scholarly journals Mechanism of Chloride Elimination from 3-Chloro- and 2,4-Dichloro-cis,cis-Muconate: New Insight Obtained from Analysis of Muconate Cycloisomerase Variant CatB-K169A

2001 ◽  
Vol 183 (15) ◽  
pp. 4551-4561 ◽  
Author(s):  
Ursula Kaulmann ◽  
Stefan R. Kaschabek ◽  
Michael Schlömann
Keyword(s):  
Pseudomonas Putida ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Significant Rate ◽  
Chloride Elimination ◽  
Muconate Cycloisomerase

ABSTRACT Chloromuconate cycloisomerases of bacteria utilizing chloroaromatic compounds are known to convert 3-chloro-cis,cis-muconate tocis-dienelactone (cis-4-carboxymethylenebut-2-en-4-olide), while usual muconate cycloisomerases transform the same substrate to the bacteriotoxic protoanemonin. Formation of protoanemonin requires that the cycloisomerization of 3-chloro-cis,cis-muconate to 4-chloromuconolactone is completed by protonation of the exocyclic carbon of the presumed enol/enolate intermediate before chloride elimination and decarboxylation take place to yield the final product. The formation ofcis-dienelactone, in contrast, could occur either by dehydrohalogenation of 4-chloromuconolactone or, more directly, by chloride elimination from the enol/enolate intermediate. To reach a better understanding of the mechanisms of chloride elimination, the proton-donating Lys169 of Pseudomonas putida muconate cycloisomerase was changed to alanine. As expected, substrates requiring protonation, such ascis,cis-muconate as well as 2- and 3-methyl-, 3-fluoro-, and 2-chloro-cis,cis-muconate, were not converted at a significant rate by the K169A variant. However, the variant was still active with 3-chloro- and 2,4-dichloro-cis,cis-muconate. Interestingly,cis-dienelactone and 2-chloro-cis-dienelactone were formed as products, whereas the wild-type enzyme forms protoanemonin and the not previously isolated 2-chloroprotoanemonin, respectively. Thus, the chloromuconate cycloisomerases may avoid (chloro-)protoanemonin formation by increasing the rate of chloride abstraction from the enol/enolate intermediate compared to that of proton addition to it.

scholarly journals Disulfide bond engineering of AppA phytase for increased thermostability requires co-expression of protein disulfide isomerase in Pichia pastoris

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Laura Navone ◽  
Thomas Vogl ◽  
Pawarisa Luangthongkam ◽  
Jo-Anne Blinco ◽  
Carlos H. Luna-Flores ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Phytic Acid ◽  
Disulfide Bond ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
E Coli ◽  
Biochemical Characterisation ◽  
Disulfide Bond Isomerase ◽  
Microbial Systems ◽  
Protein Disulfide

Abstract Background Phytases are widely used commercially as dietary supplements for swine and poultry to increase the digestibility of phytic acid. Enzyme development has focused on increasing thermostability to withstand the high temperatures during industrial steam pelleting. Increasing thermostability often reduces activity at gut temperatures and there remains a demand for improved phyases for a growing market. Results In this work, we present a thermostable variant of the E. coli AppA phytase, ApV1, that contains an extra non-consecutive disulfide bond. Detailed biochemical characterisation of ApV1 showed similar activity to the wild type, with no statistical differences in kcat and KM for phytic acid or in the pH and temperature activity optima. Yet, it retained approximately 50% activity after incubations for 20 min at 65, 75 and 85 °C compared to almost full inactivation of the wild-type enzyme. Production of ApV1 in Pichia pastoris (Komagataella phaffi) was much lower than the wild-type enzyme due to the presence of the extra non-consecutive disulfide bond. Production bottlenecks were explored using bidirectional promoters for co-expression of folding chaperones. Co-expression of protein disulfide bond isomerase (Pdi) increased production of ApV1 by ~ 12-fold compared to expression without this folding catalyst and restored yields to similar levels seen with the wild-type enzyme. Conclusions Overall, the results show that protein engineering for enhanced enzymatic properties like thermostability may result in folding complexity and decreased production in microbial systems. Hence parallel development of improved production strains is imperative to achieve the desirable levels of recombinant protein for industrial processes.

scholarly journals Engineered Phage Endolysin Eliminates Gardnerella Biofilm without Damaging Beneficial Bacteria in Bacterial Vaginosis Ex Vivo

Pathogens ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 54
Author(s):  
Christine Landlinger ◽  
Lenka Tisakova ◽  
Vera Oberbauer ◽  
Timo Schwebs ◽  
Abbas Muhammad ◽  
...  
Keyword(s):  
Bacterial Vaginosis ◽  
Bactericidal Activity ◽  
High Selectivity ◽  
Ex Vivo ◽  
Vaginal Microbiome ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Promising Alternative ◽  
First Time

Bacterial vaginosis is characterized by an imbalance of the vaginal microbiome and a characteristic biofilm formed on the vaginal epithelium, which is initiated and dominated by Gardnerella bacteria, and is frequently refractory to antibiotic treatment. We investigated endolysins of the type 1,4-beta-N-acetylmuramidase encoded on Gardnerella prophages as an alternative treatment. When recombinantly expressed, these proteins demonstrated strong bactericidal activity against four different Gardnerella species. By domain shuffling, we generated several engineered endolysins with 10-fold higher bactericidal activity than any wild-type enzyme. When tested against a panel of 20 Gardnerella strains, the most active endolysin, called PM-477, showed minimum inhibitory concentrations of 0.13–8 µg/mL. PM-477 had no effect on beneficial lactobacilli or other species of vaginal bacteria. Furthermore, the efficacy of PM-477 was tested by fluorescence in situ hybridization on vaginal samples of fifteen patients with either first time or recurring bacterial vaginosis. In thirteen cases, PM-477 killed the Gardnerella bacteria and physically dissolved the biofilms without affecting the remaining vaginal microbiome. The high selectivity and effectiveness in eliminating Gardnerella, both in cultures of isolated strains as well as in clinically derived samples of natural polymicrobial biofilms, makes PM-477 a promising alternative to antibiotics for the treatment of bacterial vaginosis, especially in patients with frequent recurrence.

scholarly journals A Novel Sucrose Isomerase Producing Isomaltulose from Raoultella terrigena

2021 ◽  
Vol 11 (12) ◽  
pp. 5521
Author(s):  
Li Liu ◽  
Shuhuai Yu ◽  
Wei Zhao
Keyword(s):  
Ph Value ◽  
Wild Type ◽  
Michaelis Constant ◽  
Wild Type Enzyme ◽  
Maximum Reaction Rate ◽  
E Coli ◽  
Maximum Reaction ◽  
Conversion Rates ◽  
Raoultella Terrigena ◽  
Sucrose Isomerase

Isomaltulose is widely used in the food industry as a substitute for sucrose owing to its good processing characteristics and physicochemical properties, which is usually synthesized by sucrose isomerase (SIase) with sucrose as substrate. In this study, a gene pal-2 from Raoultella terrigena was predicted to produce SIase, which was subcloned into pET-28a (+) and transformed to the E. coli system. The purified recombinant SIase Pal-2 was characterized in detail. The enzyme is a monomeric protein with a molecular weight of approximately 70 kDa, showing an optimal temperature of 40 °C and optimal pH value of 5.5. The Michaelis constant (Km) and maximum reaction rate (Vmax) are 62.9 mmol/L and 286.4 U/mg, respectively. The conversion rate of isomaltulose reached the maximum of 81.7% after 6 h with 400 g/L sucrose as the substrate and 25 U/mg sucrose of SIase. Moreover, eight site-directed variants were designed and generated. Compared with the wild-type enzyme, the enzyme activities of two mutants N498P and Q275R were increased by 89.2% and 42.2%, respectively, and the isomaltulose conversion rates of three mutants (Y246L, H287R, and H481P) were improved to 89.1%, 90.7%, and 92.4%, respectively. The work identified a novel SIase from the Raoultella genus and its mutants showed a potential to be used for the production of isomaltulose in the industry.

scholarly journals Active-site serine mutants of the Streptomyces albus G β-lactamase

1991 ◽  
Vol 277 (3) ◽  
pp. 647-652 ◽  
Author(s):  
F Jacob ◽  
B Joris ◽  
J M Frère
Keyword(s):  
Active Site ◽  
Specific Activity ◽  
Site Directed Mutagenesis ◽  
Beta Lactamase ◽  
Wild Type ◽  
Serine Residue ◽  
Wild Type Enzyme ◽  
Ph Values ◽  
Streptomyces Albus ◽  
Small Production

By using site-directed mutagenesis, the active-site serine residue of the Streptomyces albus G beta-lactamase was substituted by alanine and cysteine. Both mutant enzymes were produced in Streptomyces lividans and purified to homogeneity. The cysteine beta-lactamase exhibited a substrate-specificity profile distinct from that of the wild-type enzyme, and its kcat./Km values at pH 7 were never higher than 0.1% of that of the serine enzyme. Unlike the wild-type enzyme, the activity of the mutant increased at acidic pH values. Surprisingly, the alanine mutant exhibited a weak but specific activity for benzylpenicillin and ampicillin. In addition, a very small production of wild-type enzyme, probably due to mistranslation, was detected, but that activity could be selectively eliminated. Both mutant enzymes were nearly as thermostable as the wild-type.

scholarly journals Effects of Combination of Different −10 Hexamers and Downstream Sequences on Stationary-Phase-Specific Sigma Factor ςS-Dependent Transcription in Pseudomonas putida

2000 ◽  
Vol 182 (23) ◽  
pp. 6707-6713 ◽  
Author(s):  
Eve-Ly Ojangu ◽  
Andres Tover ◽  
Riho Teras ◽  
Maia Kivisaar
Keyword(s):  
Stationary Phase ◽  
Pseudomonas Putida ◽  
Sigma Factor ◽  
Sigma Factors ◽  
Deficient Mutant ◽  
Wild Type ◽  
In Vivo Experiments ◽  
Silent Genes ◽  
Rna Polymerase Holoenzyme

ABSTRACT The main sigma factor activating gene expression, necessary in stationary phase and under stress conditions, is ςS. In contrast to other minor sigma factors, RNA polymerase holoenzyme containing ςS (EςS) recognizes a number of promoters which are also recognized by that containing ς70 (Eς70). We have previously shown that transposon Tn4652 can activate silent genes in starvingPseudomonas putida cells by creating fusion promoters during transposition. The sequence of the fusion promoters is similar to the ς70-specific promoter consensus. The −10 hexameric sequence and the sequence downstream from the −10 element differ among these promoters. We found that transcription from the fusion promoters is stationary phase specific. Based on in vivo experiments carried out with wild-type and rpoS-deficient mutant P. putida, the effect of ςS on transcription from the fusion promoters was established only in some of these promoters. The importance of the sequence of the −10 hexamer has been pointed out in several published papers, but there is no information about whether the sequences downstream from the −10 element can affect ςS-dependent transcription. Combination of the −10 hexameric sequences and downstream sequences of different fusion promoters revealed that ςS-specific transcription from these promoters is not determined by the −10 hexameric sequence only. The results obtained in this study indicate that the sequence of the −10 element influences ςS-specific transcription in concert with the sequence downstream from the −10 box.

scholarly journals The importance of the interdomain hinge in intramolecular electron transfer in flavocytochrome b2

1993 ◽  
Vol 291 (1) ◽  
pp. 89-94 ◽  
Author(s):  
P White ◽  
F D C Manson ◽  
C E Brunt ◽  
S K Chapman ◽  
G A Reid
Keyword(s):  
Electron Transfer ◽  
Steady State ◽  
Cytochrome C ◽  
Kinetic Properties ◽  
Stopped Flow ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Cytochrome C Reductase ◽  
Flavocytochrome B2 ◽  
Cytochrome C Reduction

The two distinct domains of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase) are connected by a typical hinge peptide. The amino acid sequence of this interdomain hinge is dramatically different in flavocytochromes b2 from Saccharomyces cerevisiae and Hansenula anomala. This difference in the hinge is believed to contribute to the difference in kinetic properties between the two enzymes. To probe the importance of the hinge, an interspecies hybrid enzyme has been constructed comprising the bulk of the S. cerevisiae enzyme but containing the H. anomala flavocytochrome b2 hinge. The kinetic properties of this ‘hinge-swap’ enzyme have been investigated by steady-state and stopped-flow methods. The hinge-swap enzyme remains a good lactate dehydrogenase as is evident from steady-state experiments with ferricyanide as acceptor (only 3-fold less active than wild-type enzyme) and stopped-flow experiments monitoring flavin reduction (2.5-fold slower than in wild-type enzyme). The major effect of the hinge-swap mutation is to lower dramatically the enzyme's effectiveness as a cytochrome c reductase; kcat. for cytochrome c reduction falls by more than 100-fold, from 207 +/- 10 s-1 (25 degrees C, pH 7.5) in the wild-type enzyme to 1.62 +/- 0.41 s-1 in the mutant enzyme. This fall in cytochrome c reductase activity results from poor interdomain electron transfer between the FMN and haem groups. This can be demonstrated by the fact that the kcat. for haem reduction in the hinge-swap enzyme (measured by the stopped-flow method) has a value of 1.61 +/- 0.42 s-1, identical with the value for cytochrome c reduction and some 300-fold lower than the value for the wild-type enzyme. From these and other kinetic parameters, including kinetic isotope effects with [2-2H]lactate, we conclude that the hinge plays a crucial role in allowing efficient electron transfer between the two domains of flavocytochrome b2.

Substitution of murine ferrochelatase glutamate-287 with glutamine or alanine leads to porphyrin substrate-bound variants

2001 ◽  
Vol 356 (1) ◽  
pp. 217-222 ◽  
Author(s):  
Ricardo FRANCO ◽  
Alice S. PEREIRA ◽  
Pedro TAVARES ◽  
Arianna MANGRAVITA ◽  
Michael J. BARBER ◽  
...  
Keyword(s):  
Absorption Spectra ◽  
Catalytic Mechanism ◽  
Protoporphyrin Ix ◽  
Three Dimensional ◽  
Iron Chelation ◽  
Enzymic Activity ◽  
Dimensional Structure ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Flow Experiments

Ferrochelatase (EC 4.99.1.1) is the terminal enzyme of the haem biosynthetic pathway and catalyses iron chelation into the protoporphyrin IX ring. Glutamate-287 (E287) of murine mature ferrochelatase is a conserved residue in all known sequences of ferrochelatase, is present at the active site of the enzyme, as inferred from the Bacillus subtilis ferrochelatase three-dimensional structure, and is critical for enzyme activity. Substitution of E287 with either glutamine (Q) or alanine (A) yielded variants with lower enzymic activity than that of the wild-type ferrochelatase and with different absorption spectra from the wild-type enzyme. In contrast to the wild-type enzyme, the absorption spectra of the variants indicate that these enzymes, as purified, contain protoporphyrin IX. Identification and quantification of the porphyrin bound to the E287-directed variants indicate that approx. 80% of the total porphyrin corresponds to protoporphyrin IX. Significantly, rapid stopped-flow experiments of the E287A and E287Q variants demonstrate that reaction with Zn2+ results in the formation of bound Zn-protoporphyrin IX, indicating that the endogenously bound protoporphyrin IX can be used as a substrate. Taken together, these findings suggest that the structural strain imposed by ferrochelatase on the porphyrin substrate as a critical step in the enzyme catalytic mechanism is also accomplished by the E287A and E287Q variants, but without the release of the product. Thus E287 in murine ferrochelatase appears to be critical for the catalytic process by controlling the release of the product.

Molecular characterization of three mutations in katG affecting the activity of hydroperoxidase I of Escherichia coli

1990 ◽  
Vol 68 (7-8) ◽  
pp. 1037-1044 ◽  
Author(s):  
Peter C. Loewen ◽  
Jacek Switala ◽  
Mark Smolenski ◽  
Barbara L. Triggs-Raine
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Polymerase Chain Reaction Amplification ◽  
Protein A ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Gene Encoding ◽  
Reaction Amplification ◽  
Mutant Genes

Hydroperoxidase I (HPI) of Escherichia coli is a bifunctional enzyme exhibiting both catalase and peroxidase activities. Mutants lacking appreciable HPI have been generated using nitrosoguanidine and the gene encoding HPI, katG, has been cloned from three of these mutants using either classical probing methods or polymerase chain reaction amplification. The mutant genes were sequenced and the changes from wild-type sequence identified. Two mutants contained G to A changes in the coding strand, resulting in glycine to aspartate changes at residues 119 (katG15) and 314 (katG16) in the deduced amino acid sequence of the protein. A third mutant contained a C to T change resulting in a leucine to phenylalanine change at residue 139 (katG14). The Phe139-, Asp119-, and Asp314-containing mutants exhibited 13, < 1, and 18%, respectively, of the wild-type catalase specific activity and 43, 4, and 45% of the wild-type peroxidase specific activity. All mutant enzymes bound less protoheme IX than the wild-type enzyme. The sensitivities of the mutant enzymes to the inhibitors hydroxylamine, azide, and cyanide and the activators imidazole and Tris were similar to those of the wild-type enzyme. The mutant enzymes were more sensitive to high temperature and to β-mercaptoethanol than the wild-type enzyme. The pH profiles of the mutant catalases were unchanged from the wild-type enzyme.Key words: catalase, hydroperoxidase I, mutants, sequence analysis.

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