P.L. Howell

Structure of Penicillium citrinum alpha 1,2-mannosidase reveals the basis for differences in specificity of the endoplasmic reticulum and Golgi class I enzymes.

Class I α1,2-mannosidases (glycosylhydrolase family 47) are key enzymes in the maturation of N-glycans. This protein family includes two distinct enzymatically active subgroups. Subgroup 1 includes the yeast and human endoplasmic reticulum (ER) α1,2-mannosidases that primarily trim Man9GlcNAc2 to Man8GlcNAc2 isomer B whereas subgroup 2 includes mammalian Golgi α1,2-mannosidases IA, IB, and IC that trim Man9GlcNAc2 to Man5GlcNAc2 via Man8GlcNAc2 isomers A and C. The structure of the catalytic domain of the subgroup 2 α1,2-mannosidase fromPenicillium citrinum has been determined by molecular replacement at 2.2-Å resolution. The fungal α1,2-mannosidase is an (αα)7-helix barrel, very similar to the subgroup 1 yeast (Vallée, F., Lipari, F., Yip, P., Sleno, B., Herscovics, A., and Howell, P. L. (2000) EMBO J. 19, 581–588) and human (Vallée, F., Karaveg, K., Herscovics, A., Moremen, K. W., and Howell, P. L. (2000) J. Biol. Chem. 275, 41287–41298) ER enzymes. The location of the conserved acidic residues of the catalytic site and the binding of the inhibitors, kifunensine and 1-deoxymannojirimycin, to the essential calcium ion are conserved in the fungal enzyme. However, there are major structural differences in the oligosaccharide binding site between the two α1,2-mannosidase subgroups. In the subgroup 1 enzymes, an arginine residue plays a critical role in stabilizing the oligosaccharide substrate. In the fungal α1,2-mannosidase this arginine is replaced by glycine. This replacement and other sequence variations result in a more spacious carbohydrate binding site. Modeling studies of interactions between the yeast, human and fungal enzymes with different Man8GlcNAc2 isomers indicate that there is a greater degree of freedom to bind the oligosaccharide in the active site of the fungal enzyme than in the yeast and human ER α1,2-mannosidases.

The structure and metal dependent activity of Escherichia coli PgaB provides insight into the partial de-N-acetylation of poly-β-1,6-N-acetyl-D-glucosamine

Background: Polysaccharide intercellular adhesin-dependent biofilm formation in E. coli requires the de-N-acetylation of poly-β-1,6-N-acetyl-d-glucosamine by PgaB. Results: Nickel- and iron-bound structures of PgaB have been determined, and the metal-dependent de-N-acetylase activity of the enzyme has been characterized. Conclusion: PgaB has low catalytic efficiency and shows preference for Co2+, Ni2+, and Fe2+ ions. Significance: The structure of PgaB will guide inhibitor design to combat biofilm formation. Exopolysaccharides are required for the development and integrity of biofilms produced by a wide variety of bacteria. In Escherichia coli, partial de-N-acetylation of the exopolysaccharide poly-β-1,6-N-acetyl-d-glucosamine (PNAG) by the periplasmic protein PgaB is required for polysaccharide intercellular adhesin-dependent biofilm formation. To understand the molecular basis for PNAG de-N-acetylation, the structure of PgaB in complex with Ni2+ and Fe3+ have been determined to 1.9 and 2.1 Å resolution, respectively, and its activity on β-1,6-GlcNAc oligomers has been characterized. The structure of PgaB reveals two (β/α)x barrel domains: a metal-binding de-N-acetylase that is a member of the family 4 carbohydrate esterases (CE4s) and a domain structurally similar to glycoside hydrolases. PgaB displays de-N-acetylase activity on β-1,6-GlcNAc oligomers but not on the β-1,4-(GlcNAc)4 oligomer chitotetraose and is the first CE4 member to exhibit this substrate specificity. De-N-acetylation occurs in a length-dependent manor, and specificity is observed for the position of de-N-acetylation. A key aspartic acid involved in de-N-acetylation, normally seen in other CE4s, is missing in PgaB, suggesting that the activity of PgaB is attenuated to maintain the low levels of de-N-acetylation of PNAG observed in vivo. The metal dependence of PgaB is different from most CE4s, because PgaB shows increased rates of de-N-acetylation with Co2+ and Ni2+ under aerobic conditions, and Co2+, Ni2+ and Fe2+ under anaerobic conditions, but decreased activity with Zn2+. The work presented herein will guide inhibitor design to combat biofilm formation by E. coli and potentially a wide range of medically relevant bacteria producing polysaccharide intercellular adhesin-dependent biofilms.

Structural and functional characterization of Pseudomonas aeruginosa AlgX: role of AlgX in alginate acetylation

Background: AlgX is required for the biosynthesis and export of the exopolysaccharide alginate. Results: The structure of AlgX has been determined, and the functional characterization of AlgX and mutant variants has been performed. Conclusion: AlgX contains an SGNH hydrolase-like domain and carbohydrate-binding module. Mutation of the Ser-His-Asp triad in vivo results in non-acetylated alginate. Significance: This is the first structural characterization of a polysaccharide acetyltransferase. The exopolysaccharide alginate, produced by mucoid Pseudomonas aeruginosa in the lungs of cystic fibrosis patients, undergoes two different chemical modifications as it is synthesized that alter the properties of the polymer and hence the biofilm. One modification, acetylation, causes the cells in the biofilm to adhere better to lung epithelium, form microcolonies, and resist the effects of the host immune system and/or antibiotics. Alginate biosynthesis requires 12 proteins encoded by the algD operon, including AlgX, and although this protein is essential for polymer production, its exact role is unknown. In this study, we present the X-ray crystal structure of AlgX at 2.15 Å resolution. The structure reveals that AlgX is a two-domain protein, with an N-terminal domain with structural homology to members of the SGNH hydrolase superfamily and a C-terminal carbohydrate-binding module. A number of residues in the carbohydrate-binding module form a substrate recognition “pinch point” that we propose aids in alginate binding and orientation. Although the topology of the N-terminal domain deviates from canonical SGNH hydrolases, the residues that constitute the Ser-His-Asp catalytic triad characteristic of this family are structurally conserved. In vivo studies reveal that site-specific mutation of these residues results in non-acetylated alginate. This catalytic triad is also required for acetylesterase activity in vitro. Our data suggest that not only does AlgX protect the polymer as it passages through the periplasm but that it also plays a role in alginate acetylation. Our results provide the first structural insight for a wide group of closely related bacterial polysaccharide acetyltransferases.

Distributed Replica Sampling.

We present a simple and general scheme for efficient Boltzmann sampling of conformational space by computer simulation. Multiple replicas of the system differing in temperature T or reaction coordinate λ are simulated independently. In addition, occasional stochastic moves of individual replicas in T or λ space are considered one at a time on the basis of a generalized Hamiltonian containing an extra potential energy term or bias that depends on the distribution of all replicas. The algorithm is inherently suited for shared or heterogeneous computing platforms such as a distributed network.

Modification and periplasmic translocation of the biofilm exopolysaccharide poly-β-1,6-N-acetyl-d-glucosamine

Significance Extracellular polysaccharides are important for bacterial aggregation and surface attachment during the formation of a biofilm. Bacteria living within a biofilm are more resistant to antibiotics and host defenses than those living in a free planktonic state. Poly-β-1,6-N-acetyl-d-glucosamine (PNAG) is produced by a number of pathogenic bacteria but is an insoluble polymer, making it difficult to study in vitro. Polyglucosamine subunit B (PgaB) is an outer membrane lipoprotein responsible for the deacetylation of PNAG, a key modification required for biofilm formation. Herein, we address a number of key questions related to the modification and translocation of PNAG/de–N-acetylated PNAG through the periplasmic space. The study provides valuable insight for synthase-dependent exopolysaccharide systems and a brute-force molecular dynamics approach for studying insoluble polymers using monosaccharides. Poly-β-1,6-N-acetyl-d-glucosamine (PNAG) is an exopolysaccharide produced by a wide variety of medically important bacteria. Polyglucosamine subunit B (PgaB) is responsible for the de–N-acetylation of PNAG, a process required for polymer export and biofilm formation. PgaB is located in the periplasm and likely bridges the inner membrane synthesis and outer membrane export machinery. Here, we present structural, functional, and molecular simulation data that suggest PgaB associates with PNAG continuously during periplasmic transport. We show that the association of PgaB’s N- and C-terminal domains forms a cleft required for the binding and de–N-acetylation of PNAG. Molecular dynamics (MD) simulations of PgaB show a binding preference for N-acetylglucosamine (GlcNAc) to the N-terminal domain and glucosammonium to the C-terminal domain. Continuous ligand binding density is observed that extends around PgaB from the N-terminal domain active site to an electronegative groove on the C-terminal domain that would allow for a processive mechanism. PgaB’s C-terminal domain (PgaB310–672) directly binds PNAG oligomers with dissociation constants of ∼1–3 mM, and the structures of PgaB310–672 in complex with β-1,6-(GlcNAc)6, GlcNAc, and glucosamine reveal a unique binding mode suitable for interaction with de–N-acetylated PNAG (dPNAG). Furthermore, PgaB310–672 contains a β-hairpin loop (βHL) important for binding PNAG that was disordered in previous PgaB42–655 structures and is highly dynamic in the MD simulations. We propose that conformational changes in PgaB310–672 mediated by the βHL on binding of PNAG/dPNAG play an important role in the targeting of the polymer for export and its release.

Intragenic Complementation at the Human Argininosuccinate Lyase Locus IDENTIFICATION OF THE MAJOR COMPLEMENTING ALLELES

To determine the molecular and biochemical basis of intragenic complementation observed at the human argininosuccinate lyase (ASL) locus, we identified the ASL alleles in ASL-deficient cell strains with two unique complementation phenotypes: (i) frequent complementers, strains that participated in the majority of complementation events, and (ii) high activity complementers, strains in which complementation was associated with a relatively high level of restoration of ASL activity. Four mutations (Q286R, D87G, A398D, and a deletion of exon 13) were identified in the four strains examined. One of the two frequent complementers was homozygous, and the other heterozygous, for the Q286R allele. Similarly, one of the two high activity complementers was homozygous, and the other heterozygous, for the D87G allele. When the Q286R and D87G mutations were introduced by site-directed mutagenesis into wild-type ASL cDNA, each conferred loss of ASL activity in COS cell transfection assays. To test directly the hypothesis that intragenic complementation occurs at the ASL locus, one of the major complementation events observed previously, between strains carrying the Q286R and D87G alleles, was reconstructed in COS cell transfection assays. A partial restoration of ASL activity, comparable with the increase seen in the fibroblast complementation analysis, was observed on joint cotransfection of these two alleles. The results provide molecular confirmation of the major features of the ASL mutant complementation map, identify the Q286R and D87D alleles as the frequent and high activity complementing alleles, respectively, and provide direct proof of intragenic complementation at the ASL locus.

Structures of 5-methylthioribose kinase reveal substrate specificity and unusual mode of nucleotide binding.

The methionine salvage pathway is ubiquitous in all organisms, but metabolic variations exist between bacteria and mammals. 5-Methylthioribose (MTR) kinase is a key enzyme in methionine salvage in bacteria and the absence of a mammalian homolog suggests that it is a good target for the design of novel antibiotics. The structures of the apo-form of Bacillus subtilis MTR kinase, as well as its ADP, ADP-PO4, AMPPCP, and AMPPCP-MTR complexes have been determined. MTR kinase has a bilobal eukaryotic protein kinase fold but exhibits a number of unique features. The protein lacks the DFG motif typically found at the beginning of the activation loop and instead coordinates magnesium via a DXE motif (Asp250-Glu252). In addition, the glycine-rich loop of the protein, analogous to the “Gly triad” in protein kinases, does not interact extensively with the nucleotide. The MTR substrate-binding site consists of Asp233 of the catalytic HGD motif, a novel twin arginine motif (Arg340/Arg341), and a semi-conserved W-loop, which appears to regulate MTR binding specificity. No lobe closure is observed for MTR kinase upon substrate binding. This is probably because the enzyme lacks the lobe closure/inducing interactions between the C-lobe of the protein and the ribosyl moiety of the nucleotide that are typically responsible for lobe closure in protein kinases. The current structures suggest that MTR kinase has a dissociative mechanism.

Expression, purification, crystallization and preliminary X-ray analysis of Pseudomonas fluorescens AlgK

AlgK is an outer-membrane lipoprotein involved in the biosynthesis of alginate in Pseudomonads and Azotobacter vinelandii. A recombinant form of Pseudomonas fluorescens AlgK with a C-terminal polyhistidine affinity tag has been expressed and purified from the periplasm of Escherichia coli cells and diffraction-quality crystals of AlgK have been grown using the hanging-drop vapour-diffusion method. The crystals grow as flat plates with unit-cell parameters a = 79.09, b = 107.85, c = 119.15 A, beta = 96.97 degrees. The crystals exhibit the symmetry of space group P2(1) and diffract to a minimum d-spacing of 2.5 A at Station X29 of the National Synchrotron Light Source, Brookhaven National Laboratory. On the basis of the Matthews coefficient (V(M) = 2.53 A3 Da(-1)), four protein molecules are estimated to be present in the asymmetric unit.