The Hydroxyl Side Chain of a Highly Conserved Serine Residue Is Required for Cation Selectivity and Substrate Transport in the Glial Glutamate Transporter GLT-1/SLC1A2

Glutamate transporters maintain synaptic concentration of the excitatory neurotransmitter below neurotoxic levels. Their transport cycle consists of cotransport of glutamate with three sodium ions and one proton, followed by countertransport of potassium. Structural studies proposed that a highly conserved serine located in the binding pocket of the homologous Glt_Ph coordinates l-aspartate as well as the sodium ion Na1. To experimentally validate these findings, we generated and characterized several mutants of the corresponding serine residue, Ser-364, of human glutamate transporter SLC1A2 (solute carrier family 1 member 2), also known as glutamate transporter GLT-1 and excitatory amino acid transporter EAAT2. S364T, S364A, S364C, S364N, and S364D were expressed in HEK cells and Xenopus laevis oocytes to measure radioactive substrate transport and transport currents, respectively. All mutants exhibited similar plasma membrane expression when compared with WT SLC1A2, but substitutions of serine by aspartate or asparagine completely abolished substrate transport. On the other hand, the threonine mutant, which is a more conservative mutation, exhibited similar substrate selectivity, substrate and sodium affinities as WT but a lower selectivity for Na⁺ over Li⁺. S364A and S364C exhibited drastically reduced affinities for each substrate and enhanced selectivity for l-aspartate over d-aspartate and l-glutamate, and lost their selectivity for Na⁺ over Li⁺. Furthermore, we extended the analysis of our experimental observations using molecular dynamics simulations. Altogether, our findings confirm a pivotal role of the serine 364, and more precisely its hydroxyl group, in coupling sodium and substrate fluxes.     

The conserved sequence NXX[S/T]HX[S/T]QDXXXT of the lactate/pyruvate:H+ symporter subfamily defines the function of the substrate translocation pathway

In Saccharomyces cerevisiae Jen1p is a lactate/proton symporter belonging to the lactate/pyruvate:H⁺ symporter subfamily (TC#2.A.1.12.2) of the Major Facilitator Superfamily. We investigated structure-function relationships of Jen1p using a rational mutational analysis based on the identification of conserved amino acid residues. In particular, we studied the conserved sequence ³⁷⁹NXX[S/T]HX[S/T]QDXXXT³⁹¹. Substitution of amino acid residues N379, H383 or D387, even with very similar amino acids, resulted in a dramatic reduction of lactate and pyruvate uptake, but conserved measurable acetate transport. Acetate transport inhibition assays showed that these mutants conserve the ability to bind, but do not transport, lactate and pyruvate. More interestingly, the double mutation H383D/D387H, while behaving as a total loss-of-function allele for lactate and pyruvate uptake, can fully restore the kinetic parameters of Jen1p for acetate transport. Thus, residues N379, H383 or D387 affect both the transport capacity and the specificity of Jen1p. Substitutions of Q386 and T391 resulted in no or moderate changes in Jen1p transport capacities for lactate, pyruvate and acetate. On the other hand, Q386N reduces the binding affinities for all Jen1p substrates, while Q386A increases the affinity specifically for pyruvate. We also tested Jen1p specificity for a range of monocarboxylates. Several of the mutants studied showed altered inhibition constants for these acids. These results and 3D in silico modelling by homology threading suggest that the conserved motif analyzed is part of the substrate translocation pathway in the lactate/pyruvate:H⁺ symporter subfamily.     

Serine319 and 321 Are Functional in Isocitrate Lyase from Escherichia coli

With site-directed mutagenesis, Ser319 and Ser321 in conserved stretch 3 of tetrameric isocitrate lyase from Escherichia coli were each substituted with alanine, cysteine, asparagine, or threonine in addition to simultaneous alanine/alanine substitutions. Besides their absolute conservation in all aligned isocitrate lyase sequences, the location of these serine residues, which flank a completely conserved proline, had been suggested in the active site in previous research by studies of photoinactivation of the enzyme by vanadate [Ko et al. (1992) J Biol Chem 267:91]. All substitutions for Ser321 and 319 except by threonine appreciably reduced the k_cat of E. coli isocitrate lyase relative to that for wild-type (100) as follows: S319A, 0.4; S319C, 0.05; S319N, 0.01; S319T, 32.3; S321A, 2.9; S321C, 0.3; S321N, 0.1; S321T, 0.3; and S319A/S321A, 0, with little or no effect on the K _m for the substrate Mg²⁺-D_s-isocitrate. The most active variant S319T exhibited threefold less activity than the wild-type enzyme; all variants assembled into tetramers. The S319T mutant isocitrate lyase was 100-fold more active than the S321T variant. This observation suggests that the requirement for a β-hydroxymethyl group of serine in catalysis is less important at position 319 than at position 321. Although most singly substituted variants had very low isocitrate lyase activity, all variants harboring mutant isocitrate lyase of very low activity did grow on acetate as a sole carbon source albeit with longer doubling times and lag phases. Substitution of Pro320 by Ala, Asp, Gly, or His was highly detrimental to activity and increased the K _m for substrate 3.5- to 8-fold; this suggests that Pro fixes the location of adjacent Ser OH groups and facilitates substrate binding and catalysis. From these collective results, it is proposed that Ser319 and Ser321 are involved in E. coli isocitrate lyase catalysis, perhaps by stabilizing the postulated reaction intermediate succinate trianion in the aci-carboxylate form and the related transition state via hydrogen bonding. Received: 3 September 1996 / Accepted: 20 September 1996     

Transport of Charged Dipeptides by the Intestinal H+/Peptide Symporter PepT1 Expressed in Xenopus laevis Oocytes

The cloned intestinal peptide transporter is capable of electrogenic H⁺-coupled cotransport of neutral di- and tripeptides and selected peptide mimetics. Since the mechanism by which PepT1 transports substrates that carry a net negative or positive charge at neutral pH is poorly understood, we determined in Xenopus oocytes expressing PepT1 the characteristics of transport of differently charged glycylpeptides. Transport function of PepT1 was assessed by flux studies employing a radiolabeled dipeptide and by the two-electrode voltage-clamp-technique. Our studies show, that the transporter is capable of translocating all substrates by an electrogenic process that follows Michaelis Menten kinetics. Whereas the apparent K_0.5 value of a zwitterionic substrate is only moderately affected by alterations in pH or membrane potential, K_0.5 values of charged substrates are strongly dependent on both, pH and membrane potential. Whereas the affinity of the anionic dipeptide increased dramatically by lowering the pH, a cationic substrate shows only a weak affinity for PepT1 at all pH values (5.5–8.0). The driving force for uptake is provided mainly by the inside negative transmembrane electrical potential. In addition, affinity for proton interaction with PepT1 was found to depend on membrane potential and proton binding subsequently affects the substrate affinity. Furthermore, our studies suggest, that uptake of the zwitterionic form of a charged substrate contributes to overall transport and that consequently the stoichiometry of the flux-coupling ratios for peptide: H⁺/H₃O⁺ cotransport may vary depending on pH. Received: 19 August 1996/Revised: 10 October 1996     

Analysis of essential amino acid residues for catalytic activity of cis-epoxysuccinate hydrolase from Bordetella sp. BK-52

cis-Epoxysuccinate hydrolase (CESH) from Bordetella sp. BK-52, an epoxide hydrolase (EH), catalyzes the stereospecific hydrolysis of cis-epoxysuccinate to d(?)-tartrate. The enzyme, which shows no homology to other reported EHs, belongs to the DUF849 superfamily of prokaryotic proteins, which have unknown function. Metal composition analysis revealed that the CESH is a Zn²⁺-dependent enzyme with an approximately 1:1 molar ratio of zinc to enzyme. The results of an ¹⁸O-labeling study suggest that the enzyme catalyzes epoxide hydrolysis by means of a one-step mechanism. We evaluated the relationship between the structure and function of the enzyme by means of sequence alignment, modeling, substrate binding, and reaction kinetics studies. The CESH has a canonical (β/α)₈ TIM barrel fold, and we used site-directed mutagenesis to identify eight residues (H47, H49, R51, T82, Y138, N140, W164, and D251) as being localized to the active site or highly conserved. On the basis of these results and theoretical considerations, we identified H47 and H49 as zinc-binding ligands, and we propose that a zinc atom and R51, T82, Y138, N140, W164, and D251 are the catalytic residues and participate in substrate binding. In summary, the structure and catalytic mechanism of the CESH from Bordetella sp. BK-52 differ from those of classic EHs, which have an α/β hydrolase fold, act via a two-step catalytic mechanism, and do not require cofactors, prosthetic groups, or metal ions.     

The transmembrane tyrosines Y56, Y91 and Y167 play important roles in determining the affinity and transport rate of the rabbit proton-coupled peptide transporter PepT1

The mammalian proton-coupled peptide transporter PepT1 is widely accepted as the major route of uptake for dietary nitrogen, as well as being responsible for the oral absorption of a number of classes of drugs, including β-lactam antibiotics and angiotensin-converting enzyme (ACE) inhibitors. Using site-directed mutagenesis and zero-trans transport assays, we investigated the role of conserved tyrosines in the transmembrane domains (TMDs) of rabbit PepT1 as predicted by hydropathy plots.All the individual TMD tyrosines were substituted with phenylalanine and shown to retain the ability to traffic to the plasma membrane of Xenopus laevis oocytes. These single substitutions of TMD tyrosines by phenylalanine residues did not affect the proton dependence of peptide uptake, with all retaining wild-type PepT1-like pH dependence. Individual mutations of four of the nine TMD residue tyrosines (Y64, Y287, Y345 and Y587) were without measurable effect on PepT1 function, whereas the other five (Y12, Y56, Y91, Y167 and Y345) were shown to result in altered transport function compared to the wild-type PepT1.Intriguingly, the affinity of Y56F-PepT1 was found to be dramatically increased (approximately 100-fold) in comparison to that of the wild-type rabbit PepT1. Y91 mutations also affected the substrate affinity of the transporter, which increased in line with the hydrophilicity of the substituted amino acid (F > Y > Q > R). Y167 was demonstrated to play a pivotal role in rabbit PepT1 function since Y167F, Y167R and Y167Q demonstrated very little transport function. These results are discussed with regard to a proposed mechanism for PepT1 substrate binding.     

Alanine racemase of Pseudomonas. Observations on subtrate and inhibitor specificity

Systematic studies with purified alanine racemase and a number of substrate analogs permit the generalization that effective competitive inhibition is limited to 2- and 3-carbon compounds. A free α-amino group was not necessary for relatively tight binding; compounds lacking an amino group, or with an α-amino group acylated even by a bulky substituent, were bound as tightly as alanine. Substitution at the α-carbon of alanine (i.e., replacement of the α-H) eliminated binding, while substitution at the β-carbon generally reduced binding. Of several inhibitory compounds tested for substrate activity by H exchange with ³H₂O, only glycine appeared active. Covalent binding to the enzyme by halo analogs was not demonstrated.     

Mutagenesis of the Glucose-1-Phosphate-Binding Site of Potato Tuber ADP-Glucose Pyrophosphorylase

Lysine (Lys)-195 in the homotetrameric ADP-glucose pyrophosphorylase (ADPGlc PPase) from Escherichia coli was shown previously to be involved in the binding of the substrate glucose-1-phosphate (Glc-1-P). This residue is highly conserved in the ADPGlc PPase family. Site-directed mutagenesis was used to investigate the function of this conserved Lys residue in the large and small subunits of the heterotetrameric potato (Solanum tuberosum) tuber enzyme. The apparent affinity for Glc-1-P of the wild-type enzyme decreased 135- to 550-fold by changing Lys-198 of the small subunit to arginine, alanine, or glutamic acid, suggesting that both the charge and the size of this residue influence Glc-1-P binding. These mutations had little effect on the kinetic constants for the other substrates (ATP and Mg²⁺ or ADP-Glc and inorganic phosphate), activator (3-phosphoglycerate), inhibitor (inorganic phosphate), or on the thermal stability. Mutagenesis of the corresponding Lys (Lys-213) in the large subunit had no effect on the apparent affinity for Glc-1-P by substitution with arginine, alanine, or glutamic acid. A double mutant, S_K198RL_K213R, was also obtained that had a 100-fold reduction of the apparent affinity for Glc-1-P. The data indicate that Lys-198 in the small subunit is directly involved in the binding of Glc-1-P, whereas they appear to exclude a direct role of Lys-213 in the large subunit in the interaction with this substrate.     

Characterization of the cysteine protease domain of Semliki Forest virus replicase protein nsP2 by in vitro mutagenesis

The function of Semliki Forest Virus nsP2 protease was investigated by site-directed mutagenesis. Mutations were introduced in its protease domain, Pro39, and the mutated proteins were expressed in Escherichia coli, purified and their activity in vitro was compared to that of the wild type Pro39. Mutations M781T, A662T and G577R, found in temperature-sensitive virus strains, rendered the enzyme temperature-sensitive in vitro as well. Five conserved residues were required for the proteolytic activity of Pro39. Changes affecting Cys⁴⁷⁸, His⁵⁴⁸, and Trp⁵⁴⁹ resulted in complete inactivation of the enzyme, whereas the replacements N600D and N605D significantly impaired its activity. The importance of Trp⁵⁴⁹ for the proteolytic cleavage specificity is discussed and a new structural motif involved in substrate recognition by cysteine proteases is proposed.     

Structural modeling and functional analysis of the essential ribosomal processing protease Prp from Staphylococcus aureus

In Firmicutes and related bacteria, ribosomal large subunit protein L27 is encoded with a conserved N‐terminal extension that is removed to expose residues critical for ribosome function. Bacteria encoding L27 with this N‐terminal extension also encode a sequence‐specific cysteine protease, Prp, which carries out this cleavage. In this work, we demonstrate that L27 variants with an un‐cleavable N‐terminal extension, or lacking the extension (pre‐cleaved), are unable to complement an L27 deletion in Staphylococcus aureus. This indicates that N‐terminal processing of L27 is not only essential but possibly has a regulatory role. Prp represents a new clade of previously uncharacterized cysteine proteases, and the dependence of S. aureus on L27 cleavage by Prp validates the enzyme as a target for potential antibiotic development. To better understand the mechanism of Prp activity, we analyzed Prp enzyme kinetics and substrate preference using a fluorogenic peptide cleavage assay. Molecular modeling and site‐directed mutagenesis implicate several residues around the active site in catalysis and substrate binding, and support a structural model in which rearrangement of a flexible loop upon binding of the correct peptide substrate is required for the active site to assume the proper conformation. These findings lay the foundation for the development of antimicrobials that target this novel, essential pathway.     

Substrate specificities of Cl- -activated arginine aminopeptidases from human and rat origin

The substrate specificities of four Cl^?-activated arginine aminopeptidases purified from the livers and inflammatory exudates of the rat, human fetal livers, and human erythrocytes were studied using peptides and N-l-aminoacyl-2-naphthylamides as substrates. With 2-naphthylamide substrates, these aminopeptidases showed similar substrate specificity; only the derivatives of Arg and Lys were measurably hydrolyzed. Di- and tripeptides with Arg or Lys as the N-terminal residue were readily split by the enzymes from the livers and inflammatory exudates of the rat and human fetal livers but oligopeptides were not hydrolyzed. Arg- and Lys-peptides were also hydrolyzed by the erythrocyte enzyme but this enzyme additionally split several other peptides, oligopeptides being hydrolyzed at internal bonds. The following properties were similar for all four arginine aminopeptidases: Dipeptides were preferred over tripeptides both in substrate binding and catalysis. The rat and human liver, rat exudate, and human erythrocyte enzymes revealed similar K_m values for the best substrates, the values increasing in the following order: ArgPhe, ArgTrp, ArgLys < ArgVal, ArgGly, Arg-2-naphthylamide < ArgGlyGly. The k_cat values were also similar for the four arginine aminopeptidases. Arg-2-naphthylamide was by far the most rapidly hydrolyzed substrate by all enzymes followed by ArgPhe and ArgTrp. With peptide substrates the highest Cl^? activation (10–20%) was found with ArgPhe and ArgTrp. With Arg-2-naphthylamide, however, the activating effect of 0.2 m Cl^? was severalfold. The hydrophobicity of the C-terminal residue of the substrate seemed to play an important role both in the Cl^? effect and substrate catalysis. Substrate binding, however, also depended on the charged groups of the substrate. Evidently Arg-2-naphthylamide and the peptides were hydrolyzed at the same active center but the mechanisms involved in the hydrolyses of chromogenic substrates and peptides may be different. It was also concluded that the less specific Cl^?-activated enzyme from human erythrocytes does not belong to the same group of Cl^?-activated arginine aminopeptidases that show a narrow substrate specificity.     

Functional analysis and crystallographic structure of clotrimazole bound OleP,a cytochrome P450 epoxidase from Streptomyces antibioticus involved in oleandomycin biosynthesis

BackgroundOleP is a cyt P450 from Streptomyces antibioticus carrying out epoxigenation of the antibiotic oleandomycin during its biosynthesis. The timing of its reaction has not been fully clarified, doubts remain regarding its substrate and catalytic mechanism.MethodsThe crystal structure of OleP in complex with clotrimazole, an inhibitor of P450s used in therapy, was solved and the complex formation dynamics was characterized by equilibrium and kinetic binding studies and compared to ketoconazole, another azole differing for the N1-substituent.ResultsClotrimazole coordinates the heme and occupies the active site. Most of the residues interacting with clotrimazole are conserved and involved in substrate binding in MycG, the P450 epoxigenase with the highest homology with OleP. Kinetic characterization of inhibitor binding revealed OleP to follow a simple bimolecular reaction, without detectable intermediates.ConclusionsClotrimazole-bound OleP adopts an open form, held by a π-π stacking chain that fastens helices F and G and the FG loop. Affinity is affected by the interactions of the N1 substituent within the active site, given the one order of magnitude difference of the off-rate constants between clotrimazole and ketoconazole. Based on structural similarities with MycG, we propose a binding mode for both oleandomycin intermediates, that are the candidate substrates of OleP.General significanceAmong P450 epoxigenases OleP is the only one that introduces an epoxide on a non-activated C–C bond. The data here presented are necessary to understand the rare chemistry carried out by OleP, to engineer it and to design more selective and potent P450-targeted drugs.     

Thrombin mutants with altered enzymatic activity have an impaired mitogenic effect on mouse fibroblasts and are inefficient modulators of stellation of rat cortical astrocytes.

We produced recombinant human thrombin mutants to investigate the correlation between the thrombin enzyme and mitogenic activity. Single amino acid substitutions were introduced in the catalytic triad (H43N, D99N, S205A, S205T), in the oxy-anion binding site (G203A) and in the anion binding exosite-1 region (R73E). Proteins were produced as prethrombin-2 mutants secreted in the culture medium of DXB11-derived cell lines. All mutants were activated by ecarin to the corresponding thrombin mutants; the enzymatic activity was assayed on a chromogenic substrate and on the procoagulant substrate fibrinogen. Mutations S205A and G203A completely abolished the enzyme activity. Mutations H43N, D99N and S205T dramatically impaired the enzyme activity toward both substrates. The R73E mutation dissociated the amidolytic activity and the clotting activity of the protein. The ability of thrombin mutants to induce proliferation was investigated in NIH3T3 mouse fibroblasts and rat cortical astrocytes. The ability of the thrombin mutants to revert astrocyte stellation was also studied. The mitogenic activity and the effect on the astrocyte stellation of the thrombin mutants correlated with their enzymatic activity. Furthermore the receptor occupancy by the inactive S205A mutant prevented the thrombin effects providing strong evidence that a proteolytically activated receptor is involved in cellular responses to thrombin.     

Substrate preference is altered by mutations in the fifth transmembrane domain of Ptr2p,the di/tri-peptide transporter of Saccharomyces cerevisiae

The integral membrane protein Ptr2p transports di/tri-peptides into the yeast Saccharomyces cerevisiae. The sequence FYXXINXG (FYING motif) in the 5th transmembrane domain (TM5) is invariably conserved among the members of the PTR (Peptide TRansport) family ranging from yeast to human. To test the role of TM5 in Ptr2p function, Ala-scanning mutagenesis of the 22 residues comprising TM5 was completed. All mutated transporters, with the exception of the Y248A mutant, were expressed as determined by immunoblots. In peptide-dependent growth assays, ten mutants of the non-FYING residues grew as well as wild-type Ptr2p on all twelve different peptides tested. All of the FYING motif mutants, except the non-expressed Y248A, plus seven other mutants in TM5 exhibited differential growth on peptides including Leu-Leu and Met-Met-Met indicating that these mutations conferred substrate preference. In assays measuring direct uptake of the radioactive peptides ³H-Leu-Leu or ¹⁴C-Met-Met-Met, the F, I and G mutants of the FYING motif did not demonstrate accumulation of these peptides over a ten minute interval. The mutation N252A of the FYING motif, along with L240A, M250A, and L258A, exhibited differential substrate preference for Met-Met-Met over Leu-Leu. Other mutations (T239A, Q241A, N242A, M245A, and A260) resulted in preference for Leu-Leu over Met-Met-Met. These data demonstrate that TM5, in particular its conserved FYING motif, is involved in substrate preference of Ptr2p.     

Human liver alanine aminopeptidase. Inhibition by amino acids.

Human liver alanine aminopeptidase is inhibited by L-amino acids having hydrophobic side chains such as Phe, Tyr, Trp, Met, and Leu. Blocking of the amino group or the carboxyl group greatly reduces the inhibitory capacity of the amino acid. Kinetic studies demonstrate that inhibition of hydrolysis of the substrate L-Ala-beta-naphthylamide is of the noncompetitive type. Inhibition of the substrate L-Leu-L-Leu is of the mixed type. Inhibition of the substrate L-Ala-L-Ala-L-Ala is of the competitive type. These changes in the mechanism of inhibition are thought to be the result of the binding of the amino acid to the third residue binding site on the enzyme. This is the part of the active center to which the third residue from the amino end of a peptide substrate is normally bound. The inhibitor constants of several alanine oligopeptides are shown to decrease with increasing length through L-Ala-L-Ala-L-Ala-L-Ala, demonstrating that alanine aminopeptidase is a multisited enzyme with three and possibly four residue sites per active center. The inhibitor constant for Gly-Gly--Phe suggesting that indeed the third residue site preferentially binds large hydrophobic residues.     

The first transmembrane segment (TMS1) of UapA contains determinants necessary for expression in the plasma membrane and purine transport

UapA, a member of the NAT/NCS2 family, is a high affinity, high capacity, uric acid-xanthine/H⁺ symporter in Aspergillus nidulans. Determinants critical for substrate binding and transport lie in a highly conserved signature motif downstream from TMS8 and within TMS12. Here we examine the role of TMS1 in UapA biogenesis and function. First, using a mutational analysis, we studied the role of a short motif (Q⁸⁵H⁸⁶), conserved in all NATs. Q85 mutants were cryosensitive, decreasing (Q85L, Q85N, Q85E) or abolishing (Q85T) the capacity for purine transport, without affecting physiological substrate binding or expression in the plasma membrane. All H86 mutants showed nearly normal substrate binding affinities but most (H86A, H86K, H86D) were cryosensitive, a phenotype associated with partial ER retention and/or targeting of UapA in small vacuoles. Only mutant H86N showed nearly wild-type function, suggesting that His or Asn residues might act as H donors in interactions affecting UapA topology. Thus, residues Q85 and H86 seem to affect the flexibility of UapA, in a way that affects either transport catalysis per se (Q⁸⁵), or expression in the plasma membrane (H⁸⁶). We then examined the role of a transmembrane Leu Repeat (LR) motif present in TMS1 of UapA, but not in other NATs. Mutations replacing Leu with Ala residues altered differentially the binding affinities of xanthine and uric acid, in a temperature-sensitive manner. This result strongly suggested that the presence of L⁷⁷, L⁸⁴ and L⁹¹ affects the flexibility of UapA substrate binding site, in a way that is necessary for high affinity uric acid transport. A possible role of the LR motif in intramolecular interactions or in UapA dimerization is discussed.     

Functional assembly and characterization of a modular xylanosome for hemicellulose hydrolysis in yeast

Five trimeric xylanosomes were successfully assembled on the cell surface of Saccharomyces cerevisiae. Three dockerin‐tagged fungal enzymes, an endoxylanase (XynAc) from Thermomyces lanuginosus, a β‐xylosidase (XlnDt) from Aspergillus niger and an acetylxylan esterase (AwAXEf) from Aspergillus awamori, were displayed for the synergistic saccharification of birchwood xylan. The surface‐expression scaffoldins were modular constructs with or without carbohydrate binding modules from Thermotoga maritima (family 22) or Clostridium thermocellum (family 3). The synergy due to enzyme–enzyme and enzyme–substrate proximity, and the effects of binding domain choice and position on xylan hydrolysis were determined. The scaffoldin‐based enzymes (with no binding domain) showed a 1.6‐fold increase in hydrolytic activity over free enzymes; this can be attributed to enzyme–enzyme proximity within the scaffoldin. The addition of a xylan binding domain from T. maritima improved hydrolysis by 2.1‐fold relative to the scaffoldin without a binding domain (signifying enzyme–substrate synergy), and 3.3‐fold over free enzymes, with a xylose productivity of 105 mg g^?1 substrate after 72 h hydrolysis. This system was also superior to the xylanosome carrying the cellulose binding module from C. thermocellum by 1.4‐fold. Furthermore, swapping the xylan binding module position within the scaffoldin resulted in 1.5‐fold more hydrolysis when the binding domain was adjacent to the endoxylanase. These results demonstrate the applicability of designer xylanosomes toward hemicellulose saccharification in yeast, and the importance of the choice and position of the carbohydrate binding module for enhanced synergy. Biotechnol. Bioeng. 2013; 110: 275–285. © 2012 Wiley Periodicals, Inc.     

Synthesis and Biochemical Study of N2-(p-n-Butylphenyl)-2′-deoxyguanosine 5′-(α,β-imido)triphosphate (BuPdGMPNHPP): A Non-Substrate Inhibitor of B Family DNA Polymerases

Abstract

BuPdGMPNHPP was synthesized and assayed as a non-incorporable inhibitor of B family DNA polymerases. The derivative was synthesized by preparation of the imidophosphorane of BuPdG followed by reaction with orthophosphate using the imidazolide method. BuPdGMPNHPP inhibited human DNA polymerase a and T4 DNA polymerase 10 and 3.5-times more potently than BuPdGTP, respectively, and was not a substrate for either enzyme. BuPdGMPNHPP acts as an active site affinity probe that could find use in co-crystallization trials of B family DNA polymerases.     

Functional importance of the conserved N-terminal domain of the mitochondrial replicative DNA helicase

The mitochondrial replicative DNA helicase is an essential cellular protein that shows high similarity with the bifunctional primase-helicase of bacteriophage T7, the gene 4 protein (T7 gp4). The N-terminal primase domain of T7 gp4 comprises seven conserved sequence motifs, I, II, III, IV, V, VI, and an RNA polymerase basic domain. The putative primase domain of metazoan mitochondrial DNA helicases has diverged from T7 gp4 and in particular, the primase domain of vertebrates lacks motif I, which comprises a zinc binding domain. Interestingly, motif I is conserved in insect mtDNA helicases. Here, we evaluate the effects of overexpression in Drosophila cell culture of variants carrying mutations in conserved amino acids in the N-terminal region, including the zinc binding domain. Overexpression of alanine substitution mutants of conserved amino acids in motifs I, IV, V and VI and the RNA polymerase basic domain results in increased mtDNA copy number as is observed with overexpression of the wild type enzyme. In contrast, overexpression of three N-terminal mutants W282L, R301Q and P302L that are analogous to human autosomal dominant progressive external ophthalmoplegia mutations results in mitochondrial DNA depletion, and in the case of R301Q, a dominant negative cellular phenotype. Thus whereas our data suggest lack of a DNA primase activity in Drosophila mitochondrial DNA helicase, they show that specific N-terminal amino acid residues that map close to the central linker region likely play a physiological role in the C-terminal helicase function of the protein.