Spectroscopic characterization of mutations at the Phe41 position in the distal haem pocket of horseradish peroxidase C: structural and functional consequences

2002 ◽  
Vol 363 (3) ◽  
pp. 571-579 ◽  
Author(s):  
Hendrik A. HEERING ◽  
Andrew T. SMITH ◽  
Giulietta SMULEVICH
Keyword(s):  
Horseradish Peroxidase ◽  
Fold Increase ◽  
Spectroscopic Characterization ◽  
Wild Type ◽  
Compound I ◽  
Peroxidase Isoenzyme ◽  
Mutant Proteins ◽  
Aromatic Donor ◽  
Form Compound ◽  
Trp Mutant

Three mutants of horseradish peroxidase isoenzyme C (HRPC) have been constructed in which the conserved distal aromatic residue Phe41 has been substituted by Trp, Val or Ala and the properties of the mutant proteins have been compared with that of the wild-type. The ferric and ferrous states have been studied by resonance Raman, electronic absorption and Fourier-transform infrared spectroscopies, together with their respective fluoride and CO complexes as probes for the integrity of the distal haem-pocket hydrogen-bonding network. The catalytic properties of the mutants, most notably the HRPC-mutant Phe41→Trp (F41W) variant, were also affected. Structural modelling suggests that the bulky indole group of the F41W mutant blocks the distal cavity, inhibiting the binding of fluoride and CO to the haem iron, severely impairing the reaction of the enzyme with H2O2 to form Compound I. Substitution with the smaller side-chain residues Val or Ala resulted in a 2-fold increase in the affinity of the mutants for the aromatic donor benzhydroxamic acid (BHA) compared with the wild-type, whereas the sterically hindered F41W mutant was not able to bind BHA at all. All the mutations studied increased the amount of a ferric six-coordinate aquo-high-spin species. On the other hand, the similarity in the Fe—Im stretching frequencies of the mutants and wild-type protein suggests that the distal haem-pocket mutations do not cause any substantive changes on the proximal side of the haem. Spectra of the HRPC mutant Phe41→Ala—CO and the HRPC mutant Phe41→Val—CO complexes strongly suggested a weakening of the interaction between CO and Arg38 due to a secondary rearrangement of the haem relative to helix B. The effects observed for these HRP mutants were somewhat different from those noted recently for the analogous Coprinus cinereus peroxidase (CIP) mutants, particularly the Trp mutant. These differences can be reconciled in part as being due to the smaller size of the distal cavity of HRP compared with that of CIP.

scholarly journals Conversion of Escherichia coli pyruvate oxidase to an ‘α-ketobutyrate oxidase’

2000 ◽  
Vol 352 (3) ◽  
pp. 717-724 ◽  
Author(s):  
Ying-Ying CHANG ◽  
John E. CRONAN
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Random Mutagenesis ◽  
Fold Increase ◽  
Natural Substrate ◽  
Wild Type ◽  
Pyruvate Oxidase ◽  
Mutant Proteins ◽  
Essentially Normal

Escherichia coli pyruvate oxidase (PoxB), a lipid-activated homotetrameric enzyme, is active on both pyruvate and 2-oxobutanoate (‘α-ketobutyrate’), although pyruvate is the favoured substrate. By localized random mutagenesis of residues chosen on the basis of a modelled active site, we obtained several PoxB enzymes that had a markedly decreased activity with the natural substrate, pyruvate, but retained full activity with 2-oxobutanoate. In each of these mutant proteins Val-380had been replaced with a smaller residue, namely alanine, glycine or serine. One of these, PoxB V380A/L253F, was shown to lack detectable pyruvate oxidase activity in vivo; this protein was purified, studied and found to have a 6-fold increase in Km for pyruvate and a 10-fold lower Vmax with this substrate. In contrast, the mutant had essentially normal kinetic constants with 2-oxobutanoate. The altered substrate specificity was reflected in a decreased rate of pyruvate binding to the latent conformer of the mutant protein owing to the V380A mutation. The L253F mutation alone had no effect on PoxB activity, although it increased the activity of proteins carrying substitutions at residue 380, as it did that of the wild-type protein. The properties of the V380A/L253F protein provide new insights into the mode of substrate binding and the unusual activation properties of this enzyme.

scholarly journals Scavenging of oxygen radicals by heme peroxidases.

1996 ◽  
Vol 43 (4) ◽  
pp. 673-678 ◽  
Author(s):  
L Gebicka ◽  
J L Gebicki
Keyword(s):  
Horseradish Peroxidase ◽  
Hydroxyl Radicals ◽  
Superoxide Radical ◽  
Cation Radical ◽  
Compound I ◽  
Superoxide Radical Anion ◽  
Form Compound ◽  
Activity Loss ◽  
Heme Peroxidases ◽  
Compound Ii

The reactions of two heme peroxidases, horseradish peroxidase and lactoperoxidase and their compounds II (oxoferryl heme intermediates, Fe(IV) = O or ferric protein radical Fe(III)R.) and compounds III (resonance hybrids [Fe(III)-O2-. Fe(II)-O2] with superoxide radical anion generated enzymatically or radiolytically, and with hydroxyl radicals generated radiolytically, were investigated. It is suggested that only the protein radical form of compound II of lactoperoxidase reacts with superoxide, whereas compound II of horseradish peroxidase, which exists only in oxoferryl form, is unreactive towards superoxide. Compound III of the investigated peroxidases does not react with superoxide. The lactoperoxidase activity loss induced by hydroxyl radicals is closely related to the loss of the ability to form compound I (oxoferryl porphyrin pi-cation radical, Fe(IV) = O(Por+.) or oxoferryl protein radical Fe(IV) = O(R.)). On the other hand, the modification of horseradish peroxidase induced by hydroxyl radicals has been reported to cause also restrictions in substrate binding (Gebicka, L. & Gebicki, J.L., 1996, Biochimie 78, 62-65). Nevertheless, it has been found that only a small fraction of hydroxyl radicals generated homogeneously does inactivate the enzymes.

scholarly journals Specificity Mutants of the Binding Protein of the Oligopeptide Transport System of Lactococcus lactis

2000 ◽  
Vol 182 (6) ◽  
pp. 1600-1608 ◽  
Author(s):  
Antonia Picon ◽  
Edmund R. S. Kunji ◽  
Frank C. Lanfermeijer ◽  
Wil N. Konings ◽  
Bert Poolman
Keyword(s):  
Lactococcus Lactis ◽  
Fold Increase ◽  
Binding Activity ◽  
Kinetic Properties ◽  
Wild Type ◽  
Mutant Proteins ◽  
Serovar Typhimurium ◽  
Peptide Binding Site ◽  
Oligopeptide Transport

ABSTRACT The kinetic properties of wild-type and mutant oligopeptide binding proteins of Lactococcus lactis were determined. To observe the properties of the mutant proteins in vivo, the oppAgene was deleted from the chromosome of L. lactis to produce a strain that was totally defective in oligopeptide transport. Amplified expression of the oppA gene resulted in an 8- to 12-fold increase in OppA protein relative to the wild-type level. The amplified expression was paralleled by increased bradykinin binding activity, but had relatively little effect on the overall transport of bradykinin via Opp. Several site-directed mutants were constructed on the basis of a comparison of the primary sequences of OppA fromSalmonella enterica serovar Typhimurium and L. lactis, taking into account the known structure of the serovar Typhimurium protein. Putative peptide binding-site residues were mutated. All the mutant OppA proteins exhibited a decreased binding affinity for the high-affinity peptide bradykinin. Except for OppA(D471R), the mutant OppA proteins displayed highly defective bradykinin uptake, whereas the transport of the low-affinity substrate KYGK was barely affected. Cells expressing OppA(D471R) had a similarKm for transport, whereas theV max was increased more than twofold as compared to the wild-type protein. The data are discussed in the light of a kinetic model and imply that the rate of transport is determined to a large extent by the donation of the peptide from the OppA protein to the translocator complex.

scholarly journals The Histone Fold Domain of Cse4 Is Sufficient for CEN Targeting and Propagation of Active Centromeres in Budding Yeast

2004 ◽  
Vol 3 (6) ◽  
pp. 1533-1543 ◽  
Author(s):  
Lisa Morey ◽  
Kelly Barnes ◽  
Yinhuai Chen ◽  
Molly Fitzgerald-Hayes ◽  
Richard E. Baker
Keyword(s):  
Budding Yeast ◽  
Fold Increase ◽  
Chromosome Loss ◽  
Wild Type ◽  
Mutant Proteins ◽  
Histone Fold ◽  
Kinetochore Assembly ◽  
Histone Fold Domain ◽  
Terminal Amino

ABSTRACT Centromere-specific H3-like proteins (CenH3s) are conserved across the eukaryotic kingdom and are required for packaging centromere DNA into a specialized chromatin structure required for kinetochore assembly. Cse4 is the CenH3 protein of the budding yeast Saccharomyces cerevisiae. Like all CenH3 proteins, Cse4 consists of a conserved histone fold domain (HFD) and a divergent N terminus (NT). The Cse4 NT contains an essential domain designated END (for essential N-terminal domain); deletion of END is lethal. To investigate the role of the Cse4 NT in centromere targeting, a series of deletion alleles (cse4ΔNT) were analyzed. No part of the Cse4 NT was required to target mutant proteins to centromere DNA in the presence of functional Cse4. A Cse4 degron strain was used to examine targeting of a Cse4ΔNT protein in the absence of wild-type Cse4. The END was not required for centromere targeting under these conditions, confirming that the HFD confers specificity of Cse4 centromere targeting. Surprisingly, overexpression of the HFD bypassed the requirement for the END altogether, and viable S. cerevisiae strains in which the cells express only the Cse4 HFD and six adjacent N-terminal amino acids (Cse4Δ129) were constructed. Despite the complete absence of the NT, mitotic chromosome loss in the cse4Δ129 strain increased only 6-fold compared to a 15-fold increase in strains overexpressing wild-type Cse4. Thus, when overexpressed, the Cse4 HFD is sufficient for centromere function in S. cerevisiae, and no posttranslational modification or interaction of the NT with other kinetochore component(s) is essential for accurate chromosome segregation in budding yeast.

scholarly journals Lecithin:cholesterol acyltransferase: role of N-linked glycosylation in enzyme function

1993 ◽  
Vol 294 (3) ◽  
pp. 879-884 ◽  
Author(s):  
K O ◽  
J S Hill ◽  
X Wang ◽  
R McLeod ◽  
P H Pritchard
Keyword(s):  
Enzyme Activity ◽  
Specific Activity ◽  
Consensus Sequence ◽  
Fold Increase ◽  
Glycosylation Site ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Lecithin:Cholesterol Acyltransferase ◽  
Mutant Proteins ◽  
Glycosylation Sites

Lecithin:cholesterol acyltransferase (LCAT; phosphatidylcholine-sterol acyltransferase, EC 2.3.1.43) is a glycoprotein which is responsible for the formation of cholesteryl ester in plasma. The carbohydrate content has been estimated to be approx. 25% of the total LCAT mass, and four potential N-linked glycosylation sites have been predicted at residues 20, 84, 272 and 384 of the LCAT protein sequence. In the present study, we have examined which of these sites are utilized and how the N-glycosylation affects the secretion and function of the enzyme. Site-directed mutagenesis was performed to eliminate the glycosylation consensus sequence at each of the four potential sites, and the mutant proteins were expressed in COS cells. The amount of each mutant LCAT secreted was similar to that of the wild-type enzyme but the molecular mass was decreased by 3-4 kDa. The specific activity of each mutant LCAT was significantly different from the wild-type; however, the magnitude and direction of the change depended on the glycosylation site mutagenized. Loss of carbohydrate at position 20, 84 or 272 resulted in a decrease in the specific activity of the mutant enzymes by 18%, 82%, and 62% respectively. In contrast, the mutant protein without glycosylation at position 384 displayed a 2-fold increase in enzyme activity. In addition, a quadruple mutant was constructed such that all four potential glycosylation sites were eliminated. The amount of the unglycosylated LCAT secreted into the culture medium was less than 10% of the wild-type level and the specific activity of this enzyme was decreased to 5% of that of the wild type. The results demonstrate that all four potential N-glycosylation sites in LCAT are used and the presence of carbohydrate at each site has diverse effects on the enzyme activity.

scholarly journals Rab 7: an important regulator of late endocytic membrane traffic.

1995 ◽  
Vol 131 (6) ◽  
pp. 1435-1452 ◽  
Author(s):  
Y Feng ◽  
B Press ◽  
A Wandinger-Ness
Keyword(s):  
Horseradish Peroxidase ◽  
G Protein ◽  
Dominant Negative ◽  
Endocytic Pathway ◽  
Dominant Negative Mutant ◽  
Wild Type ◽  
Small Molecular Weight ◽  
Mutant Proteins ◽  
Late Endosomes ◽  
Normal Transport

Rab5 and rab7 proteins belong to a superfamily of small molecular weight GTPases known to be associated with early and late endosomes, respectively. The rab5 protein plays an important regulatory role in early endocytosis, yet the function of rab7 protein was previously uncharacterized. This question was addressed by comparing the kinetics of vesicular stomatitis virus (VSV) G protein internalization in baby hamster kidney cells overexpressing wild-type or dominant negative mutant forms of the rab7 protein (rab7N125I and rab7T22N). Overexpression of wild-type rab7 protein allowed normal transport to late endosomes (mannose 6-phosphate receptor positive), while the rab7N125I mutant caused the VSV G protein to accumulate specifically in early (transferrin receptor positive) endosomes. Horseradish peroxidase and paramyxovirus SV5 hemagglutinin-neuraminidase (HN) were used in quantitative biochemical assays to further demonstrate that rab7 function was not required for early internalization events, but was crucial in downstream degradative events. The characteristic cleavage of SV5 HN in the late endosome distinguishes internalization from transport to later stages of the endocytic pathway. Mutant rab7N125I or rab7T22N proteins had no effect on the internalization of either horseradish peroxidase or SV5 HN protein. In contrast, the mutant proteins markedly inhibited the subsequent cleavage of the SV5 HN protein. Taken together, these data support a key role for rab7, downstream of rab5, in regulating membrane transport leading from early to late endosomes. We compare our findings to those obtained for the yeast homologues Ypt51p, Ypt52p, Ypt53p, and Ypt7p.

scholarly journals Functional expression of a yeast mitochondrial intron-encoded protein requires RNA processing at a conserved dodecamer sequence at the 3' end of the gene.

1989 ◽  
Vol 9 (4) ◽  
pp. 1507-1512 ◽  
Author(s):  
H Zhu ◽  
H Conrad-Webb ◽  
X S Liao ◽  
P S Perlman ◽  
R A Butow
Keyword(s):  
Genetic Analysis ◽  
Rna Processing ◽  
Fold Increase ◽  
Functional Expression ◽  
Rrna Gene ◽  
Yeast Mitochondria ◽  
Wild Type ◽  
Site Specific ◽  
Var1 Gene ◽  
A Site

All mRNAs of yeast mitochondria are processed at their 3' ends within a conserved dodecamer sequence, 5'-AAUAAUAUUCUU-3'. A dominant nuclear suppressor, SUV3-I, was previously isolated because it suppresses a dodecamer deletion at the 3' end of the var1 gene. We have tested the effects of SUV3-1 on a mutant containing two adjacent transversions within a dodecamer at the 3' end of fit1, a gene located within the 1,143-base-pair intron of the 21S rRNA gene, whose product is a site-specific endonuclease required in crosses for the quantitative transmission of that intron to 21S alleles that lack it. The fit1 dodecamer mutations blocked both intron transmission and dodecamer cleavage, neither of which was suppressed by SUV3-1 when present in heterozygous or homozygous configurations. Unexpectedly, we found that SUV3-1 completely blocked cleavage of the wild-type fit1 dodecamer and, in SUV3-1 homozygous crosses, intron conversion. In addition, SUV3-1 resulted in at least a 40-fold increase in the amount of excised intron accumulated. Genetic analysis showed that these phenotypes resulted from the same mutation. We conclude that cleavage of a wild-type dodecamer sequence at the 3' end of the fit1 gene is essential for fit1 expression.

scholarly journals The TonB system in Aeromonas hydrophila NJ-35 is essential for MacA2B2 efflux pump-mediated macrolide resistance

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Yuhao Dong ◽  
Qing Li ◽  
Jinzhu Geng ◽  
Qing Cao ◽  
Dan Zhao ◽  
...  
Keyword(s):  
Aeromonas Hydrophila ◽  
Efflux Pump ◽  
Fold Increase ◽  
Activity Level ◽  
Macrolide Resistance ◽  
Wild Type ◽  
Iron Deprivation ◽  
Pump Activity ◽  
Tonb System ◽  
Increased Sensitivity

AbstractThe TonB system is generally considered as an energy transporting device for the absorption of nutrients. Our recent study showed that deletion of this system caused a significantly increased sensitivity of Aeromonas hydrophila to the macrolides erythromycin and roxithromycin, but had no effect on other classes of antibiotics. In this study, we found the sensitivity of ΔtonB123 to all macrolides tested revealed a 8- to 16-fold increase compared with the wild-type (WT) strain, but this increase was not related with iron deprivation caused by tonB123 deletion. Further study demonstrated that the deletion of tonB123 did not damage the integrity of the bacterial membrane but did hinder the function of macrolide efflux. Compared with the WT strain, deletion of macA2B2, one of two ATP-binding cassette (ABC) types of the macrolide efflux pump, enhanced the sensitivity to the same levels as those of ΔtonB123. Interestingly, the deletion of macA2B2 in the ΔtonB123 mutant did not cause further increase in sensitivity to macrolide resistance, indicating that the macrolide resistance afforded by the MacA2B2 pump was completely abrogated by tonB123 deletion. In addition, macA2B2 expression was not altered in the ΔtonB123 mutant, indicating that any influence of TonB on MacA2B2-mediated macrolide resistance was at the pump activity level. In conclusion, inactivation of the TonB system significantly compromises the resistance of A. hydrophila to macrolides, and the mechanism of action is related to the function of MacA2B2-mediated macrolide efflux.

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