Utilization of acidic amino acids and their amides by pseudomonads: role of periplasmic glutaminase-asparaginase

The acidic amino acids (Asp, Glu) and their amides (Asn, Gln) support rapid growth of a variety of Pseudomonas strains when provided as the sole source of carbon and nitrogen. All key enzymes of glutamate metabolism were detected in P. fluorescence, with glutaminase and asparaginase showing the highest specific activities. A periplasmic glutaminase/asparaginase activity (PGA) was found in all pseudomonads examined, including a number of root-colonizing biocontrol strains. The enzyme was purified and shown to be identical with the ansB gene product described previously. In addition to PGA, P. fluorescens contains a cytoplasmic asparaginase with marked specificity for Asn. PGA is strongly and specifically induced by its substrates (Asn, Gln) but also by the reaction products (Asp, Glu). In addition, PGA is subject to efficient carbon catabolite repression by glucose and by citrate cycle metabolites. A mutant of P. putida KT2440 with a disrupted ansB gene was unable to utilize Gln, whereas growth of the mutant on other amino acids was normal.     

A functional<Emphasis Type="Italic"> gltB</Emphasis> gene is essential for utilization of acidic amino acids and expression of periplasmic glutaminase/asparaginase (PGA) by<Emphasis Type="Italic"> Pseudomonas putida</Emphasis> KT2440

Pseudomonas putida KT2440, a root-colonizing fluorescent pseudomonad, is capable of utilizing acidic amino acids (Asp and Glu) and their amides (Asn and Gln) as its sole source of carbon and nitrogen. The uptake of Gln and Asn is facilitated by a periplasmic glutaminase/asparaginase (PGA), which hydrolyses Asn and Gln to the respective dicarboxylates. Here, we describe transposon mutagenesis of P. putida KT2440 with a self-cloning promoter probe vector, Tn5-OT182. Transconjugants defective in Glu-mediated PGA induction were selected for further studies. In most clones the transposon was found to have integrated into the gltB gene, which encodes the major subunit of the glutamate synthase (GOGAT). The transconjugants were nonmotile, no longer showed a chemotactic response towards amino acids, and could not survive prolonged periods of starvation. The acidic amino acids and their amides supported growth of the transconjugants only when supplied together with glucose, suggesting that the gltB-mutants had lost the ability to utilize amino acids as a carbon source. To confirm that gltB inactivation was the cause of this phenotype, we constructed a mutant with a targeted disruption of gltB. This strain behaved like the clones obtained by random mutagenesis, and failed to express not only PGA but also a number of other Glu-induced proteins. In contrast to wild-type cells, the gltB ^- strain accumulated considerable amounts of both Glu and Gln during long-term incubation.Communicated by A. Kondorosi     

Mutation of active site residues in synthetic T4-lysozyme gene and their effect on lytic activity

The active site amino acids (Glu11 and Asp20) in T4-lysozyme have been mutated to their isosteric residues Gln or Asn and/or acidic residues such as Glu----Asp or Asp----Glu by the oligonucleotide-replacement method. Out of eight mutants so generated the mutant T4-lysozyme obtained from pTLY.Asp11 retains maximum amount of activity (approximately 16%), pTLY.Asn20 the least (0.9%) whereas pTLY.Gln11 lost completely. A systematic study of the active and inactive mutants thus generated supports the important role of Glu11 and Asp20 in T4-lysozyme activity as predicted in earlier studies.     

台湾家白蚁内切葡聚糖酶活性中心氨基酸的饱和突变

对内切葡聚糖酶的功能改进一直是纤维素酶研究领域的焦点。本研究对台湾家白蚁内切葡聚糖酶(CfEG)的活性位点做了饱和突变。首先,以PDB数据库中高山象白蚁内切葡聚糖酶(NtEG)的三维结构(PDB id=1ks8)为模板,对CfEG进行三维结构同源建模,二者序列一致性高达79%。位于CfEG活性中心的D53、D56、E411,分别与NtEG的催化残基D54、D57、E412重合。用简并引物对CfEG的假定活性位点D53、D56、E411进行定点饱和突变。在位点D53、D56各筛选到羧甲基纤维素酶活有一定提高的突变子D53E、D56C,其中D56C的Km值减小为原始酶的三分之一。双突变子D53L/D56I的比活比原始酶提高了近2倍,同时Km值减小至原始酶的一半。而E411的饱和突变子库均没有活性,进一步将其替换为近似氨基酸的E411D、E411Q定点突变子也丧失了酶活。由突变结果可推断,位点E411为该酶行使功能的必需残基。     

A glutamate/glutamine/aspartate/asparagine transport operon in Rhodobacter capsulatus

A mutant of Rhodobacter capsulatus was identified in which an operon encoding a binding-protein-dependent transporter was interrupted by Tn5 transposition. Cloning and sequence analysis of the wild-type operon revealed a four-gene cluster with similarities to genes encoding periplasmic binding proteins (BztA), integral membrane proteins (BztB and BztC), and ATP-binding proteins (BztD). To assess the function of this putative binding-protein-dependent transport system, a mutant was constructed in which most of the bztABCD operon was deleted and replaced by an antibiotic-resistance marker. The deletion mutant grew more slowly than the wild type in NH-free medium supplemented by glutamate, glutamine, aspartate or asparagine; it was resistant to toxic analogues of Glu, Asp, and Asn at concentrations that inhibited growth of the wild type; and it was defective in the uptake of Glu, Gin, and Asp. A complementing plasmid containing the wild-type copy of bztABCD was able to rescue all the mutant phenotypes. Taken together, these results indicate that the proteins encoded by bztABCD are active in the uptake of Glu, Gin, Asp, and Asn. In addition, competition experiments, in which the ability of each of the four amino acids to compete for the transport of one another was examined, demonstrated that all four substrates share at least one component of this transport system.     

Modification of Glu 58, an amino acid of the active center of ribonuclease T1, to Gln and Asp

Glu 58 is one of the amino acids which participates in its catalytic action of ribonuclease T1. We mutated this residue to Gln 58 or Asp 58 by genetic engineering using chemically synthesized genes. The mutant enzymes were expressed in E. coli as fused proteins and purified to homogeniety on SDS-PAGE after cleavage with cyanogen bromide. Their activities in hydrolyzing pGpC were reduced to 10% in the Asp 58 mutant and about 1% in the Gln 58 mutant compared to that of the wild-type enzyme. These results suggest that Glu 58 is important but not essential for catalysis of ribonuclease T1.     

Functional analysis of polar amino-acid residues in membrane associated regions of the NHE1 isoform of the mammalian Na+/H+ exchanger.

The NHE1 isoform of the Na+/H+ exchanger is a ubiquitous plasma membrane protein that regulates intracellular pH in mammalian cells. Site-specific mutagenesis was used to examine the functional role of conserved, polar amino-acid residues occurring in segments of the protein associated with the membrane. Seventeen mutant proteins were assessed by characterization of intracellular pH changes in stably transfected cells that lacked an endogenous Na+/H+ exchanger. All of the mutant proteins were targeted correctly to the plasma membrane and were expressed at similar levels. Amino-acid residues Glu262 and Asp267 were critical to Na+/H+ exchanger activity while mutation of Glu391 resulted in only a partial reduction in activity. The Glu262-->Gln mutant was expressed partially as a deglycosylated protein with increased sensitivity to trypsin treatment in presence of Na+. Substitution of mutated Glu262, Asp267 and Glu391 with alternative acidic residues restored Na+/H+ exchanger activity. The Glu262-->Asp mutant had a decreased affinity for Li+, but its activity for Na+ and H+ ions was unaffected. The results support the hypothesis that side-chain oxygen atoms in a few, critically placed amino acids are important in Na+/H+ exchanger activity and the acidic amino-acid residues at positions 262, 267 and 391 are good candidates for being involved in Na+ coordination by the protein.     

5'-methylthioadenosine nucleosidase from yellow lupine (Lupinus luteus): molecular characterization and mutational analysis

This is report of mutational analysis of higher plant 5'-methylthioadenosine nucleosidase (MTAN). We identified and characterized the gene encoding yellow lupine (Lupinus luteus) MTAN (LlMTAN). The role of active site amino acids residues Glu24, Phe134, Glu188 and Asp211 was analyzed by site-directed mutagenesis. The Glu24Gln and Asp211Asn substitutions completely abolished the enzyme activity. The Glu188Gln mutant showed only trace activity toward 5'-methylthioadenosine. These results indicate that these three amino acid residues are necessary for enzyme activity. Furthermore, as the result of replacement of Phe134 by less bulky leucine, LlMTAN acquired the ability to bind and hydrolyze S-adenosylhomocysteine. We also analyzed the sequence of the LlMTAN promoter region. It appeared that there may be a direct link between LlMTAN expression regulation and sulfate metabolism.     

Catalytic and binding mutants of the junction-resolving enzyme endonuclease I of bacteriophage t7: role of acidic residues.

Endonuclease I is a 149 amino acid protein of bacteriophage T7 that is a Holliday junction-resolving enzyme, i.e. a four-way junction-selective nuclease. We have performed a systematic mutagenesis study of this protein, whereby all acidic amino acids have been individually replaced by other residues, mainly alanine. Out of 21 acidic residues, five (Glu20, Glu35, Glu65, Asp55 and Asp74) are essential. Replacement of these residues by other amino acids leads to a protein that is inactive in the cleavage of DNA junctions, but which nevertheless binds selectively to DNA junctions. The remaining 16 acidic residues can be replaced without loss of activity. The five critical amino acids are located within one section of the primary sequence. It is rather likely that their function is to bind one or more metal ions that coordinate the water molecule that brings about hydrolysis of the phosphodiester bond. We have also constructed a mutant of endonuclease I that lacks nine amino acids (six of which are arginine or lysine) at the C-terminus. Unlike the acidic point mutants, the C-terminal truncation is unable to bind to DNA junctions. It is therefore likely that the basic C-terminus is an important element in binding to the DNA junction.     

Characterization of the active site and thermostability regions of endoxylanase from Thermoanaerobacterium saccharolyticum B6A-RI.

Deletion mutants were constructed from pZEP12, which contained the intact Thermoanaerobacterium saccharolyticum endoxylanase gene (xynA). Deletion of 1.75 kb from the N-terminal end of xynA resulted in a mutant enzyme that retained activity but lost thermostability. Deletion of 1.05 kb from the C terminus did not alter thermostability or activity. The deduced amino acid sequence of T. saccharolyticum B6A-RI endoxylanase XynA was aligned with five other family F beta-glycanases by using the PILEUP program of the Genetics Computer Group package. This multiple alignment of amino acid sequences revealed six highly conserved motifs which included the consensus sequence consisting of a hydrophobic amino acid, Ser or Thr, Glu, a hydrophobic amino acid, Asp, and a hydrophobic amino acid in the catalytic domain. Endoxylanase was inhibited by EDAC [1-(3-dimethylamino propenyl)-3-ethylcarbodiimide hydrochloride], suggesting that Asp and/or Glu was involved in catalysis. Three aspartic acids, two glutamic acids, and one histidine were conserved in all six enzymes aligned. Hydrophobic cluster analysis revealed that two Asp and one Glu occur in the same hydrophobic clusters in T. saccharolyticum B6A-RI endoxylanase and two other enzymes belonging to family F beta-glycanases and suggests their involvement in a catalytic triad. These two Asp and one Glu in XynA from T. saccharolyticum were targeted for analysis by site-specific mutagenesis. Substitution of Asp-537 and Asp-602 by Asn and Glu-600 by Gln completely destroyed endoxylanase activity. These results suggest that these three amino acids form a catalytic triad that functions in a general acid catalysis mechanism.     

Role of glutamic acid 216 in cytochrome P450 2D6 substrate binding and catalysis

Human cytochrome P450 (P450) 2D6 is an important enzyme involved in the metabolism of drugs, many of which are amines or contain other basic nitrogen atoms. Asp301 has generally been considered to be involved in electrostatic docking with the basic substrates, on the basis of previous modeling studies and site-directed mutagenesis. Substitution of Glu216 with a residue other than Asp strongly attenuated the binding of quinidine, bufuralol, and several other P450 2D6 ligands. Catalytic activity with the substrates bufuralol and 4-methoxyphenethylamine was strongly inhibited by neutral or basic mutations at Glu216 (>95%), to the same extent as the substitution of Asn at Asp301. Unlike the Asp301 mutants, the Gln216 mutant (E216Q) retained 40% enzyme efficiency with the substrate spirosulfonamide, devoid of basic nitrogen, suggesting that the substitutions at Glu216 affect binding of amine substrates more than other catalytic steps. Attempts to induce catalytic specificity toward new substrates by substitutions at Asp301 and Glu216 were unsuccessful. Collectively, the results provide evidence for electrostatic interaction of amine substrates with Glu216, and we propose that both of these acidic residues plus at least another residue(s) is (are) involved in binding the repertoire of P450 2D6 ligands.     

Electrophysiological analysis of the mutated Na,K-ATPase cation binding pocket

Na,K-ATPase mediates net electrogenic transport by extruding three Na+ ions and importing two K+ ions across the plasma membrane during each reaction cycle. We mutated putative cation coordinating amino acids in transmembrane hairpin M5-M6 of rat Na,K-ATPase: Asp776 (Gln, Asp, Ala), Glu779 (Asp, Gln, Ala), Asp804 (Glu, Asn, Ala), and Asp808 (Glu, Asn, Ala). Electrogenic cation transport properties of these 12 mutants were analyzed in two-electrode voltage-clamp experiments on Xenopus laevis oocytes by measuring the voltage dependence of K+-stimulated stationary currents and pre-steady-state currents under electrogenic Na+/Na+ exchange conditions. Whereas mutants D804N, D804A, and D808A hardly showed any Na+/K+ pump currents, the other constructs could be classified according to the [K+] and voltage dependence of their stationary currents; mutants N776A and E779Q behaved similarly to the wild-type enzyme. Mutants E779D, E779A, D808E, and D808N had in common a decreased apparent affinity for extracellular K+. Mutants N776Q, N776D, and D804E showed large deviations from the wild-type behavior; the currents generated by mutant N776D showed weaker voltage dependence, and the current-voltage curves of mutants N776Q and D804E exhibited a negative slope. The apparent rate constants determined from transient Na+/Na+ exchange currents are rather voltage-independent and at potentials above -60 mV faster than the wild type. Thus, the characteristic voltage-dependent increase of the rate constants at hyperpolarizing potentials is almost absent in these mutants. Accordingly, dislocating the carboxamide or carboxyl group of Asn776 and Asp804, respectively, decreases the extracellular Na+ affinity.     

Engineering PQQ glucose dehydrogenase with improved substrate specificity. Site-directed mutagenesis studies on the active center of PQQ glucose dehydrogenase

Site-directed mutagenesis was carried out on the active site of water-soluble PQQ glucose dehydrogenase (PQQGDH-B) to improve its substrate specificity. Amino acid substitution of His168 resulted in a drastic decrease in the enzyme's catalytic activity, consistent with its putative catalytic role. Substitutions were also carried out in neighboring residues, Lys166, Asp167, and Gln169, in an attempt to alter the enzyme's substrate binding site. Lys166 and Gln169 mutants showed only minor changes in substrate specificity profiles. In sharp contrast, mutants of Asp167 showed considerably altered specificity profiles. Of the numerous Asp167 mutants characterized, Asp167Glu showed the best substrate specificity profile, while retaining most of its catalytic activity for glucose and stability. We also investigated the cumulative effect of combining the Asp167Glu substitution with the previously reported Asn452Thr mutation. Interpretation of the effect of the replacement of Asp167 to Glu on the alteration of substrate specificity in relation with the predicted 3D model of PQQGDH-B is also discussed.     

Study of the importance of Glu361 in the active site of cholesterol oxidase from<Emphasis Type="Italic">Rhodococcus</Emphasis> sp. PTCC 1633 by site-directed mutagenesis

Cholesterol oxidase is a monomeric flavoenzyme which catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one. ThechoR gene was cloned in pET23a and used as the starting plasmid for Glu361Asn, Glu361Gln and Glu361Asp site-directed mutagenesis. The purified mutant proteins like the wild-type have a molecular mass of 55 kD. The specific activities of Glu361Gln and Glu361Asn mutants were 28 and 35 times less than the wild-type. Glu361Asp mutant showed nearly no catalytic activity and was not purified. These experiments clearly demonstrated the importance of Glu361 for the enzymatic reactions of cholesterol oxidaseRhodococcus sp.     

Roles of glutamine in neurotransmission

Glutamine (Gln) is found abundantly in the central nervous system (CNS) where it participates in a variety of metabolic pathways. Its major role in the brain is that of a precursor of the neurotransmitter amino acids: the excitatory amino acids, glutamate (Glu) and aspartate (Asp), and the inhibitory amino acid, γ-amino butyric acid (GABA). The precursor-product relationship between Gln and Glu/GABA in the brain relates to the intercellular compartmentalization of the Gln/Glu(GABA) cycle (GGC). Gln is synthesized from Glu and ammonia in astrocytes, in a reaction catalyzed by Gln synthetase (GS), which, in the CNS, is almost exclusively located in astrocytes (Martinez-Hernandez et al., 1977). Newly synthesized Gln is transferred to neurons and hydrolyzed by phosphate-activated glutaminase (PAG) to give rise to Glu, a portion of which may be decarboxylated to GABA or transaminated to Asp. There is a rich body of evidence which indicates that a significant proportion of the Glu, Asp and GABA derived from Gln feed the synaptic, neurotransmitter pools of the amino acids. Depolarization-induced-, calcium- and PAG activity-dependent releases of Gln-derived Glu, GABA and Asp have been observed in CNS preparations in vitro and in the brain in situ. Immunocytochemical studies in brain slices have documented Gln transfer from astrocytes to neurons as well as the location of Gln-derived Glu, GABA and Asp in the synaptic terminals. Patch-clamp studies in brain slices and astrocyte/neuron co-cultures have provided functional evidence that uninterrupted Gln synthesis in astrocytes and its transport to neurons, as mediated by specific carriers, promotes glutamatergic and GABA-ergic transmission. Gln entry into the neuronal compartment is facilitated by its abundance in the extracellular spaces relative to other amino acids. Gln also appears to affect neurotransmission directly by interacting with the NMDA class of Glu receptors. Transmission may also be modulated by alterations in cell membrane polarity related to the electrogenic nature of Gln transport or to uncoupled ion conductances in the neuronal or glial cell membranes elicited by Gln transporters. In addition, Gln appears to modulate the synthesis of the gaseous messenger, nitric oxide (NO), by controlling the supply to the cells of its precursor, arginine. Disturbances of Gln metabolism and/or transport contribute to changes in Glu-ergic or GABA-ergic transmission associated with different pathological conditions of the brain, which are best recognized in epilepsy, hepatic encephalopathy and manganese encephalopathy.     

Molecular characterization of a novel family-46 chitosanase from Pseudomonas sp. A-01

Pseudomonas sp. A-01, isolated as a strain with chitosan-degrading activity, produced a 28 kDa chitosanase. Following purification of the chitosanase (Cto1) and determination of its N-terminal amino acid sequence, the corresponding gene (cto1) was cloned by a reverse-genetic technique. The gene encoded a protein, composed of 266 amino acids, including a putative signal sequence (1-28), that showed an amino acid sequence similar to known family-46 chitosanases. Cto1 was successfully overproduced and was secreted by a Brevibacillus choshinensis transformant carrying the cto1 gene on expression plasmid vector pNCMO2. The purified recombinant Cto1 protein was stable at pH 5-8 and showed the best chitosan-hydrolyzing activity at pH 5. Replacement of two acidic amino acid residues, Glu23 and Asp41, which correspond to previously identified active centers in Streptomyces sp. N174 chitosanase, with Gln and Asn respectively caused a defect in the hydrolyzing activity of the enzyme.     

A pair of membrane-embedded acidic residues in the NuoK subunit of Escherichia coli NDH-1, a counterpart of the ND4L subunit of the mitochondrial complex I, are required for high ubiquinone reductase activity

The ND4L subunit of mitochondrial NADH:ubiquinone oxidoreductase (complex I) is an integral membrane protein that contains two highly conserved glutamates within putative trans-membrane helices. We employed complex I from Escherichia coli (NDH-1) to study the role of these residues by site-directed mutagenesis. The conserved glutamates of the NuoK subunit, E36 and E72, were replaced by either Asp or Gln residues, and the effects of the mutations on cell growth and catalysis of electron transfer from deamino-NADH to ubiquinone analogues were examined. Additional mutants that carried acidic residues at selected positions within this domain were also prepared and analyzed. The results indicated that two closely located membrane-embedded acidic residues in NuoK are essential for high rates of ubiquinone reduction, a prerequisite for the growth of cytochrome bo-deficient E. coli cells on malate as the main carbon source. The two acidic residues do not have to be on adjacent helices, and mutual location on the same helix, either helix 2 or 3, at an interval of three amino acids (about one turn of the putative helix), resulted in high activity and good growth phenotypes. Nevertheless, shifting only one of them, either E36 or E72, toward the periplasmic side of the membrane by about one turn of the helix severely hampered activity and growth, whereas moving both acidic residues together to that deeper membrane position stimulated the ubiquinone reductase activity of the enzyme but not cell growth on malate, suggesting impaired energy conservation in this mutant.     

A study of the Asp110-Glu112 region of EcoRII restriction endonuclease by site-directed mutagenesis

Site-directed mutagenesis of the ecoRII gene has been used to search for the active site of the EcoRII restriction endonuclease. Plasmids with point mutations in ecoRII gene resulting in substitutions of amino acid residues in the Asp110-Glu112 region of the EcoRII endonuclease (Asp110 --> Lys, Asn, Thr, Val, or Ile; Pro111 --> Arg, His, Ala, or Leu; Glu112 --> Lys, Gln, or Asp) have been constructed. When expressed in E. coli, all these plasmids displayed EcoRII endonuclease activity. We also constructed a plasmid containing a mutant ecoRII gene with deletion of the sequence coding the Gln109-Pro111 region of the protein. This mutant protein had no EcoRII endonuclease activity. The data suggest that Asp110, Pro111, and Glu112 residues do not participate in the formation of the EcoRII active site. However, this region seems to be relevant for the formation of the tertiary structure of the EcoRII endonuclease.     

Aspartic acid-66 is the only essential negatively charged residue in the putative hydrophilic loop region of the metal-tetracycline/H+ antiporter encoded by transposon Tn10 of Escherichia coli.

Of the 16 acidic amino acid residues located in the hydrophilic region of the metal-tetracycline/H+ antiporter of transposon Tn10, five glutamic acids and three aspartic acids are conserved among the tetracycline/H+ antiporters of Gram-negative bacteria. When these conserved acidic residues were each replaced by a neutral polar residue, glutamine or asparagine, only the Asp66 substitution mutants completely lost their transport activity. The substitution of Glu274, Asp120, Glu181, or Asp38 caused significant reduction of the transport activity, whereas the substitution of the other three residues had no detectable effect on the activity. These findings led to the conclusion that only Asp66 is essential for the transport function.