher order hexameric or helical oligomeric structures in the crystal correlating well with the aggregation properties of these domains observed in solution. Understanding the structural basis for domain‐swapped dimerization and oligomerization will support engineering efforts of the Tencon domain to produce variants with desired biophysical properties. Proteins 2014; 82:1359–1369. © 2013 Wiley Periodicals, Inc.     

In situ immobilization of β‐galactosidase from Bacillus circulans in silica by sol‐gel process: Application in prebiotic synthesis

The enzyme encapsulation is a very well‐known stabilization pathway. However, there are some challenges in order to avoid the enzyme denaturation under encapsulation conditions. The β‐galactosidase from Bacillus circulans was immobilized through sol‐gel encapsulation route assisted by Triton X‐100 surfactant and sugars. The effects of sugar presence in the immobilization process and the gelation time on the biocatalyst activity/stability were explained taking into account the characteristics of the formed silica matrix and the changes of the enzyme environment. The enzyme was effectively immobilized by this strategy, with high immobilization yield in terms of activity (29%) and expressed activity (47 IU/g). The immobilization through silica sol‐gel in the presence of 1×10^?3 M Triton X‐100 and fructose conferred 28.4‐fold higher stability to the enzyme compared with the soluble form. This is an advantage for its use in the synthesis of the galacto‐oligosaccharides at 50ºC. The total lactose conversion to galacto‐oligosaccharides was 26%wt, which is comparable with that reported in the literature. The obtained biocatalyst is useful for the synthesis of galacto‐oligosaccharides and its catalytic behavior is rationalized in this work.     

Heterologous expression and periplasmic secretion of an antifungal Bacillus amyloliquefaciensBLB369 endo‐β‐1,3‐1,4‐glucanase in Escherichia coli

The endo‐β‐1,3‐1,4‐glucanases are glycoside hydrolases involved in the enzymatic depolymerization of 1,3‐1,4 β‐glucans and showed an antifungal activity against some fungi. Bacillus amyloliquefaciensBLB369 has a high antagonistic activity against phytopathogenic fungi. Its glu369 full‐coding sequence of the endo‐β‐1,3‐1,4‐glucanase gene (732 bp) was sequenced, cloned and successfully expressed in Escherichia coli Top10. The encoded protein (243 amino acids) has a calculated molecular mass of 27.3 kDa. To simplify the purification procedure, the glu369 coding sequence was cloned into the vector pKJD4. The produced OmpA‐His‐Glu369 harboured OmpA signal sequence for E. coli periplasmic localization and followed by a 6His residues for its purification. The purified His‐tagged proteins revealed two bands on SDS‐PAGE analysis with molecular masses of about 30.5 (His‐Glu369) and 32.5 kDa (OmpA‐His‐Glu369). They had the ability to inhibit the growth of phytopathogenic fungus Alternaria alternata. These favourable properties make the endo‐β‐1,3‐1,4‐glucanase a good candidate for biotechnological applications.     

BGAL1 depletion boosts the level of β‐galactosylation of N‐ and O‐glycans in N. benthamiana

Glyco‐design of proteins is a powerful tool in fundamental studies of structure–function relationship and in obtaining profiles optimized for efficacy of therapeutic glycoproteins. Plants, particularly Nicotiana benthamiana, are attractive hosts to produce recombinant glycoproteins, and recent advances in glyco‐engineering facilitate customized N‐glycosylation of plant‐derived glycoproteins. However, with exception of monoclonal antibodies, homogenous human‐like β1,4‐galactosylation is very hard to achieve in recombinant glycoproteins. Despite significant efforts to optimize the expression of β1,4‐galactosyltransferase, many plant‐derived glycoproteins still exhibit incomplete processed N‐glycans with heterogeneous terminal galactosylation. The most obvious suspects to be involved in trimming terminal galactose residues are β‐galactosidases (BGALs) from the glycosyl hydrolase family GH35. To elucidate the so far uncharacterized mechanisms leading to the trimming of terminal galactose residues from glycans of secreted proteins, we studied a N. benthamiana BGAL known to be active in the apoplast (NbBGAL1). Here, we determined the NbBGAL1 subcellular localization, substrate specificity and in planta biological activity. We show that NbBGAL1 can remove β1,4‐ and β1,3‐galactose residues on both N‐ and O‐glycans. Transient BGAL1 down‐regulation by RNA interference (RNAi) and BGAL1 depletion by genome editing drastically reduce β‐galactosidase activity in N. benthamiana and increase the amounts of fully galactosylated complex N‐glycans on several plant‐produced glycoproteins. Altogether, our data demonstrate that NbBGAL1 acts on galactosylated complex N‐glycans of plant‐produced glycoproteins.     

QM/MM studies on the calcium‐assisted β‐elimination mechanism of pectate lyase from bacillus subtilis

Pectate lyase utilizes the anti‐β‐elimination chemistry to catalyze the cleavage of α‐1,4 glycosidic bond between _D‐galacturonate regions during the degradation of plant polysaccharide pectin. We report here detailed mechanistic studies of the Bacillus subtilis pectate lyase (BsPel) using QM/MM calculations. It was found that the residue Arg279 serves as the catalytic base to abstract the α‐proton from C₅₂ atom of substrate Ada2 subsite, forming an unstable carbanion intermediate. The glycosidic bond of this intermediate is scissile to generate the 4,5‐unsaturated digalacturonate product and a negatively charged β‐leaving group. Two active site residues (Lys247 and Arg279) and two Ca²⁺ ions (Ca2 and Ca3) form hydrogen‐bonding and coordination interactions with C₅₂? COO^? of Ada2, respectively, which facilitate the proton abstraction and stabilize the generated carbanion intermediates. Arg284 is not the potential proton donor to saturate the leaving group. Actually, the proton source of leaving group is the solvent water molecule rather than any active site acidic residues. In addition, the calculation results suggest that careful selections of QM‐ and Active‐regions are essential to accurately explore the enzymatic reactions. Proteins 2016; 84:1606–1615. © 2016 Wiley Periodicals, Inc.     

Insight into a strategy for attenuating AmpC‐mediated β‐lactam resistance: Structural basis for selective inhibition of the glycoside hydrolase NagZ

NagZ is an exo‐N‐acetyl‐β‐glucosaminidase, found within Gram‐negative bacteria, that acts in the peptidoglycan recycling pathway to cleave N‐acetylglucosamine residues off peptidoglycan fragments. This activity is required for resistance to cephalosporins mediated by inducible AmpC β‐lactamase. NagZ uses a catalytic mechanism involving a covalent glycosyl enzyme intermediate, unlike that of the human exo‐N‐acetyl‐β‐glucosaminidases: O‐GlcNAcase and the β‐hexosaminidase isoenzymes. These latter enzymes, which remove GlcNAc from glycoconjugates, use a neighboring‐group catalytic mechanism that proceeds through an oxazoline intermediate. Exploiting these mechanistic differences we previously developed 2‐N‐acyl derivatives of O‐(2‐acetamido‐2‐deoxy‐D ‐glucopyranosylidene)amino‐N‐phenylcarbamate (PUGNAc), which selectively inhibits NagZ over the functionally related human enzymes and attenuate antibiotic resistance in Gram‐negatives that harbor inducible AmpC. To understand the structural basis for the selectivity of these inhibitors for NagZ, we have determined its crystallographic structure in complex with N‐valeryl‐PUGNAc, the most selective known inhibitor of NagZ over both the human β‐hexosaminidases and O‐GlcNAcase. The selectivity stems from the five‐carbon acyl chain of N‐valeryl‐PUGNAc, which we found ordered within the enzyme active site. In contrast, a structure determination of a human O‐GlcNAcase homologue bound to a related inhibitor N‐butyryl‐PUGNAc, which bears a four‐carbon chain and is selective for both NagZ and O‐GlcNAcase over the human β‐hexosamnidases, reveals that this inhibitor induces several conformational changes in the active site of this O‐GlcNAcase homologue. A comparison of these complexes, and with the human β‐hexosaminidases, reveals how selectivity for NagZ can be engineered by altering the 2‐N‐acyl substituent of PUGNAc to develop inhibitors that repress AmpC mediated β‐lactam resistance.     

A novel enzymatic microreactor with Aspergillus oryzae β‐galactosidase immobilized on silicon dioxide nanosprings

The use of silicon dioxide (SiO₂) nanosprings as supports for immobilized enzymes in a continuous microreactor is described. A nanospring mat (2.2 cm² × 60 μm thick) was functionalized with γ‐aminopropyltriethoxysilane, then treated with N‐succinimidyl‐3‐(2‐pyridyldithio)‐propionate (SPDP) and dithiothreitol (DTT) to produce surface thiol (? SH) groups. SPDP‐modified β‐galactosidase from Aspergillus oryzae was immobilized on the thiolated nanosprings by reversible disulfide linkages. The enzyme‐coated nanospring mat was placed into a 175‐μm high microchannel, with the mat partially occluding the channel. The kinetics and steady‐state conversion of hydrolysis of o‐nitrophenyl β‐D ‐galactosylpyranoside at various substrate flow rates and concentrations were measured. Substantial flow was observed through the nanosprings, for which the Darcy permeability κ ≈ 3 × 10^?6 cm². A simple, one‐parameter numerical model coupling Navier‐Stokes and Darcy flow with a pseudo‐first‐order reaction was used to fit the experimental data. Simulated reactor performance was sensitive to changes in κ and the height of the nanospring mat. Permeabilities lower than 10^?8 cm² practically eliminated convective flow through the nanosprings, and substantially decreased conversion. Increasing the height of the mat increased conversion in simulations, but requires more enzymes and could cause sealing issues if grown above channel walls. Preliminary results indicate that in situ regeneration by reduction with DTT and incubation with SPDP‐modified β‐galactosidase is possible. Nanosprings provide high solvent‐accessible surface area with good permeability and mechanical stability, can be patterned into existing microdevices, and are amenable to immobilization of biomolecules. Nanosprings offer a novel and useful support for enzymatic microreactors, biosensors, and lab‐on‐chip devices. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

p‐Coumaric acid decarboxylase from Lactobacillus plantarum: Structural insights into the active site and decarboxylation catalytic mechanism

p‐Coumaric acid decarboxylases (PDCs) catalyze the nonoxidative decarboxylation of hydroxycinnamic acids to generate the corresponding vinyl derivatives. Despite the biotechnological relevance of PDCs in food industry, their catalytic mechanism remains largely unknown. Here, we report insights into the structural basis of catalysis for the homodimeric PDC from Lactobacillus plantarum (LpPDC). The global fold of LpPDC is based on a flattened β‐barrel surrounding an internal cavity. Crystallographic and functional analyses of single‐point mutants of residues located within this cavity have permitted identifying a potential substrate‐binding pocket and also to provide structural evidences for rearrangements of surface loops so that they can modulate the accessibility to the active site. Finally, combination of the structural and functional data with in silico results enables us to propose a two‐step catalytic mechanism for decarboxylation of p‐coumaric acid by PDCs where Glu71 is involved in proton transfer, and Tyr18 and Tyr20 are involved in the proper substrate orientation and in the release of the CO₂ product. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Acyl acceptor recognition by Enterococcus faecium l,d‐transpeptidase Ldtfm

In Mycobacterium tuberculosis and ampicillin‐resistant mutants of Enterococcus faecium, the classical target of β‐lactam antibiotics is bypassed by l ,d ‐transpeptidases that form unusual 3 → 3 peptidoglycan cross‐links. β‐lactams of the carbapenem class, such as ertapenem, are mimics of the acyl donor substrate and inactivate l ,d ‐transpeptidases by acylation of their catalytic cysteine. We have blocked the acyl donor site of E. faecium l ,d ‐transpeptidase Ldt_fm by ertapenem and identified the acyl acceptor site based on analyses of chemical shift perturbations induced by binding of peptidoglycan fragments to the resulting acylenzyme. An nuclear magnetic resonance (NMR)‐driven docking structure of the complex revealed key hydrogen interactions between the acyl acceptor and Ldt_fm that were evaluated by site‐directed mutagenesis and development of a cross‐linking assay. Three residues are reported as critical for stabilisation of the acceptor in the Ldt_fm active site and proper orientation of the nucleophilic nitrogen for the attack of the acylenzyme carbonyl. Identification of the catalytic pocket dedicated to the acceptor substrate opens new perspectives for the design of inhibitors with an original mode of action that could act alone or in synergy with β‐lactams.     

Crystal structure of a GH1 β‐glucosidase from Hamamotoa singularis

A GH1 β‐glucosidase from the fungus Hamamotoa singularis (HsBglA) has high transgalactosylation activity and efficiently converts lactose to galactooligosaccharides. Consequently, HsBglA is among the most widely used enzymes for industrial galactooligosaccharide production. Here, we present the first crystal structures of HsBglA with and without 4′‐galactosyllactose, a tri‐galactooligosaccharide, at 3.0 and 2.1 Å resolutions, respectively. These structures reveal details of the structural elements that define the catalytic activity and substrate binding of HsBglA, and provide a possible interpretation for its high catalytic potency for transgalactosylation reaction.