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Site-directed mutagenesis of the Klebsiella pneumoniae nitrogenase. Effects of modifying conserved cysteine residues in the alpha- and beta-subunits.

ABSTRACT: The five conserved cysteine residues present in the alpha-subunit and the three conserved cysteine residues present in the beta-subunit of nitrogenase component 1 were individually changed to alanine. Mutations in the alpha-subunit at positions 63, 89, 155 and 275 and in the beta-subunit at positions 69, 94 and 152 all resulted in a loss of diazotrophic growth and component 1 activity and loss of the normal e.p.r. signal of the component 1 protein. Component 2 activity was retained. Replacement of cysteine-184 in the alpha-subunit with alanine greatly diminished, but did not eliminate, diazotrophic growth and component 1 activity. Substitution of serine for cysteine at position 152 in the beta-subunit, in contrast with the substitution of alanine at this position, resulted in the formation of active component 1. Replacement of the non-conserved cysteine-112 in the beta-subunit with alanine did not greatly perturb diazotrophic growth or the activity of component 1. Extracts prepared from a mutant, with cysteine-275 of the alpha-subunit replaced by alanine, complemented extracts of a mutant unable to synthesize the iron-molybdenum cofactor of nitrogenase, indicating that the alanine-275 substitution increases the availability of cofactor. Furthermore extracts of this mutant exhibited an e.p.r. signal similar to that of extracted iron-molybdenum cofactor. These data suggest a role for cysteine-275 as a ligand to the cofactor.

SUBMITTER: Kent HM

PROVIDER: S-EPMC1133571 | biostudies-other | 1989 Nov

REPOSITORIES: biostudies-other

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Project description:Glutathione transferase (GST) epsilon (also known as GST2 or GST B1B1), the major Class Alpha GST in human liver has been subjected to oligonucleotide-directed site-specific mutagenesis. Four arginine residues, R13, R20, R69 and R187, of which all but R69 are strictly conserved through GST Classes Alpha, Mu and Pi have been replaced by Ala. The mutant enzymes have been expressed in Escherichia coli, purified by affinity chromatography and characterised. Compared with the wild-type enzyme, all mutant GSTs had altered catalytic properties. All mutants had decreased specific activity with 1-chloro-2,4-dinitrobenzene (CDNB). Mutants R13A, R69A and R187A also showed decreased activities with other substrates such as cumene hydroperoxide (CuOOH) and androstenedione. In contrast, mutant R20A had an increased peroxidase activity and an isomerase activity essentially the same as that of the wild-type GST. With the substrates used, kcat./Km values were decreased for all mutant GSTs. Increases in the [S0.5] values were most significant for glutathione (GSH), while values for CDNB and CuOOH were less markedly affected. Thus, various kinetic data indicate that the GSH affinity has been reduced by the mutations and that this loss of affinity is linked to the decreased specific activities. Inhibition studies showed an increased sensitivity towards S-hexyl-GSH; this was particularly marked for mutant R69A. Mutant R20A had a lowered [I50] value but, in contrast, also the highest [I80] value as compared with the wild-type enzyme. Towards bromosulphophthalein, mutants R20A and R69A had a markedly increased sensitivity, about 35-fold in comparison with the wild-type. The inhibition properties of mutant R187A were similar to those of the wild-type enzyme and the properties of mutant R13A were in between. The increased sensitivity to S-hexyl-GSH, in contrast with the decreased affinity for GSH, was suggested to be due to an altered distribution between conformational states of the enzyme induced by the mutations. The arginine residues in positions 13, 20 and 69 all seem to be important for the catalytic properties of GST. Further, the inhibition studies indicate a role of arginine residues in the stabilisation of conformational states of the enzyme.

Project description:1. Nitrogenase from the facultative anaerobe Klebsiella pneumoniae was resolved into two protein components resembling those obtained from other nitrogen-fixing bacteria. 2. Both proteins were purified to homogeneity as shown by the criteria of disc electrophoresis and ultracentrifugal analysis. 3. The larger component had a mol.wt. of 218000 and contained one Mo atom, 17Fe atoms and 17 acid-labile sulphide groups/mol; it contained two types of subunit, present in equal amounts, of mol.wts. 50000 and 60000. All the common amino acids were present, with a predominance of acidic residues. The apparent partial specific volume was 0.73; ultracentrifugal analysis gave s(0) (20,w)=11.0S and D(0) (20,w)=4.94x10(-7)cm(2)/s. The specific activities (nmol of product formed/min per mg of protein) when assayed with the second nitrogenase component were 1500 for H(2) evolution, 380 for N(2) reduction, 1200 for acetylene reduction and 5400 for ATP hydrolysis. The reduced protein showed electron-paramagnetic-resonance signals at g=4.3, 3.7 and 2.015; the Mössbauer spectrum of the reduced protein consisted of at least three doublets. The u.v. spectra of the oxidized and reduced proteins were identical. On oxidation the absorbance increased generally throughout the visible region and a shoulder at 430nm appeared. The circular-dichroism spectra of both the oxidized and reduced proteins were the same, consisting mainly of a negative trough at 220nm. 4. The smaller component had mol.wt. 66800 and contained four Fe atoms and four acid-labile sulphide groups in a molecule comprising two subunits each of mol.wt. 34600. All common amino acids except tryptophan were present, with a predominance of acidic residues. The apparent partial specific volume calculated from the amino acid analysis was 0.732, which was significantly higher than that obtained from density measurements (0.69); ultracentrifugal analysis gave s(0) (20,w)=4.8S and D(0) (20,w)=5.55x10(-7)cm(2)/s. The specific activities (nmol of product formed/min per mg of protein) were 1050 for H(2) evolution, 275 for N(2) reduction, 980 for acetylene reduction and 4350 for ATP hydrolysis. The protein was not cold-labile. The reduced protein showed electron-paramagnetic-resonance signals in the g=1.94 region. The Mössbauer spectrum of the reduced protein consisted of a doublet at 77 degrees K. The u.v. spectra of reduced and O(2)-inactivated proteins were identical, and inactivation by O(2) generally increased the absorbance in the visible region and resulted in a shoulder at 460nm. The circular-dichroism spectra exhibited a negative trough at 220nm and inactivation by O(2) decreased the depth of the trough. 5. The reduction of N(2) and acetylene, and H(2) evolution, were maximal at a 1:1 molar ratio of the Fe-containing protein to the Mo-Fe-containing protein; excess of the Mo-Fe-containing protein was inhibitory. All reductions were accompanied by H(2) evolution. The combined proteins had no ATP-independent hydrogenase activity.

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Site-directed mutagenesis of the Klebsiella pneumoniae nitrogenase. Effects of modifying conserved cysteine residues in the alpha- and beta-subunits.

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