Structure and Function of the DUF2233 Domain in Bacteria and in the Human Mannose 6-Phosphate Uncovering Enzyme

DUF2233, a domain of unknown function (DUF), is present in many bacterial and several viral proteins and was also identified in the mammalian transmembrane glycoprotein N-acetylglucosamine-1-phosphodiester α-N-acetylglucosaminidase (“uncovering enzyme” (UCE)). We report the crystal structure of BACOVA_00430, a 315-residue protein from the human gut bacterium Bacteroides ovatus that is the first structural representative of the DUF2233 protein family. A notable feature of this structure is the presence of a surface cavity that is populated by residues that are highly conserved across the entire family. The crystal structure was used to model the luminal portion of human UCE (hUCE), which is involved in targeting of lysosomal enzymes. Mutational analysis of several residues in a highly conserved surface cavity of hUCE revealed that they are essential for function. The bacterial enzyme (BACOVA_00430) has ∼1% of the catalytic activity of hUCE toward the substrate GlcNAc-P-mannose, the precursor of the Man-6-P lysosomal targeting signal. GlcNAc-1-P is a poor substrate for both enzymes. We conclude that, for at least a subset of proteins in this family, DUF2233 functions as a phosphodiester glycosidase.     

Lysosomal Hydrolase Mannose 6-Phosphate Uncovering Enzyme Resides in the trans-Golgi Network

A crucial step in lysosomal biogenesis is catalyzed by "uncovering" enzyme (UCE), which removes a covering N-acetylglucosamine from the mannose 6-phosphate (Man-6-P) recognition marker on lysosomal hydrolases. This study shows that UCE resides in the trans-Golgi network (TGN) and cycles between the TGN and plasma membrane. The cytosolic domain of UCE contains two potential endocytosis motifs: (488)YHPL and C-terminal (511)NPFKD. YHPL is shown to be the more potent of the two in retrieval of UCE from the plasma membrane. A green-fluorescent protein-UCE transmembrane-cytosolic domain fusion protein colocalizes with TGN 46, as does endogenous UCE in HeLa cells, showing that the transmembrane and cytosolic domains determine intracellular location. These data imply that the Man-6-P recognition marker is formed in the TGN, the compartment where Man-6-P receptors bind cargo and are packaged into clathrin-coated vesicles.     

Three-dimensional structure of gurmarin,a sweet taste-suppressing polypeptide

Summary The solution structure of gurmarin was studied by two-dimensional proton NMR spectroscopy at 600 MHz. Gurmarin, a 35-amino acid residue polypeptide recently discovered in an Indian-originated tree Gymnema sylvestre, selectively suppresses the neural responses of rat to sweet taste stimuli. Sequence-specific protons. The three-dimensional solution structure was determined by simulated-annealing calculations on the basis of 135 interproton distance constraints derived from NOEs, six distance constraints for three hydrogen bonds and 16 dihedral angle constraints derived from coupling constants. A total of 10 structures folded into a well-defined structure with a triple-stranded antiparallel -sheet. The average rmsd values between any two structures were 1.65±0.39 Å for the backbone atoms (N, C, C) and 2.95±0.27 Å for all heavy atoms. The positions of the three disulfide bridges, which could not be deterermined chemically, were estimated to be Cys³–Cys¹⁸, Cys¹⁰–Cys²³ and Cys¹⁷–Cys³³ on the basis of the NMR distance constraints. This disulfide bridge pattern in gurmarin turned out to be analogous to that in -conotoxin and Momordica charantia trypsin inhibitor-II, and the topology of folding was the same as that in -conotoxin.Abbreviations DQF-COSY double-quantum-filtered correlated spectroscopy - HOHAHA homonuclear Hartmann-Hahn spectroscopy - NOESY nuclear Overhauser enhancement spectroscopy - ppm parts per million; rmsd, root-mean-square deviation - TSP 3-(trimethylsilyl)-2,2,3,3-tetradeutero-propionate     

Promiscuous and specific phospholipid binding by domains in ZAC,a membrane-associated Arabidopsis protein with an ARF GAP zinc finger and a C2 domain

Arabidopsis proteins were predicted which share an 80 residue zinc finger domain known from ADP-ribosylation factor GTPase-activating proteins (ARF GAPs). One of these is a 37 kDa protein, designated ZAC, which has a novel domain structure in which the N-terminal ARF GAP domain and a C-terminal C2 domain are separated by a region without homology to other known proteins. Zac promoter/-glucuronidase reporter assays revealed highest expression levels in flowering tissue, rosettes and roots. ZAC protein was immuno-detected mainly in association with membranes and fractionated with Golgi and plasma membrane marker proteins. ZAC membrane association was confirmed in assays by a fusion between ZAC and the green fluorescence protein and prompted an analysis of the in vitro phospholipid-binding ability of ZAC. Phospholipid dot-blot and liposome-binding assays indicated that fusion proteins containing the ZAC-C2 domain bind anionic phospholipids non-specifically, with some variance in Ca²⁺ and salt dependence. Similar assays demonstrated specific affinity of the ZAC N-terminal region (residues 1–174) for phosphatidylinositol 3-monophosphate (PI-3-P). Binding was dependent in part on an intact zinc finger motif, but proteins containing only the zinc finger domain (residues 1–105) did not bind PI-3-P. Recombinant ZAC possessed GTPase-activating activity on Arabidopsis ARF proteins. These data identify a novel PI-3-P-binding protein region and thereby provide evidence that this phosphoinositide is recognized as a signal in plants. A role for ZAC in the regulation of ARF-mediated vesicular transport in plants is discussed.     

Interactions among Stalk Modules of Thrombospondin-1

Thrombospondin-1 is a trimeric, modular calcium-binding glycoprotein. The subunit is composed of an N-terminal module; oligomerization domain; stalk modules including a von Willebrand factor type C module, three properdin or thrombospondin type 1 repeat (TSR) modules, and two thrombospondin-type EGF-like modules; and a C-terminal signature domain comprising single copies of the epidermal growth factor (EGF)-like, wire, and lectin-like modules. Conformational changes in the signature domain influence ligand binding to the N-terminal modules. Interactions have been demonstrated among the modules of the signature domain and the thrombospondin-type EGF-like modules. We have extended this analysis to the rest of the stalk modules. Differential scanning calorimetry revealed interactions between the most C-terminal TSR module and the EGF-like modules. Calorimetry and differences in expression levels of single versus tandem modules indicated that the three TSRs interact with each other as well. No evidence of interactions between the von Willebrand factor type C and TSR modules were detected by differential scanning calorimetry, circular dichroism, or intrinsic fluorescence. These results indicate that the TSR and thrombospondin-type EGF-like stalk modules act as a unit that may relay conformational information between the N-terminal and C-terminal parts of the protein.Thrombospondin-1 (TSP-1)² is a major secreted protein of platelets that plays multiple roles after vascular injury (1, 2). TSPs are a family of multimodular, calcium-binding, extracellular glycoproteins. There are five family members in tetropods, each of which has a specific pattern of expression in embryonic and adult tissues (3). TSPs have two unique features, a signature domain comprising single copies of EGF-like, Ca²⁺-binding wire, and lectin-like modules and the TSP-type EGF-like module in which Cys⁴ and Cys⁵ are separated by two rather than one residue (3, 4). The family falls into two groups: A or trimeric TSPs, TSP-1 and TSP-2; and B or pentameric TSPs, TSP-3, TSP-4, and TSP-5. As depicted in Fig. 1, a subunit of the group A TSPs is composed of an N-terminal module tethered to an oligomerization domain, a von Willebrand Factor type C (vWF-C) module, three properdin or TSP type 1 repeat (TSR) modules, two TSP-type EGF-like modules, and the signature domain (3, 4). Subunits of group B TSPs lack vWF-C and TSR modules and have an extra TSP-type EGF-like module (4). Multiple interactions have been demonstrated among the modules of the signature domain of Ca²⁺-replete TSP-2 and TSP-5 (5, 6) and between the signature domain wire and second TSP-type EGF-like module of Ca²⁺-replete TSP-2 (5, 7).Open in a separate windowFIGURE 1.Schematics of (A) TSP-1 stalk modules studied in this paper, (B) TSP-1 in its Ca²⁺-depleted conformation, and (C) TSP-1 in its Ca²⁺-replete formation. Parts of TSP-1 in panels A and B are labeled as follows: N, N-terminal module; T, tether; C, vWF-C module; P, properdin or TSR module, E, EGF-like module; wire, Ca²⁺-binding repeats with 26 Ca²⁺-binding sites; and L, lectin-like module. The TSP-type EGF-like modules, E1 and E2, contain central shading. Sites of binding to heparin sulfate proteoglycan (HSPG), latent transforming growth factor-β (TGF), and CD36 are indicated in panel C. The schematics have been drawn based on structures described in the text. Sites of fucosylation of TSRs are indicated by open diamonds, and inter-module CPIXG sequences between P2 and P3 and between P3 and E1 are indicated with dots. As per the “Discussion,” changes in conformation and charge density of the signature domain due to gain or loss of Ca²⁺ are proposed to be propagated throughout trimeric TSP-1 by the stalk modules.TSP-1 has a distinctive appearance when examined by rotary shadowing electron microscopy: three bunched globules, which are thought to be the N-terminal modules, are connected by three stalks to three larger globules thought to be the C-terminal signature domains (4). Rotary shadowing electron microscopy demonstrates a striking conformational change upon removal of Ca²⁺ from the C-terminal signature domain with apparent lengthening of the stalk and loss of size of the C-terminal globules (8–10). Considerations of structures of the parts of TSP-1 indicate that the vWF-C, TSR, and TSP-type EGF-like modules form the stalk in Ca²⁺-replete TSP-1 (4), as depicted in Fig. 1. Immunochemical studies suggest that lengthening of the stalk is due, at least in part, to unraveling of two of the 13 Ca²⁺-binding repeats of the wire module (11).Removal of Ca²⁺ from binding sites on the C-terminal signature domain impacts binding of ligands or antibodies to the N-terminal modules of TSP-1 (12). The N700S polymorphism in TSP-1 that alters coordination of Ca²⁺ by the first Ca²⁺-binding wire repeat (13) also impacts interactions of the N-terminal modules with ligands (14). These observations indicate that TSP-1 possesses an allosteric mechanism whereby changes in the C-terminal signature domain are transmitted to the N-terminal modules. We have reported that the two TSP-type EGF-like modules and the signature domain EGF-like module interact with each other, suggesting a mechanism by which conformational changes in the signature domain can be propagated N-terminal as far as the first TSP-type EGF-like module (15). We have now explored the potential of EGF-like modules to work with TSR and vWF-C modules to transmit conformational information between the two ends of TSP-1.     

Differential zinc and DNA binding by partial peptides of human protamine HP2

The Zn(II) binding by partial peptides of human protamine HP2: HP2_1–15; HP2_1–25, HP2_26–40, HP2_37–47, and HP2_43–57 was studied by circular dichroism (CD). Precipitation of a 20mer DNA by these partial peptides and the effects of Zn(II) thereon were investigated using polyacrylamide gel electrophoresis (GE). The results of this study suggest that reduced HP2 (thiol groups intact) can bind Zn(II) at various parts of the molecule. In the absence of DNA, the primary Zn(II) binding site in reduced HP2 is located in the 37–47 sequence (involving Cys37, His39, His43, and Cys47), while in the presence of DNA, the strongest Zn(II) binding is provided by sequences 12–22 (by His12, Cys13, His19, and His22) and 43–57 (His43, Cys47, Cys53, and His57). In its oxidized form, HP2 can bind zinc through His residues of the 7–22 sequence. Zn(II) markedly enhances DNA binding by all partial peptides. These findings suggest that Zn(II) ions may be a regulatory factor for sperm chromatin condensation processes.     

An InCytes from MBC Selection: Mice Lacking Mannose 6-Phosphate Uncovering Enzyme Activity Have a Milder Phenotype than Mice Deficient for N-Acetylglucosamine-1-Phosphotransferase Activity

The mannose 6-phosphate (Man-6-P) lysosomal targeting signal on acid hydrolases is synthesized by the sequential action of uridine 5′-diphosphate-N-acetylglucosamine: lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase) and GlcNAc-1-phosphodiester α-N-acetylglucosaminidase (“uncovering enzyme” or UCE). Mutations in the two genes that encode GlcNAc-1-phosphotransferase give rise to lysosomal storage diseases (mucolipidosis type II and III), whereas no pathological conditions have been associated with the loss of UCE activity. To analyze the consequences of UCE deficiency, the UCE gene was inactivated via insertional mutagenesis in mice. The UCE −/− mice were viable, grew normally and lacked detectable histologic abnormalities. However, the plasma levels of six acid hydrolases were elevated 1.6- to 5.4-fold over wild-type levels. These values underestimate the degree of hydrolase hypersecretion as these enzymes were rapidly cleared from the plasma by the mannose receptor. The secreted hydrolases contained GlcNAc-P-Man diesters, exhibited a decreased affinity for the cation-independent mannose 6-phosphate receptor and failed to bind to the cation-dependent mannose 6-phosphate receptor. These data demonstrate that UCE accounts for all the uncovering activity in the Golgi. We propose that in the absence of UCE, the weak binding of the acid hydrolases to the cation-independent mannose 6-phosphate receptor allows sufficient sorting to lysosomes to prevent the tissue abnormalities seen with GlcNAc-1-phosphotranferase deficiency.     

Identification of the Matriptase Second CUB Domain as the Secondary Site for Interaction with Hepatocyte Growth Factor Activator Inhibitor Type-1

Matriptase is a type II transmembrane serine protease comprising 855 amino acid residues. The extracellular region of matriptase comprises a noncatalytic stem domain (containing two tandem repeats of complement proteases C1r/C1s-urchin embryonic growth factor-bone morphogenetic protein (CUB) domain) and a catalytic serine protease domain. The stem domain of matriptase contains site(s) for facilitating the interaction of this protease with the endogenous inhibitor, hepatocyte growth factor activator inhibitor type-1 (HAI-1). The present study aimed to identify these site(s). Analyses using a secreted variant of recombinant matriptase comprising the entire extracellular domain (MAT), its truncated variants, and a recombinant HAI-1 variant with an entire extracellular domain (HAI-1–58K) revealed that the second CUB domain (CUB domain II, Cys³⁴⁰–Pro⁴⁵²) likely contains the site(s) of interest. We also found that MAT undergoes cleavage between Lys³⁷⁹ and Val³⁸⁰ within CUB domain II and that the C-terminal residues after Val³⁸⁰ are responsible for facilitating the interaction with HAI-1–58K. A synthetic peptide corresponding to Val³⁸⁰–Asp³⁹⁰ markedly increased the matriptase-inhibiting activity of HAI-1–58K, whereas the peptides corresponding to Val³⁸⁰–Val³⁸⁹ and Phe³⁸²–Asp³⁹⁰ had no effect. HAI-1–58K precipitated with immobilized streptavidin resins to which a synthetic peptide Val³⁸⁰–Pro³⁹² with a biotinylated lysine residue at its C terminus was bound, suggesting direct interaction between CUB domain II and HAI-1. These results led to the identification of the matriptase CUB domain II, which facilitates the primary inhibitory interaction between this protease and HAI-1.     

Evaluation by site-directed mutagenesis of active site amino acid residues of Anaerobiospirillum succiniciproducens phosphoenolpyruvate carboxykinase

Anaerobiospirillum succiniciproducens phosphoenolpyruvate (PEP) carboxykinase catalyzes the reversible formation of oxaloacetate and adenosine triphosphate from PEP, adenosine diphosphate, and carbon dioxide, and uses Mn²⁺ as the activating metal ion. The enzyme is a monomer and presents 68% identity with Escherichia coli PEP carboxykinase. Comparison with the crystalline structure of homologous E. coli PEP carboxykinase [Tari, L. W., Matte, A., Goldie, H., and Delbaere, L. T. J. (1997). Nature Struct. Biol. 4, 990–994] suggests that His225, Asp262, Asp263, and Thr249 are located in the active site of the protein, interacting with manganese ions. In this work, these residues were individually changed to Gln (His225) or Asn. The mutated enzymes present 3–6 orders of magnitude lower values of V _max/K _m, indicating high catalytic relevance for these residues. The His225Gln mutant showed increased K _m values for Mn²⁺ and PEP as compared with wild-type enzyme, suggesting a role of His²²⁵ in Mn²⁺ and PEP binding. From 1.5–1.6 Kcal/mol lower affinity for the 3(2)-O-(N-methylantraniloyl) derivative of adenosine diphosphate was observed for the His225Gln and Asp263Asn mutant A. succiniciproducens PEP carboxykinases, implying a role of His²²⁵ and Asp²⁶³ in nucleotide binding.     

Interaction of the Human Prostacyclin Receptor with Rab11: CHARACTERIZATION OF A NOVEL Rab11 BINDING DOMAIN WITHIN α-HELIX 8 THAT IS REGULATED BY PALMITOYLATION*

The human prostacyclin receptor (hIP) undergoes agonist-induced internalization and subsequent recyclization in slowly recycling endosomes involving its direct physical interaction with Rab11a. Moreover, interaction with Rab11a localizes to a 22-residue putative Rab11 binding domain (RBD) within the carboxyl-terminal tail of the hIP, proximal to the transmembrane 7 (TM7) domain. Because the proposed RBD contains Cys³⁰⁸ and Cys³¹¹, in addition to Cys³⁰⁹, that are known to undergo palmitoylation, we sought to identify the structure/function determinants of the RBD, including the influence of palmitoylation, on agonist-induced trafficking of the hIP. Through complementary approaches in yeast and mammalian cells along with computational structural studies, the RBD was localized to a 14-residue domain, between Val²⁹⁹ and Leu³¹², and proposed to be organized into an eighth α-helical domain (α-helix 8), comprising Val²⁹⁹–Val³⁰⁷, adjacent to the palmitoylated residues at Cys³⁰⁸–Cys³¹¹. From mutational and [³H]palmitate metabolic labeling studies, it is proposed that palmitoylation at Cys³¹¹ in addition to agonist-regulated deacylation at Cys³⁰⁹ > Cys³⁰⁸ may dynamically position α-helix 8 in proximity to Rab11a, to regulate agonist-induced intracellular trafficking of the hIP. Moreover, Ala-scanning mutagenesis identified several hydrophobic residues within α-helix 8 as necessary for the interaction with Rab11a. Given the diverse membership of the G protein-coupled receptor superfamily, of which many members are also predicted to contain an α-helical 8 domain proximal to TM7 and, often, adjacent to palmitoylable cysteine(s), the identification of a functional role for α-helix 8, as exemplified as an RBD for the hIP, is likely to have broader significance for certain members of the superfamily.     

Diurnal variation in physical-chemical properties and primary production in the interconnected marine,mangrove and freshwater biotopes of Kakinada coast,Andhra Pradesh,India

Diurnal variation in hydrological variables and dissolved inorganic nutrients such as PO _inf4 ^sup3– -P, N O _inf2 ^sup– -N, NO _inf3 ^sup– -N and NH _inf4 ^sup+ -N were studied in three interconnected biotopes including freshwater, marine and mangrove brackish water of the Kakinada coastal zone, Andhra Pradesh. Samples were collected at intervals of 3 hours, for a period of 24 hours. In the marine environment salinity varied from 26 to 32 whereas in the mangrove waters it fluctuated from 12 to 20 and in both biotopes salinity showed bimodal type of oscillation. Dissolved oxygen content was high in the mangrove waters during day time but decreased rapidly during the night hours. In the marine environment PO_f4 ^p3–-P concentration varied from 0.345 to 1.195 g at l^–1, NO _inf3 ^sup– -N from 1.03 to 6.62 g at l^–1 and NO _inf2 ^sup– -N from 0.086 to 0.506 g at l^–1. The highest and the lowest concentrations of PO _inf4 ^sup3– -P, NO _inf3 ^sup– -N, NO _inf2 ^sup– -N recorded in the mangrove waters were 0.790 and 0.325 g at l^–1, 7.10 and 1.60 g at l^–1 and 0.278 and 0.060 g at l^–1, respectively. The concentration of PO _inf4 ^sup3– -P, NO _inf3 ^sup– -N and NO _inf2 ^sup– -N were high in the freshwater canal, the maximum and minimum values being 1.110 and 0.730 g at l^–1, 26.40 and 9.98 g at l^–1 and 0.520 and 0.252 g at l^–1 respectively. The concentration of ammonia was relatively high in the mangrove water. Gross and net primary production in the mangrove water was 4 times higher than in the marine biotope. There was no export of dissolved nutrients from the mangrove environment to the adjacent marine waters.     

The activation mechanism of human porphobilinogen synthase by 2-mercaptoethanol: intrasubunit transfer of a reserve zinc ion and coordination with three cysteines in the active center

Human porphobilinogen synthase [EC.4.2.1.24] is a homo-octamer enzyme. In the active center of each subunit, four cysteines are titrated with 5,5-dithiobis(2-nitrobenzoic acid). Cys¹²², Cys¹²⁴ and Cys¹³² are placed near two catalytic sites, Lys¹⁹⁹ and Lys²⁵², and coordinate a zinc ion, referred to as a proximal zinc ion, and Cys²²³ is placed at the orifice of the catalytic cavity and coordinates a zinc ion, referred to as a distal zinc ion, with His¹³¹ . When the wild-type enzymes C122A (Cys¹²²Ala), C124A (Cys¹²⁴Ala), C132A (Cys¹³²Ala) and C223A (Cys²²³Ala) were oxidized by hydrogen peroxide, the levels of activity were decreased. Two cysteines were titrated with 5,5-dithiobis(2-nitrobenzoic acid) in the wild-type enzyme, while on the other hand, one cysteine was titrated in the mutant enzymes. When wild-type and mutant enzymes were reduced by 2-mercaptoethanol, the levels of activity were increased: four and three cysteines were titrated, respectively, suggesting that a disulfide bond was formed among Cys¹²², Cys¹²⁴ and Cys¹³² under oxidizing conditions. We analyzed the enzyme-bound zinc ion of these enzymes using inductively coupled plasma mass spectrometry with gel-filtration chromatography. The results for C223A showed that the number of proximal zinc ions correlated to the level of enzymatic activity. Furthermore, zinc-ion-free 2-mercaptoethanol increased the activity of the wild-type enzyme without a change in the total number of zinc ions, but C223A was not activated. These findings suggest that a distal zinc ion moved to the proximal binding site when a disulfide bond among Cys¹²², Cys¹²⁴ and Cys¹³² was reduced by reductants. Thus, in the catalytic functioning of the enzyme, the distal zinc ion does not directly contribute but serves rather as a reserve as the next proximal one that catalyzes the enzyme reaction. A redox change of the three cysteines in the active center accommodates the catch and release of the reserve distal zinc ion placed at the orifice of the catalytic cavity.     

Analysis of the Global Architecture of Hemoglobin A2 by Heme Binding Studies and Molecular Modeling

The kinetics of CNProto- and CNDeutero-hemin binding to apohemoglobin A₂ was investigated in a stopped-flow device in 0.05 M potassium phosphate buffer, pH 7, at 10°C. The overall kinetic profile exhibited multiple phases: Phases I–IV corresponding with heme insertion (8.5–13 × 10⁷ M^–1 s^–1), local structural rearrangement (0.21–0.23 s^–1), global structural event (0.071–0.098 s^–1), and formation of the Fe–His bond (0.009–0.012 s^–1), respectively. Kinetic differences observed between apohemoglobin A₂ and apohemoglobin A (previously studied) prompted an analysis of the structures of and chains through molecular modeling. This revealed a structural repositioning of the residues not only at, but also distant from the site of the amino acid substitutions, specifically those involved in the heme contact and subunit interface. A significant global change was observed in the structure of the exon-coded 3 region and provided additional evidence for the designation of this as the subunit assembly domain.     

Study of Secondary Specificity of Enteropeptidase in Comparison with Trypsin

A comparative study of secondary specificities of enteropeptidase and trypsin was performed using peptide substrates with general formula A-(Asp/Glu) _n -Lys(Arg)--B, where n = 1-4. This was the first study to demonstrate that, similar to other serine proteases, enteropeptidase has an extended secondary binding site interacting with 6-7 amino acid residues surrounding the peptide bond to be hydrolyzed. However, in the case of typical enteropeptidase substrates containing four negatively charged Asp/Glu residues at positions P2-P5, electrostatic interaction between these residues and the secondary site Lys99 of the enteropeptidase light chain is the main factor that determines hydrolysis efficiency. The secondary specificity of enteropeptidase differs from the secondary specificity of trypsin. The chromophoric synthetic enteropeptidase substrate G₅DK-F(NO₂)G (k _cat/K _m = 2380 mM^–1·min^–1) is more efficient than the fusion protein PrAD₄K-P26 (k _cat/K _m = 1260 mM^–1·min^–1).     

Asparagine and glutamine side-chain conformation in solution and crystal: A comparison for hen egg-white lysozyme using residual dipolar ouplings

Experimental ¹⁵N–¹H and ¹H–¹H residual dipolar couplings (RDCs) for the asparagine (Asn) and glutamine (Gln) side chains of hen egg-white lysozyme are measured and analysed in conjunction with ¹N relaxation data, information about ¹ torsion angles in solution and molecular dynamics simulations. The RDCs are compared to values predicted from 16 high-resolution crystal structures. Two distinct groups of Asn and Gln side chains are identified. The first contains residues whose side chains show a fixed, relatively rigid, conformation in solution. For these residues there is good agreement between the experimental and predicted RDCs. This agreement improves when the experimental order parameter, S, is included in the calculation of the RDCs from the crystal structures. The comparison of the experimental RDCs with values calculated from the X-ray structures shows that the similarity between the oxygen and nitrogen electron densities is a limitation to the correct assignment of the Asn and Gln side-chain orientation in X-ray structures. In the majority of X-ray structures a 180° rotation about ² or ³, leading to the swapping of N ² and O ¹, is necessary for at least one Asn or Gln residue in order to achieve good agreement between experimental and predicted RDCs. The second group contains residues whose side chains do not adopt a single, well-defined, conformation in solution. These residues do not show a correlation between the experimental and predicted RDCs. In many cases the family of crystal structures shows a range of orientations for these side chains, but in others the crystal structures show a well-defined side-chain position. In the latter case, this is found to arise from crystallographic contacts and does not represent the behaviour of the side chain in solution.     

Direct evidence of structural changes in reaction centers of Rb. sphaeroides containing suppressor mutations for Asp L213 → Asn: A FTIR study of QB photoreduction

In bacterial reaction centers (RCs), changes of protonation state of carboxylic groups, of quinone-protein interactions as well as backbone rearrangements occuring upon Q_B photoreduction can be revealed by FTIR difference spectroscopy. The influence of compensatory mutations to the detrimental Asp L213 Asn replacement on Q_B ^–/Q_B FTIR spectra of Rb. sphaeroides RCs was studied in three double mutants carrying a Asn M44 Asp, Arg M233 Cys, or Arg H177 His suppressor mutation. The proton uptake by Glu L212 upon Q_B ^– formation, as reflected by the positive band at 1728 cm^–1, is increased in the Asn M44 Asp and Arg H177 His suppressor RCs with respect to native RCs, and remains comparable to that observed in Asp L213 Asn mutant RCs. Only the Arg M233 Cys suppressor mutation affected the 1728 cm^–1 band, reducing its amplitude to near native level. Thus, there is no clear correlation between the apparent extent of proton uptake by Glu L212 and the recovery of the proton transfer RC function. In all of the mutant spectra, several protein (amide I and amide II) and quinone anion (C...O/C...C) modes are perturbed compared to the spectrum of native RCs. These IR data show that all of the compensatory mutations alter the semiquinone-protein interactions and the backbone providing direct evidence of structural changes accompanying the restoration of efficient proton transfer in RCs containing the Asp L213 Asn lesion.     

The Metabolic Origins of Mannose in Glycoproteins

Mannose in N-glycans is derived from glucose through phosphomannose isomerase (MPI, Fru-6-P ↔ Man-6-P) whose deficiency causes a congenital disorder of glycosylation (CDG)-Ib (MPI-CDG). Mannose supplements improve patients'' symptoms because exogenous mannose can also directly contribute to N-glycan synthesis through Man-6-P. However, the quantitative contributions of these and other potential pathways to glycosylation are still unknown. We developed a sensitive GC-MS-based method using [1,2-¹³C]glucose and [4-¹³C]mannose to measure their contribution to N-glycans synthesized under physiological conditions (5 mm glucose and 50 μm mannose). Mannose directly provides ∼10–45% of the mannose found in N-glycans, showing up to a 100-fold preference for mannose over exogenous glucose based on their exogenous concentrations. Normal human fibroblasts normally derive 25–30% of their mannose directly from exogenous mannose, whereas MPI-deficient CDG fibroblasts with reduced glucose flux secure 80% of their mannose directly. Thus, both MPI activity and exogenous mannose concentration determine the metabolic flux into the N-glycosylation pathway. Using various stable isotopes, we found that gluconeogenesis, glycogen, and mannose salvaged from glycoprotein degradation do not contribute mannose to N-glycans in fibroblasts under physiological conditions. This quantitative assessment of mannose contribution and its metabolic fate provides information that can help bolster therapeutic strategies for treating glycosylation disorders with exogenous mannose.     

Comparative analysis of the two-step reaction catalyzed by prokaryotic and eukaryotic phytochelatin synthase by an ion-pair liquid chromatography assay

Genes encoding phytochelatin (PC) synthase have been found in higher plants, fission yeast and worm. Recently, kinetic and mutagenic analyses of recombinant PC synthase have been revealing the molecular mechanisms underlying PC synthesis, however, a conclusive model has not been established. To clarify the mechanism of PC synthase found in eukaryotes, we have compared the two-step reactions catalyzed by the prokaryotic Nostoc PC synthase (NsPCS) and the eukaryotic Arabidopsis PC synthase (AtPCS1). Comparative analysis shows that in the first step of PC synthesis corresponding to the cleavage of -glutamylcysteine (-EC) from glutathione (GSH), free GSH or PCs acts as a donor molecule to supply a -EC unit for elongation of the PC chain, and heavy metal ions are required to carry out the cleavage. Furthermore, functional analyses of various mutants of NsPCS and AtPCS1, selected by comparing the sequences of NsPCS and AtPCS1, indicate that the N-terminal region (residues 1–221) in AtPCS1 is the catalytic domain, and in this region, the Cys⁵⁶ residue is associated with the PC synthesis reaction. These results enable us to propose an advanced model of PC synthesis, describing substrate specificity, heavy metal requirement, and the active site in the enzyme.     

Characterization of second site mutations show that fast proton transfer to QB − is restored in bacterial reaction centers of Rhodobacter sphaeroides containing the Asp-L213 → Asn lesion

The structural basis for proton coupled electron transfer to Q_B in bacterial reaction centers (RCs) was studied by investigating RCs containing second site suppressor mutations (Asn M44 Asp, Arg M233 Cys, Arg H177 His) that complement the effects of the deleterious Asp L213 Asn mutation [DN(L213)]. The suppressor RCs all showed an increased proton coupled electron transfer rate k _AB ⁽²⁾(Q_A ^–Q_B ^– + H⁺ Q_AQ_BH^–) by at least 10³ (pH 7.5) and a recombination rate k _BD (D⁺Q_AQ_B ^– DQ_AQ_B) 15–40 times larger than the value found in DN(L213) RCs. Proton transfer was studied by measuring the dependence of k _AB ⁽²⁾ on the free energy for electron transfer (G_et). k _AB ⁽²⁾ was independent of G_et in DN(L213) RCs, but dependent on G_et in native and all suppressor RCs. This shows that proton transfer limits the k _AB ⁽²⁾ reaction with a rate of 0.1s^–1 in DN(L213) RCs but is not rate limiting and at least 10⁸-fold faster in native and 10⁵-fold faster in the suppressor RCs. The increased rate of proton transfer by the suppressor mutations are proposed to be due to: (i) a reduction in the barrier to proton transfer by providing a more negative electrostatic potential near Q_B ^–; and/or (ii) structural changes that permit fast proton transfer through the network of protonatable residues and water molecules near Q_B.     

Cell volume distribution dynamics of <Emphasis Type="Italic">Chlorella</Emphasis><Emphasis Type="Italic">vulgaris</Emphasis> Beij. in batch cultures under continuous light

Cell volume distribution in Chlorella vulgaris cultures coming out of senescence was measured by flow cytometry every 6 h for 114 h in a full-factorial experiment with initial nitrate (420–4200 g NO₃-N l^–1), phosphate (9–186 g PO₄-P l^–1), and continuous light (50–330 E m^–2 s^–1) as treatments. The maxima in median and median absolute deviation (MAD) of cell volume were achieved within 6 h of each other and their timing was not affected by any treatment. Population specific growth rate during the first 66 h calculated from volume distribution changes was significantly affected by light treatment only (p=0.002).Revisions requested 4 November 2004; Revisions received 17 January 2005