Amino acid substitutions in the ε-subunit of the F1F0-ATPase of Escherichia coli

A mutant strain of Escherichia coli was isolated in which Gly-48 of the mature ε-subunit of the energy-transducing adenosine triphosphatase was replaced by Asp. This amino acid substitution caused inhibition of ATPase activity (about 70%), loss of ATP-dependent proton translocation and lowered oxidative phosphorylation, but did not affect proton translocation through the F₀. Purified F₁-ATPase from the mutant strain bound to stripped membranes with the same affinity as the normal F₁-ATPase. Partial revertant strains were isolated in which Pro-47 of the ε-subunit was replaced by Ser or Thr. Pro-47 and Gly-48 are predicted to be residues 2 and 3 in a Type II β-turn and the Gly-48 to Asp substitution is predicted to cause a change from a Type II to a Type I or III β-turn. Space-filling models of the β-turn (residues 46–49) in the normal, mutant and partial revertant ε-subunits indicate that the peptide oxygen between Pro-47 and Gly-48 is in a different position to the peptide oxygen between Pro-47 and Asp-48 and that the substitution of Pro-47 by either Ser or Thr restores an oxygen close to the original position. It is suggested that the peptide oxygen between Pro-47 and Gly-48 of the ε-subunit is involved either structurally in inter-subunit H-bonding or directly in proton movements through the F₁-ATPase.     

Mutations in three of the putative transmembrane helices of subunit a of the Escherichia coli F1F0-ATPase disrupt ATP-driven proton translocation

Three missense mutants in subunit a of the Escherichia coli F1F0-ATPase were isolated and characterized after hydroxylamine mutagenesis of a plasmid carrying the uncB (subunit a) gene. The mutations resulted in Asp119----His, Ser152----Phe, or Gly197----Arg substitutions in subunit a. Function was not completely abolished by any of the mutations. The F0 membrane sector was assembled in all three cases as judged by restoration of dicyclohexylcarbodiimide sensitivity to the F1F0-ATPase. The H+ translocation capacity of F0 was reduced in all three mutants. ATP-driven H+-translocation was also reduced, with the response in the Gly197----Arg mutant being almost nil and that in the Asp119----His and Ser152----Phe mutants less severely affected. The substituted residues are predicted to lie in the second, third, and fourth transmembrane helices suggested in most models for subunit a. The Gly197----Arg mutation lies in a very conserved region of the protein and the substitution may disrupt a structure that is critical to function. The Asp119----His and Ser152----Phe mutations also lie in areas with sequence conservation. A further analysis of randomly generated mutants may provide more information on regions of the protein that are crucial to function. Heterodiploid transformants, carrying plasmids with either the wild-type uncB gene or mutant uncB genes in an uncB (Trp231----stop) background, were characterized biochemically. The truncated subunit a was not detected in membranes of the background strain by Western blotting, and the uncB+ plasmid complemented strain showed normal biochemistry. The uncB mutant genes were shown to cause equivalent defects in either the heterodiploid background configuration, or after incorporation into an otherwise wild-type unc operon. The subunit a (Trp231----stop) background strain was shown to bind F1-ATPase nearly normally despite lacking subunit a in its membrane.     

The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.     

A homologous sequence between H+-ATPase (F0F1) and cation-transporting ATPases. Thr-285----Asp replacement in the beta subunit of Escherichia coli F1 changes its catalytic properties

A sequence of 10 amino acids (I-C-S-D-K-T-G-T-L-T) of ion motive ATPases such as Na+/K+-ATPase is similar to the sequence of the beta subunit of H+-ATPases, including that of Escherichia coli (I-T-S-T-K-T-G-S-I-T) (residues 282-291). The Asp (D) residue phosphorylated in ion motive ATPase corresponds to Thr (T) of the beta subunit. This substitution may be reasonable because there is no phosphoenzyme intermediate in the catalytic cycle of F1-ATPase. We replaced Thr-285 of the beta subunit by an Asp residue by in vitro mutagenesis and reconstituted the alpha beta gamma complex from the mutant (or wild-type) beta and wild-type alpha and gamma subunits. The uni- and multisite ATPase activities of the alpha beta gamma complex with mutant beta subunits were about 20 and 30% of those with the wild-type subunit. The rate of ATP binding (k1) of the mutant complex under uni-site conditions was about 10-fold less than that of the wild-type complex. These results suggest that Thr-285, or the region in its vicinity, is essential for normal catalysis of the H+-ATPase. The mutant complex could not form a phosphoenzyme under the conditions where the H+/K+-ATPase is phosphorylated, suggesting that another residue(s) may also be involved in formation of the intermediate in ion motive ATPase. The wild-type alpha beta gamma complex had slightly different kinetic properties from the wild-type F1, possibly because it did not contain the epsilon subunit.     

The distribution of heavy-chain isoforms of myosin in airways smooth muscle from adult and neonate humans.

ATP synthesis by oxidative phosphorylation in Escherichia coli occurs in catalytic sites on the beta-subunits of F1-ATPase. Random mutagenesis of the beta-subunit combined with phenotypic screening is potentially important for studies of the catalytic mechanism. However, when applied to haploid strains, this approach is hampered by a preponderance of mutants in which assembly of F1-ATPase in vivo is defective, precluding enzyme purification. Here we mutagenized plasmids carrying the uncD (beta-subunit) gene with hydroxylamine or N-methyl-N'-nitro-N-nitrosoguanidine and isolated, by phenotypic screening and complementation tests, six plasmids carrying mutant uncD alleles. When the mutant plasmids were used to transform a suitable uncD- strain, assembly of F1-ATPase in vivo occurred in each case. Moreover, in one case (beta Gly-223----Asp) F1-ATPase assembly proceeded although it had previously been reported that this mutation, when present on the chromosome of a haploid strain, prevented assembly of the enzyme in vivo. Therefore, this work demonstrates an improved approach for random mutagenesis of the F1-beta-subunit. Six new mutant uncD alleles were identified: beta Cys-137----Tyr; beta Gly-142----Asp; beta Gly-146----Ser; beta Gly-207----Asp; beta-Gly-223----Asp; and a double mutant beta Pro-403----Ser,Gly-415----Asp which we could not separate. The first five of these lie within or very close to the predicted catalytic nucleotide-binding domain of the beta-subunit. The double mutant lies outside this domain; we speculate that the region around residues beta 403-415 is part of an alpha-beta intersubunit contact surface. Membrane ATPase and ATP-driven proton pumping activities were impaired by all six mutations. Purified F1-ATPase was obtained from each mutant and shown to have impaired specific ATPase activity.     

Interaction between Glu-219 and His-245 within the a subunit of F1F0-ATPase in Escherichia coli

Oligonucleotide-directed mutagenesis was used to generate mutations in the a subunit gene (uncB) altering the glutamic acid 219 and the histidine 245 codons. Substitutions of aspartic acid, glutamine, histidine, and leucine for glutamic acid at position 219 neither alter the hydrolytic activity of membrane-bound F1 nor the association of F1 with F0. However, the efficiency of F0-mediated proton translocation was reduced to varying degrees. Replacement of glutamic acid 219 with leucine reduced the ATP-driven proton pumping activity of intact F1F0 to undetectable levels. Roughly 5% of normal activity was observed when glutamine occupied position 219. Surprisingly higher activity, approaching 20% of wild type levels, is seen when histidine replaced glutamic acid 219. The aspartic acid substitution resulted in little loss of enzyme function. Substitution of glutamic acid for histidine 245 results in a reduction to about 45% of normal proton translocation. Construction of the double mutant with substitution of histidine at position 219 and glutamic acid at position 245 yields a complex with better proton translocation than with either mutant separately. The possibility that a functionally important interaction between histidine 245 and glutamic acid 219 of the a subunit may be directly involved in the proton translocation mechanism of F1F0-ATP synthase is discussed.     

Construction and characterization of mutant iso-2-cytochromes c with replacement of conserved prolines

Oligonucleotide-directed mutagenesis has been used to construct two mutant forms of iso-2-cytochrome c. In one, Pro-30 is replaced by threonine; in the other, Pro-76 is replaced by glycine. Both prolines are fully conserved among mitochondrial cytochromes c and play important structural and functional roles. Yeast with either the Pro-30 or the Gly-76 mutation has appreciable levels of mutant protein in vivo and grows on media containing nonfermentable carbon sources. Thus, neither mutation blocks protein targeting to mitochondria, uptake by mitochondria, covalent attachment of heme, or in vivo function. As judged by ultraviolet-visible spectrophotometry and proton nuclear magnetic resonance spectroscopy, the nativelike conformation of purified Gly-76 iso-2 at pH 6 is almost indistinguishable from that of the normal protein at pH 6. Ultraviolet second-derivative spectrophotometry, however, suggests an increase in the average number of exposed tyrosine side chains, with 2.25 out of 5 residues exposed for the mutant compared to 1.95 for normal iso-2. Above neutral pH, the protein folds to a mutant conformation possibly related to alkaline cytochrome c. Nuclear Overhauser difference spectroscopy of the reduced nativelike conformation allows assignment of several proton resonances and comparison of side-chain conformations of the heme ligand Met-80 in the mutant and the normal proteins. The proton chemical shifts for the assigned resonances are the same within errors for Gly-76 iso-2 and normal iso-2 at pD 6, 20 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Study of the yeast Saccharomyces cerevisiae F1F0-ATPase epsilon-subunit.

The yeast Saccharomyces cerevisiae F1F0-ATPase epsilon-subunit (61 residues) was synthesized by the solid-phase peptide approach under both acidic and basic strategies. Only the latter strategy allowed us to obtain a pure epsilon-subunit. The strong propensity of the protein to produce few soluble dimeric species depending on pH has been proved by size-exclusion chromatography, electrophoresis and mass spectrometry. A circular dichroism study showed that an aqueous solution containing 30% trifluoroethanol or 200 mM sodium dodecyl sulphate is required for helical folding. In both solvents at acidic pH, the epsilon-subunit is soluble and monomeric.     

Growth control strength and active site of yeast plasma membrane ATPase studied by site-directed mutagenesis

Several amino acids which are conserved in cation-pumping ATPases with phosphorylated intermediate have been mutagenized in the yeast plasma membrane H+-ATPase. The mutant genes have been selectively expressed in a yeast strain where the wild-type ATPase is only expressed in galactose medium. A series of mutants with decreasing levels of activity demonstrates that the ATPase is rate-limiting for growth and that decreased ATPase activity correlates with decreased intracellular pH. Enzymatic and transport studies of mutant ATPases indicate that (a) Lys474 is the target for the inhibitor fluorescein 5'-isothiocyanate and this residue can be replaced by either arginine or histidine with partial retention of activity; (b) the sensitivity to inhibition by vanadate is affected by the mutations Thr231----Gly, Cys376----Leu, Lys379----Gln and Asp634----Asn; (c) the mutation Ser234----Ala causes uncoupling between ATP hydrolysis and proton transport and reduces the ATP content of the cells; (d) the mutation Asp730----Asn, which affects a polar residue conserved in hydrophobic stretches of H+-ATPases, abolishes ATPase activity and proton transport but not the formation of a phosphorylated intermediate.     

Mutagenesis of the alpha subunit of the F1Fo-ATPase from Escherichia coli. Mutations at Glu-196, Pro-190, and Ser-199

In an attempt to identify amino acid residues involved in proton translocation by the Fo sector of the Escherichia coli F1Fo-ATPase, 16 mutations at the carboxyl-terminal third of the a subunit have been isolated, and their phenotypes have been partially characterized. Thirteen mutations were constructed by "cassette" mutagenesis at two highly conserved residues, aglu196 and apro190. Two mutations were products of oligonucleotide-directed mutagenesis of a portion of of oligonucleotide-directed mutagenesis of a portion of the uncB gene cloned into an M13 vector. One mutation was isolated after in vitro mutagenesis of the entire uncB gene in a plasmid vector with hydroxylamine. Amino acid substitutions for aglu196 (Asp, Gln, His, Asn, Lys, Ala, Ser, Pro) affect ATP-driven proton translocation and passive proton permeability by Fo to varying extents, but do not prevent growth on minimal succinate media. Amino acid substitutions of glutamine or arginine for apro190 affect F1Fo-ATPase assembly and eliminate ATP-driven proton translocation, while the substitution of asparagine at this position does not significantly affect either assembly or proton translocation. The substitution of amino acids threonine or alanine for aser199 causes no detectable phenotypic change from wild type. These and other mutations are discussed in terms of the assembly, structure, and function of the a subunit. It is concluded that aglu196 and apro190 are not obligate components of the proton channel, but that they affect proton translocation indirectly.     

The inhibitor peptide of the mitochondrial F1.F0-ATPase interacts with calmodulin and stimulates the calmodulin-dependent Ca2+-ATPase of erythrocytes

The binding of calmodulin to the mitochondrial F1.F0-ATPase has been studied. [125I]Iodoazidocalmodulin binds to the epsilon-subunit and to the endogeneous ATPase inhibitor peptide in a Ca2+-dependent reaction. The effect of the mitochondrial ATPase inhibitor peptide on the purified Ca2+-ATPase of erythrocytes has also been analyzed. The inhibitor peptide stimulates the ATPase when pre-incubated with the enzyme. The activation of the Ca2+-ATPase by calmodulin is not influenced by the inhibitor peptide, indicating that the two mechanisms of activation are different. These in vitro effects of the two regulatory proteins may reflect a common origin of the two ATPases considered and/or of the regulatory proteins.     

Alterations of the carboxyl-terminal amino acid residues of Escherichia coli lipoprotein affect the formation of murein-bound lipoprotein.

Mutations in the Escherichia coli lpp gene resulting in the alterations of the COOH-terminal region of the lipoprotein have been isolated by oligonucleotide-directed mutagenesis. As might be expected, substitution of Lys78 with Arg78 completely abolished the formation of murein-bound lipoprotein. Each of the following single amino acid substitutions did not significantly affect the formation of bound-form lipoprotein: Asp70 to Glu70 or Gly70; Lys75 to Thr75; and Tyr76 to His76, Ile76, or Leu76. In contrast, mutational alterations of Tyr76 to Cys76, Gly76, Asn76, Pro76, or Ser76 resulted in a reduction of the bound-form lipoprotein to levels of 14-32% of that in the wild-type strain. A common feature of these lpp COOH-terminal mutations affecting the formation of bound-form lipoprotein is the presence of a beta-turn secondary structure at the COOH-terminal region of all these mutant lipoproteins. In addition, substitution of Tyr76 to Asp76 or Glu76, and Arg77 to Asp77 or Leu77 also resulted in a reduced formation of the bound-form lipoprotein. These results suggest that the formation of murein-bound lipoprotein requires a COOH-terminal Lys residue and a positively charged COOH-terminal region. Furthermore, a beta-turn secondary structure in the COOH-terminal random coil region interferes with the attachment of the lipoprotein to the peptidoglycan.     

The F1F0-ATPase of Escherichia coli. The substitution of alanine by threonine at position 25 in the c-subunit affects function but not assembly

A mutant strain of Escherichia coli carrying a mutation in the uncE gene which codes for the c-subunit of the F1F0-ATPase has been isolated and examined. The mutant allele, designated uncE513, results in alanine at position 25 of the c-subunit being replaced by threonine. The mutant F1F0-ATPase appears to be fully assembled and is partially functional with respect to oxidative phosphorylation. The ATPase activity of membranes from the mutant strain is resistant to the inhibitor dicyclohexylcarbodiimide, but this is due to the F1-ATPase being lost from the membranes in the presence of the inhibitor. Mutant membranes from which the F1-ATPase has been removed have a greatly reduced proton permeability compared with similarly treated normal membranes. The results are discussed in relation to a previously proposed mechanism of oxidative phosphorylation.     

Complementation between uncF alleles affecting assembly of the F1F0-ATPase complex of Escherichia coli.

A mutant affected in the b subunit (coded by the uncF gene) of the F1F0-ATPase in Escherichia coli was isolated by a localized mutagenesis procedure in which a plasmid carrying the unc genes was mutagenized in vivo. The biochemical properties of cells carrying the uncF515 allele were examined in a strain carrying the allele on a multicopy plasmid and a mutator-induced polar unc mutation on the chromosome. The strain carrying the mutant unc allele was uncoupled with respect to oxidative phosphorylation. Membrane-bound ATPase activity was very low or absent, and membranes were somewhat proton permeable. It was concluded that the F0 sector was assembled. Determination of the DNA sequence of the uncF515 allele showed it differed from wild type in that a G----A substitution occurred at position 392, resulting in glycine being replaced by aspartate at position 131. Genetic complementation tests indicated that the uncF515 allele complemented the uncF476 allele (Gly 9----Asp). Two-dimensional gel electrophoresis of membrane preparations indicated that the uncF515 and uncF476 alleles interrupted assembly of the F1F0-ATPase at different stages.     

The defective proton-ATPase of uncD mutants of Escherichia coli. Identification by DNA sequencing of residues in the beta-subunit which are essential for catalysis or normal assembly

Six mutant uncD alleles, affecting essential residues of the beta-subunit of Escherichia coli proton-ATPase, have been identified by intragenic complementation mapping, cloning, and DNA sequencing. Five of the mutations impair catalysis but do not cause structural perturbation of F1-ATPase. The amino acid substitutions found were as follows: uncD412, Gly-142----Ser; uncD430 and uncD431, both Arg-246----Cys; uncD478, Ser-174----Phe; and uncD484, Met-209----Ile. Kinetic characteristics of each corresponding mutant F1-ATPase are described or reviewed. In each case, the major determinant of impaired catalysis appears to be an attenuation of positive catalytic site cooperativity. Additionally, each mutation affects intrinsic properties of the catalytic site, including affinity for ATP, the ratio between unisite-bound substrate and products, and the rate of release of product inorganic phosphate under unisite ATP hydrolysis conditions. These effects are discussed in terms of a structural model of the catalytic nucleotide-binding domain of beta-subunit proposed recently (Duncan, T.M., Parsonage, D., and Senior, A.E. (1986) FEBS Lett. 208, 1-6). Each of the mutations lies within that domain. The uncD409 allele abolishes normal assembly of F1-ATPase. The amino acid substitution is Gly-214----Arg, which is suggested to affect a beta-turn connecting a beta-strand and an alpha-helix in the predicted nucleotide-binding domain of the beta-subunit.     

Effects of mutations of conserved Lys-155 and Thr-156 residues in the phosphate-binding glycine-rich sequence of the F1-ATPase beta subunit of Escherichia coli.

beta Lys-155 in the glycine-rich sequence of the beta subunit of Escherichia coli F1-ATPase has been shown to be near the gamma-phosphate moiety of ATP by affinity labeling (Ida, K., Noumi, T., Maeda, M., Fukui, T., and Futai, M. (1991) J. Biol. Chem. 266, 5424-5429). For examination of the roles of beta Lys-155 and beta Thr-156, mutants (beta Lys-155-->Ala, Ser, or Thr; beta Thr-156-->Ala, Cys, Asp, or Ser; beta Lys-155/beta Thr-156-->beta Thr-155/beta Lys-156; and beta Thr-156/beta Val-157-->beta Ala-156/beta Thr-157) were constructed, and their properties were studied extensively. The beta Ser-156 mutant was active in ATP synthesis and had approximately 1.5-fold higher membrane ATPase activity than the wild type. Other mutants were defective in ATP synthesis, had < 0.1% of the membrane ATPase activity of the wild type, and showed no ATP-dependent formation of an electrochemical proton gradient. The mutants had essentially the same amounts of F1 in their membranes as the wild type. Purified mutant enzymes (beta Ala-155, beta Ser-155, beta Ala-156, and beta Cys-156) showed low rates of multisite (< 0.02% of the wild type) and unisite (< 1.5% of the wild type) catalyses. The k1 values of the mutant enzymes for unisite catalysis were lower than that of the wild type: not detectable with the beta Ala-156 and beta Cys-156 enzymes and 10(2)-fold lower with the beta Ala-155 and beta Ser-155 enzymes. The beta Thr-156-->Ala or Cys enzyme showed an altered response to Mg2+, suggesting that beta Thr-156 may be closely related to Mg2+ binding. These results suggest that beta Lys-155 and beta Thr-156 are essential for catalysis and are possibly located in the catalytic site, although beta Thr-156 could be replaced by a serine residue.     

Mutations in the beta-subunit Thr(159) and Glu(184) of the Rhodospirillum rubrum F(0)F(1) ATP synthase reveal differences in ligands for the coupled Mg(2+)- and decoupled Ca(2+)-dependent F(0)F(1) activities

In the crystal structure of the mitochondrial F(1)-ATPase, the beta-Thr(163) residue was identified as a ligand to Mg(2+) and the beta-Glu(188) as directly involved in catalysis. We replaced the equivalent beta-Thr(159) of the chromatophore F(0)F(1) ATP synthase of Rhodospirillum rubrum with Ser, Ala, or Val and the Glu(184) with Gln or Lys. The mutant beta subunits were isolated and tested for their capacity to assemble into a beta-less chromatophore F(0)F(1) and restore its lost activities. All of them were found to bind into the beta-less enzyme with the same efficiency as the wild type beta subunit, but only the beta-Thr(159) --> Ser mutant restored the activity of the assembled enzyme. These results indicate that both Thr(159) and Glu(184) are not required for assembly and that Glu(184) is indeed essential for all the membrane-bound chromatophore F(0)F(1) activities. A detailed comparison between the wild type and the beta-Thr(159) --> Ser mutant revealed a rather surprising difference. Although this mutant restored the wild type levels and all specific properties of this F(0)F(1) proton-coupled ATP synthesis as well as Mg- and Mn-dependent ATP hydrolysis, it did not restore at all the proton-decoupled CaATPase activity. This clear difference between the ligands for Mg(2+) and Mn(2+), where threonine can be replaced by serine, and Ca(2+), where only threonine is active, suggests that the beta-subunit catalytic site has different conformational states when occupied by Ca(2+) as compared with Mg(2+). These different states might result in different interactions between the beta and gamma subunits, which are involved in linking F(1) catalysis with F(0) proton-translocation and can thus explain the complete absence of Ca-dependent proton-coupled F(0)F(1) catalytic activity.     

Mutagenesis of nisin’s leader peptide proline strongly modulates export of precursor nisin

The lantibiotic nisin is produced by Lactococcus lactis as a precursor peptide comprising a 23 amino acid leader peptide and a 34 amino acid post-translationally modifiable core peptide. We previously demonstrated that the conserved FNLD part of the leader is essential for intracellular enzyme-catalyzed introduction of lanthionines in the core peptide and also for transporter-mediated export, whereas other positions are subject to large mutational freedom. We here demonstrate that, in the absence of the extracellular leader peptidase, NisP, export of precursor nisin via the modification and transporter enzymes, NisBTC, is strongly affected by multiple substitutions of the leader residue at position -2, but not by substitution of positions in the vicinity of this site. Export levels of precursor nisin increased by more than 70% for position -2 mutants Asp, Thr, Ser, Trp, Lys, Val and decreased more than 70% for Cys, His, Met. In a strain with leader peptidase, the Pro-2Lys and Pro-2Asp precursor nisins were less efficiently cleaved by NisP than wild type precursor nisin. Taken together, the wild type precursor nisin with a proline at position -2 allows balanced export and cleavage efficiencies by precursor nisin’s transporter and leader peptidase.     

Mutations in the conserved proline 43 residue of the uncE protein (subunit c) of Escherichia coli F1F0-ATPase alter the coupling of F1 to F0

The conserved Pro43 residue of the uncE protein (subunit c) of the Escherichia coli F1F0-ATPase was changed to Ser or Ala by oligonucleotide-directed mutagenesis, and the mutations were incorporated into the chromosome. The resultant mutant strains were capable of oxidative phosphorylation as indicated by their ability to grow on succinate and had growth yields on glucose that were 80-90% of wild type. Membrane vesicles from the mutants were slightly less efficient than wild type vesicles in ATP-driven proton pumping as indicated by ATP-dependent quenching of quinacrine fluorescence. The decreased quenching response was not due to increased H+ leakiness of the mutant membranes or to loss of F1-ATPase activity from the membrane. These results indicate that the mutant F1F0-ATPases are defective in coupling ATP hydrolysis to H+ translocation. The membrane ATPase activity of the mutants was inhibited less by dicyclohexylcarbodiimide than that of wild type. The decrease in sensitivity to inhibition by dicyclohexylcarbodiimide was caused primarily by dissociation of the F1-ATPase from the mutant F0 in the ATPase assay mixture. These results support the idea that Pro43, and neighboring conserved polar residues play an important role in the binding and functional coupling of F1 to F0. Although a Pro residue is found at position 43 in all species of subunit c studied, surprisingly, it is not absolutely essential to function.     

Molecular basis of altered enzyme specificities in a family of mutant amidases from Pseudomonas aeruginosa.

A family of mutant amidases has been derived by experimental evolution of the aliphatic amidase of Pseudomonas aeruginosa strain PAC1. Mutation amiE16, in the structural gene for the enzyme, results in the production of the mutant B amidase by strain B6. This strain, unlike the wild-type, can utilize butyramide for growth. Strain B6 gave rise by a single mutational event to strain V9, utilizing valeramide, and strain PhB3, utilizing phenylacetamide. Strain V9 was not itself able to utilize phenylacetamide but gave rise by mutation to the phenylacetamide-utilizing mutant PhV1. Peptide 108 was isolated from chymotryptic digests of mutant amidases from strains B6, PhB3 and PhV1, but could not be detected in chymotryptic digests of the wild-type amidase. The sequence of peptide 108 was established as Met-Arg-His-Gly-Asp-Ile-Phe. Thermolytic digests of mutant amidases from strains B6, PhB3, PhV1 and V9 were compared with digests of the wild-type amidase. A peptide of the composition Met, Arg, His, Gly2, Asp3, Ile, Ser3, Thr, Val was found in the digest of the wild-type amidase and was replaced in the digests of the mutant amidases by a peptide of the composition Met, Arg, His, Gly2, Asp3, Ile, Ser3, Thr, Val, Phe. Mutation amiE16 is common to the four mutant enzymes and can be accounted for by the mutation Ser leads to Phe. The sequence of the chymotryptic peptide corresponds with the N-terminal sequence of the amidase protein, and can also be related to the thermolysin peptides. It is concluded that mutation amiE16 is a Ser leads to Phe change at position 7 from the N-terminus and the effect of this on the enzyme conformation is discussed.