Comparative substrate recognition by bacterial and fungal purine transporters of the NAT/NCS2 family

We compared the interactions of purines and purine analogues with representative fungal and bacterial members of the widespread Nucleobase-Ascorbate Transporter (NAT) family. These are: UapA, a well-studied xanthine-uric acid transporter of A. nidulans, Xut1, a novel transporter from C. albicans, described for the first time in this work, and YgfO, a recently characterized xanthine transporter from E. coli. Using transport inhibition experiments with 64 different purines and purine-related analogues, we describe a kinetic approach to build models on how NAT proteins interact with their substrates. UapA, Xut1 and YgfO appear to bind several substrates via interactions with both the pyrimidine and imidazol rings. Fungal homologues interact with the pyrimidine ring of xanthine and xanthine analogues via H-bonds, principally with N1-H and =O6, and to a lower extent with =O2. The E. coli homologue interacts principally with N3-H and =O2, and less strongly with N1-H and =O6. The basic interaction with the imidazol ring appears to be via a H-bond with N9. Interestingly, while all three homologues recognize xanthines with similar high affinities, interaction with uric acid or/and oxypurinol is transporter-specific. UapA recognizes uric acid with high affinity, principally via three H-bonds with =O2, =O6 and =O8. Xut1 has a 13-fold reduced affinity for uric acid, based on a different set of interactions involving =O8, and probably H atoms from positions N1, N3, N7 or N9. YgfO does not recognize uric acid at all. Both Xut1 and UapA recognize oxypurinol, but use different interactions reflected in a nearly 26-fold difference in their affinities for this drug, while YgfO interacts with this analogue very inefficiently.     

Comparative substrate recognition by bacterial and fungal purine transporters of the NAT/NCS2 family

We compared the interactions of purines and purine analogues with representative fungal and bacterial members of the widespread Nucleobase-Ascorbate Transporter (NAT) family. These are: UapA, a well-studied xanthine-uric acid transporter of A. nidulans, Xut1, a novel transporter from C. albicans, described for the first time in this work, and YgfO, a recently characterized xanthine transporter from E. coli. Using transport inhibition experiments with 64 different purines and purine-related analogues, we describe a kinetic approach to build models on how NAT proteins interact with their substrates. UapA, Xut1 and YgfO appear to bind several substrates via interactions with both the pyrimidine and imidazol rings. Fungal homologues interact with the pyrimidine ring of xanthine and xanthine analogues via H-bonds, principally with N1-H and =O6, and to a lower extent with =O2. The E. coli homologue interacts principally with N3-H and =O2, and less strongly with N1-H and =O6. The basic interaction with the imidazol ring appears to be via a H-bond with N9. Interestingly, while all three homologues recognize xanthines with similar high affinities, interaction with uric acid or/and oxypurinol is transporter-specific. UapA recognizes uric acid with high affinity, principally via three H-bonds with =O2, =O6 and =O8. Xut1 has a 13-fold reduced affinity for uric acid, based on a different set of interactions involving =O8, and probably H atoms from positions N1, N3, N7 or N9. YgfO does not recognize uric acid at all. Both Xut1 and UapA recognize oxypurinol, but use different interactions reflected in a nearly 26-fold difference in their affinities for this drug, while YgfO interacts with this analogue very inefficiently.     

Choline sulfatase from Ensifer (Sinorhizobium) meliloti: Characterization of the unmodified enzyme

Ensifer (Sinorhizobium) meliloti is a nitrogen-fixing α-proteobacterium able to biosynthesize the osmoprotectant glycine betaine from choline sulfate through a metabolic pathway that starts with the enzyme choline-O-sulfatase. This protein seems to be widely distributed in microorganisms and thought to play an important role in their sulfur metabolism. However, only crude extracts with choline sulfatase activity have been studied. In this work, Ensifer (Sinorhizobium) meliloti choline-O-sulfatase was obtained in a high degree of purity after expression in Escherichia coli. Gel filtration and dynamic light scattering experiments showed that the recombinant enzyme exists as a dimer in solution. Using calorimetry, its catalytic activity against its natural substrate, choline-O-sulfate, gave a k_cat=2.7×10^?1 s^?1 and a K_M=11.1 mM. For the synthetic substrates p-nitrophenyl sulfate and methylumbelliferyl sulfate, the k_cat values were 3.5×10^?2 s^?1 and 4.3×10^?2 s^?1, with K_M values of 75.8 and 11.8 mM respectively. The low catalytic activity of the recombinant sulfatase was due to the absence of the formylglycine post-translational modification in its active-site cysteine 54. Nevertheless, unmodified Ensifer (Sinorhizobium) meliloti choline-O-sulfatase is a multiple-turnover enzyme with remarkable catalytic efficiency.     

Salt Bridge Swapping in the EXXERFXYY Motif of Proton-coupled Oligopeptide Transporters

Proton-coupled oligopeptide transporters (POTs) couple the inward transport of di- or tripeptides with an inwardly directed transport of protons. Evidence from several studies of different POTs has pointed toward involvement of a highly conserved sequence motif, E₁XXE₂RFXYY (from here on referred to as E₁XXE₂R), located on Helix I, in interactions with the proton. In this study, we investigated the intracellular substrate accumulation by motif variants with all possible combinations of glutamate residues changed to glutamine and arginine changed to a tyrosine, the latter being a natural variant found in the Escherichia coli POT YjdL. We found that YjdL motif variants with E₁XXE₂R, E₁XXE₂Y, E₁XXQ₂Y, or Q₁XXE₂Y were able to accumulate peptide, whereas those with E₁XXQ₂R, Q₁XXE₂R, or Q₁XXQ₂Y were unable to accumulate peptide, and Q₁XXQ₂R abolished uptake. These results suggest a mechanism that involves swapping of an intramotif salt bridge, i.e. R-E₂ to R-E₁, which is consistent with previous structural studies. Molecular dynamics simulations of the motif variants E₁XXE₂R and E₁XXQ₂R support this mechanism. The simulations showed that upon changing conformation arginine pushes Helix V, through interactions with the highly conserved FYING motif, further away from the central cavity in what could be a stabilization of an inward facing conformation. As E₂ has been suggested to be the primary site for protonation, these novel findings show how protonation may drive conformational changes through interactions of two highly conserved motifs.     

Molecular Determinants of Substrate Specificity in Sodium-coupled Glutamate Transporters

Crystal structures of the archaeal homologue Glt_Ph have provided important insights into the molecular mechanism of transport of the excitatory neurotransmitter glutamate. Whereas mammalian glutamate transporters can translocate both glutamate and aspartate, Glt_Ph is only one capable of aspartate transport. Most of the amino acid residues that surround the aspartate substrate in the binding pocket of Glt_Ph are highly conserved. However, in the brain transporters, Thr-352 and Met-362 of the reentrant hairpin loop 2 are replaced by the smaller Ala and Thr, respectively. Therefore, we have studied the effects of T352A and M362T on binding and transport of aspartate and glutamate by Glt_Ph. Substrate-dependent intrinsic fluorescence changes were monitored in transporter constructs containing the L130W mutation. Glt_Ph-L130W/T352A exhibited an ∼15-fold higher apparent affinity for l-glutamate than the wild type transporter, and the M362T mutation resulted in an increased affinity of ∼40-fold. An even larger increase of the apparent affinity for l-glutamate, around 130-fold higher than that of wild type, was observed with the T352A/M362T double mutant. Radioactive uptake experiments show that Glt_Ph-T352A not only transports aspartate but also l-glutamate. Remarkably, Glt_Ph-M362T exhibited l-aspartate but not l-glutamate transport. The double mutant retained the ability to transport l-glutamate, but its kinetic parameters were very similar to those of Glt_Ph-T352A alone. The differential impact of mutation on binding and transport of glutamate suggests that hairpin loop 2 not only plays a role in the selection of the substrate but also in its translocation.     

苜蓿根瘤菌自生与共生状态下核糖体蛋白的比较

苜蓿根瘤菌在与宿主植物建立共生关系的过程中,以自生状态进入宿主植物细胞,经过分化发育转变为共生状态的类菌体(Bacteroid)。由于生存环境发生了变化,类菌体在形态、结构和功能方面都产生了很大的改变,其中最为明显的改变是类菌体获得了共生固氮的能力。此时,类菌体中许多与共生相关的基因被激活,蛋白的表达量显著增加。为了探明这种改变是否与合成蛋白质的细胞器-核糖体有关,比较分析了苜蓿根瘤菌在自生和共生状态下核糖体蛋白的表达谱。蛋白质双向电泳结果显示二者之间没有明显的差别,说明类菌体的分化发育过程中核糖体蛋白的形成没有改变。     

Colonization behaviour of Pseudomonas fluorescens and Sinorhizobium meliloti in the alfalfa (Medicago sativa) rhizosphere

The colonization ability of Pseudomonas fluorescens F113rif in alfalfa rhizosphere and its interactions with the alfalfa microsymbiont Sinorhizobium meliloti EFB1 has been analyzed. Both strains efficiently colonize the alfalfa rhizosphere in gnotobiotic systems and soil microcosms. Colonization dynamics of F113rif on alfalfa were similar to other plant systems previously studied but it is displaced by S. meliloti EFB1, lowering its population by one order of magnitude in co-inoculation experiments. GFP tagged strains used to study the colonization patterns by both strains indicated that P. fluorescens F113rif did not colonize root hairs while S. meliloti EFB1 extensively colonized this niche. Inoculation of F113rif had a deleterious effect on plants grown in gnotobiotic systems, possibly because of the production of HCN and the high populations reached in these systems. This effect was reversed by co-inoculation. Pseudomonas fluorescens F113 derivatives with biocontrol and bioremediation abilities have been developed in recent years. The results obtained support the possibility of using this bacterium in conjunction with alfalfa for biocontrol or rhizoremediation technologies.     

The role of arginine 143 in the electrostatics and mechanism of Cu,Zn superoxide dismutase: Computational and experimental evaluation by mutational analysis

Cu, Zn superoxide dismutase protects cells from oxidative damage by removing superoxide radicals in one of the fastest enzyme reactions known. The redox reaction at the active-site Cu ion is rate-limited by diffusion and enhanced by electrostatic guidance. To quantitatively define the electrostatic and mechanistic contributions of sequence-invariant Arg-143 in human Cu, Zn superoxide dismutase, single-site mutants at this position were investigated experimentally and computationally. Rate constants for several Arg-143 mutants were determined at different pH and ionic strength conditions using pulse radiolytic methods and compared to results from Brownian dynamics simulations. At physiological pH, substitution of Arg-143 by Lys caused a 2-fold drop in rate, neutral substitutions (Ile, Ala) reduced the rate about 10-fold, while charge-reversing substitutions (Asp, Glu) caused a 100-fold decrease. Position 143 mutants showed pH dependencies not seen in other mutants. At low pH, the acidic residue mutations exhibited pro-tonation/deprotonation effects. At high pH, all enzymes showed typical decreases in rate except the Lys mutant in which the rate dropped off at an unusually low pH. Increasing ionic strength at acidic pH decreased the rates of the wild-type enzyme and Lys mutant, while the rate of the Glu mutant was unaffected. Increasing ionic strength at higher pH (>10) increased the rates of the Lys and Glu mutants while the rate of the wild-type enzyme was unaffected. Reaction simulations with Brownian dynamics incorporating electrostatic effects tested computational predictability of ionic strength dependencies of the wild-type enzyme and the Lys, Ile, and Glu mutants. The calculated and experimental ionic strength profiles gave similar slopes in all but the Glu mutant, indicating that the electrostatic attraction of the substrate is accurately modeled. Differences between the calculated and experimental rates for the Glu and Lys mutants reflect the mechanistic contribution of Arg-143. Results from this joint analysis establish that, aside from the Cu ligands, Arg-143 is the single most important residue in Cu, Zn superoxide dismutase both electrostatically and mechanistically, and provide an explanation for the evolutionary selection of arginine at position 143. © 1994 Wiley-Liss, Inc.     

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.     

Genetic and biochemical characterization of a pathway for the degradation of 2-aminoethylphosphonate in Sinorhizobium meliloti 1021

A variety of microorganisms have the ability to use phosphonic acids as sole sources of phosphorus. Here, a novel pathway for degradation of 2-aminoethylphosphonate in the bacterium Sinorhizobium meliloti 1021 is proposed based on the analysis of the genome sequence. Gene deletion experiments confirmed the involvement of the locus containing phnW, phnA, and phnY genes in the conversion of 2-aminoethylphosphonate to inorganic phosphate. Biochemical studies of the recombinant PhnY and PhnA proteins verified their roles as phosphonoacetaldehyde dehydrogenase and phosphonoacetate hydrolase, respectively. This pathway is likely not limited to S. meliloti as suggested by the presence of homologous gene clusters in other bacterial genomes.     

Cloning and functional characterization of two bacterial members of the NAT/NCS2 family in Escherichia coli

The coding potential of the genome of E. coli K-12 includes YgfO and YicE, two members of the evolutionarily conserved NAT/NCS2 transporter family that are highly homologous to each other (45% residue identity) and closely related to UapA of Aspergillus nidulans, a most extensively studied microbial member of this family. YgfO and yicE were cloned from the genome, over-expressed extrachromosomally and assayed for uptake of [³H]xanthine and other nucleobases, in E. coli K-12, under conditions of negligible activity of the corresponding endogenous systems. Alternative, essentially equivalent functional versions of YgfO and YicE were engineered by C-terminal tagging with an epitope from the E. coli lactose permease and a biotin-acceptor domain from Klebsiella pneumoniae. Both YgfO and YicE were shown to be present in the plasma membrane of E. coli and function as specific, high-affinity transporters for xanthine (K_m 4.2–4.6 µM for YgfO, or 2.9–3.8 µM for YicE), in a proton motive force-dependent manner; they display no detectable transport of uracil, hypoxanthine, or uric acid at external concentrations of up to 0.1 mM. Both YgfO and YicE are inefficient in recognizing uric acid or xanthine analogues modified at position 8 of the purine ring (8-methylxanthine, 8-azaxanthine, oxypurinol, allopurinol), which distinguishes them from their fungal homologues UapA and Xut1.     

Heterologous expression of Anabaena sp. PCC7120 cyanophycin metabolism genes cphA1 and cphB1 in Sinorhizobium (Ensifer) meliloti 1021

Sinorhizobium meliloti infects leguminous plants resulting in a nitrogen-fixing symbiosis. Free living cells accumulate poly(3-hydroxybutyrate) (PHB) as carbon and energy source under imbalanced growth conditions. The cphA1 ₇₁₂₀ gene encoding a cyanophycin (CGP) synthetase of Anabaena sp. PCC7120 in plasmids pVLT31::cphA1 ₇₁₂₀ and pBBR1MCS-3::cphA1 ₇₁₂₀ was expressed in the wild-type S. meliloti 1021 and in a phbC-negative mutant generated in this study. Expression of cphA1 ₇₁₂₀ and accumulation of CGP in cells were studied in various media. Yeast mannitol broth (YMB) and pBBR1MCS-3::cphA1 ₇₁₂₀ yielded the highest CGP contents in both S. meliloti 1021 strains. Supplying the YMB medium with isopropyl-β-D-thiogalactopyranoside, aspartic acid, and arginine enhanced CGP contents about 2.5- and 2.8-fold in S. meliloti 1021 (pBBR1MCS-3::cphA1 ₇₁₂₀) and S. meliloti 1021 phbCΩKm (pBBR1MCS-3::cphA1 ₇₁₂₀), respectively. Varying the nitrogen-to-carbon ratio in the medium enhanced the CGP content further to 43.8% (w/w) of cell dry weight (CDW) in recombinant cells of S. meliloti 1021 phbCΩKm (pBBR1MCS-3::cphA1 ₇₁₂₀). Cells of S. meliloti 1021 (pBBR1MCS-3::cphA1 ₇₁₂₀) accumulated CGP up to 39.6% in addition to 12.1% PHB (w/w, of CDW). CGP from the S. meliloti strains consisted of equimolar amounts of aspartic acid and arginine and contained no other amino acids even if the medium was supplemented with glutamic acid, citrulline, ornithine, or lysine. CGP isolated from cells of S. meliloti 1021 (pBBR1MCS-3::cphA1 ₇₁₂₀) and S. meliloti 1021 phbCΩKm (pBBR1MCS-3::cphA1 ₇₁₂₀) exhibited average molecular weights between 20 and 25 kDa, whereas CGP isolated from Escherichia coli S17-1 (pBBR1MCS-3::cphA1 ₇₁₂₀) exhibited average molecular weight between 22 and 30 kDa. Co-expression of cyanophycinase from Anabaena sp. PCC7120 encoded by cphB1 ₇₁₂₀ in cphA1 ₇₁₂₀-positive E. coli S17-1, S. meliloti 1021, and its phbC-negative mutant gave cyanophycinase activities in crude extracts, and no CGP granules occurred. A higher PHB content in S. meliloti 1021 (pBBR1MCS-3::cphB1 ₇₁₂₀::cphA1 ₇₁₂₀) in comparison to the control indicated that the cells used CGP degradation product (β-aspartate-arginine dipeptide) to fuel PHB biosynthesis.     

Genetic basis of enterobacterial repetitive intergenic consensus (ERIC)-PCR fingerprint pattern in Sinorhizobium meliloti and identification of S. meliloti employing PCR primers derived from an ERIC-PCR fragment

The enterobacterial repetitive intergenic consensus (ERIC)-PCR method was employed to generate genomic amplification products of Sinorhizobium meliloti strain 2011. Eleven distinctive PCR fragments obtained in PCR reactions by using the ERIC2 primer were cloned and their partial or complete nucleotide sequences established. DNA sequences that extended past the ERIC2 primer region were not conserved among the 11 PCR fragments and showed no sequence similarity to the enterobacterial ERIC consensus sequence. Thus, repetitive ERIC or ERIC-like sequences seem not to be an integral part of the S. meliloti genome. An amplification product of S. meliloti 2011 was identified which was present in S. meliloti strains but absent in other rhizobial species. Based on the nucleotide sequence information, a pair of PCR primers was designed and used for PCR amplification of sequences of S. meliloti laboratory strains 2011, L5–30, AK631 and 102F34. Nucleotide sequence analysis of the amplification products revealed a 100% DNA sequence conservation. Database searches showed that the DNA fragment putatively encodes the C-terminal part of a protein displaying similarity to 2-hydroxyacid dehydrogenases of various organisms. The newly designed PCR primers should be useful for the rapid identification of S. meliloti isolates. Received: 17 February 1999 / Accepted: 9 April 1999     

The effects of mutations on motions of side-chains in protein L studied by 2H NMR dynamics and scalar couplings

Recently developed 2H spin relaxation experiments are applied to study the dynamics of methyl-containing side-chains in the B1 domain of protein L and in a pair of point mutants of the domain, F22L and A20V. X-ray and NMR studies of the three variants of protein L studied here establish that their structures are very similar, despite the fact that the F22L mutant is 3.2kcal/mol less stable. Measurements of methyl 2H spin relaxation rates, which probe dynamics on a picosecond-nanosecond time scale, and three-bond 3J(Cgamma-CO), 3J(Cgamma-N) and 3J(Calpha-Cdelta) scalar coupling constants, which are sensitive to motion spanning a wide range of time-scales, reveal changes in the magnitude of side-chain dynamics in response to mutation. Observed differences in the time-scale of motions between the variants have been related to changes in energetic barriers. Of interest, several of the residues with different motional properties across the variants are far from the site of mutation, suggesting the presence of long-range interactions within the protein that can be probed through studies of dynamics.     

Computational,pulse-radiolytic,and structural investigations of lysine-136 and its role in the electrostatic triad of human C u,Z n superoxide dismutase

Key charged residues in Cu,Zn superoxide dismutase (Cu,Zn SOD) promote electrostatic steering of the superoxide substrate to the active site Cu ion, resulting in dismutation of superoxide to oxygen and hydrogen peroxide. Lys-136, along with the adjacent residues Glu-132 and Glu-133, forms a proposed electrostatic triad contributing to substrate recognition. Human Cu,Zn SODs with single-site replacements of Lys-136 by Arg, Ala, Gln, or Glu or with a triple-site substitution (Glu-132 and Glu-133 to Gln and Lys-136 to Ala) were made to test hypotheses regarding contributions of these residues to Cu,Zn SOD activity. The structural effects of these mutations were modeled computationally and validated by the X-ray crystallographic structure determination of Cu,Zn SOD having the Lys-136-to-Glu replacement. Brownian dynamics simulations and multiple-site titration calculations predicted mutant reaction rates as well as ionic strength and pH effects measured by pulse-radiolytic experiments. Lys-136-to-Glu charge reversal decreased dismutation activity 50% from 2.2 × 10⁹ to 1.2 × 10⁹ M⁻¹ s⁻¹ due to repulsion of negatively charged superoxide, whereas charge-neutralizing substitutions (Lys-136 to Gln or Ala) had a less dramatic influence. In contrast, the triple-mutant Cu,Zn SOD (all three charges in the electrostatic triad neutralized) surprisingly doubled the reaction rate compared with wild-type enzyme but introduced phosphate inhibition. Computational and experimental reaction rates decreased with increasing ionic strength in all of the Lys-136 mutants, with charge reversal having a more pronounced effect than charge neutralization, implying that local electrostatic effects still govern the dismutation rates. Multiple-site titration analysis showed that deprotonation events throughout the enzyme are likely responsible for the gradual decrease in SOD activity above pH 9.5 and predicted a pK_a value of 11.7 for Lys-136. Overall, Lys-136 and Glu-132 make comparable contributions to substrate recognition but are less critical to enzyme function than Arg-143, which is both mechanistically and electrostatically essential. Thus, the sequence-conserved residues of this electrostatic triad are evidently important solely for their electrostatic properties, which maintain the high catalytic rate and turnover of Cu,Zn SOD while simultaneously providing specificity by selecting against binding by other anions. Proteins 29:103–112, 1997. © 1997 Wiley-Liss, Inc.     

Assembly of a Tyr122 Hydrophobic Cluster in Sarcoplasmic Reticulum Ca2+-ATPase Synchronizes Ca2+ Affinity Reduction and Release with Phosphoenzyme Isomerization

The mechanism whereby events in and around the catalytic site/head of Ca²⁺-ATPase effect Ca²⁺ release to the lumen from the transmembrane helices remains elusive. We developed a method to determine deoccluded bound Ca²⁺ by taking advantage of its rapid occlusion upon formation of E1PCa₂ and of stabilization afforded by a high concentration of Ca²⁺. The assay is applicable to minute amounts of Ca²⁺-ATPase expressed in COS-1 cells. It was validated by measuring the Ca²⁺ binding properties of unphosphorylated Ca²⁺-ATPase. The method was then applied to the isomerization of the phosphorylated intermediate associated with the Ca²⁺ release process E1PCa₂ → E2PCa₂ → E2P + 2Ca²⁺. In the wild type, Ca²⁺ release occurs concomitantly with EP isomerization fitting with rate-limiting isomerization (E1PCa₂ → E2PCa₂) followed by very rapid Ca²⁺ release. In contrast, with alanine mutants of Leu¹¹⁹ and Tyr¹²² on the cytoplasmic part of the second transmembrane helix (M2) and Ile¹⁷⁹ on the A domain, Ca²⁺ release in 10 μm Ca²⁺ lags EP isomerization, indicating the presence of a transient E2P state with bound Ca²⁺. The results suggest that these residues function in Ca²⁺ affinity reduction in E2P, likely via a structural rearrangement at the cytoplasmic part of M2 and a resulting association with the A and P domains, therefore leading to Ca²⁺ release.     

Tryparedoxin Peroxidase of Leishmania donovani: Molecular Cloning, Heterologous Expression, Specificity, and Catalytic Mechanism

Tryparedoxin peroxidase (TXNPx) of Trypanosomatidae is the terminal peroxidase of a complex redox cascade that detoxifies hydroperoxides by NADPH (Nogoceke et al., Biol. Chem. 378, 827-836, 1997). A gene putatively coding for a peroxiredoxin-type TXNPx was identified in L. donovani and expressed in Escherichia coli to yield an N-terminally His-tagged protein (LdH6TXNPx). LdH6TXNPx proved to be an active peroxidase with tryparedoxin (TXN) 1 and 2 of Crithidia fasciculata as cosubstrates. LdH6TXNPx efficiently reduces H₂O₂, is moderately active with t-butyl and cumene hydroperoxide, but only marginally with linoleic acid hydroperoxide and phosphatidyl choline hydroperoxide. The enzyme displays ping-pong kinetics with a k_cat of 11.2 s⁻¹ and limiting K_m values for t-butyl hydroperoxide and CfTXN1 of 50 and 3.6 μM, respectively. Site-directed mutagenesis confirmed that C52 and C173, as in related peroxiredoxins, are involved in catalysis. Exchanges of R128 against D and T49 against S and V, supported by molecular modelling, further disclose that the SH group of C52 builds the center of a novel catalytic triad. By hydrogen bonding with the OH of T49 and by the positive charge of R128 the solvent-exposed thiol of C52 becomes deprotonated to react with ROOH. Molecular models of oxidized TXNPx show C52 disulfide-bridged with C173′ that can be attacked by C41 of TXN2. By homology, the deduced mechanism may apply to most peroxiredoxins and complements current views of peroxiredoxin catalysis.     

抗体重链可变区框架Ⅰ区对抗体分泌的影响

抗体重链可变区框架Ⅰ区(FR-Ⅰ)对抗体在原核细胞中的分泌表达具有显著的影响,单个氨基酸的改变即可导致抗体分子分泌能力的丧失.为了探索抗体在哺乳动物细胞中的高效表达,我们对一株不能有效分泌的人源抗狂犬病病毒抗体pCMV-RV/VH的FR-Ⅰ区氨基酸编码基因进行定点突变研究.实验显示,抗体重链可变区FR-Ⅰ区H6位的氨基酸由Glu突变为Gin之后,抗体的分泌表达水平得到了显著的提高,并且与抗原特异性结合的能力也明显增强.通过免疫荧光法对抗体在细胞内的转运过程进行了初步的探讨,发现能够有效分泌的抗体与无分泌表达的抗体都能够在细胞内进行正常的转录和翻译,进入内质网并转运至高尔基体,而且胞内表达水平基本一致.我们认为分泌能力的不同可能是FR-Ⅰ区影响抗体分子的折叠与装配所致,其中该区H6位氨基酸的性质能够显著影响抗体在哺乳动物细胞中的分泌.本研究以抗体重链可变区FR-Ⅰ区氨基酸为焦点,对基因工程抗体在真核细胞中高效表达的影响因素进行了探索,为改进抗体规模化生产提供了依据.