scholarly journals Substitutions for Glu-537 of β-galactosidase from Escherichia coli cause large decreases in catalytic activity

1994 ◽  
Vol 299 (2) ◽  
pp. 527-531 ◽  
Author(s):  
J Yuan ◽  
M Martinez-Bilbao ◽  
R E Huber
Keyword(s):  
Wild Type ◽  
Inhibitory Effects ◽  
Wild Type Enzyme ◽  
Enzyme Preparations ◽  
Transition State Analogues ◽  
Normal Enzyme ◽  
Enzyme Molecules ◽  
The Stability ◽  
Beta Galactosidase ◽  
Synthetic Oligonucleotides

Glu-537 of beta-galactosidase (EC 3.2.1.23) was replaced by Asp, Gln and Val using synthetic oligonucleotides. The kcat values of the purified enzyme mixtures were reduced by about 100-fold for the Asp mutant, 30,000-60,000-fold for the Val mutant and 160,000-300,000-fold for the Gln mutant. The greatest differences in properties from the wild-type enzyme were found for the Asp-substituted enzyme: the Km values increased (from 0.12 to 0.42 mM for o-nitrophenyl beta-D-galactopyranoside), and from 0.04 to 0.37 mM for p-nitrophenyl beta-D-galactopyranoside), the Ki value for isopropyl beta-D-galactopyranoside increased (from 0.11 to 0.30 mM), the stability to heat decreased and methanol did not act as an acceptor. The enzymes with the other two substitutions had properties similar to those of the wild-type. For all three substituted enzymes, the inhibitory effects of the transition-state analogues (2-deoxy-2-amino-D-galactose and L-ribose) and the Mg2+ effects were similar to those of the normal enzyme. As all of the properties (except the kcat values) of the Gln- and Val-substituted enzyme preparations were similar to those of the wild-type enzyme, the activities in those preparations were probably due to the presence of a few wild-type enzyme molecules (formed from misreads) among the substituted enzymes. The enzymes with Gln and Val substitutions appear to be totally inactive. The results obtained support a recent suggestion that Glu-537 is an important catalytic residue of beta-galactosidase.

scholarly journals Enhancement of the Stability of a Prolipase from Rhizopus oryzae toward Aldehydes by Saturation Mutagenesis

2007 ◽  
Vol 73 (22) ◽  
pp. 7291-7299 ◽  
Author(s):  
Mirella Di Lorenzo ◽  
Aurelio Hidalgo ◽  
Rafael Molina ◽  
Juan A. Hermoso ◽  
Domenico Pirozzi ◽  
...  
Keyword(s):  
Staining Method ◽  
Rhizopus Oryzae ◽  
Specific Activity ◽  
Oxidation Products ◽  
Saturation Mutagenesis ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Lipid Oxidation Products ◽  
The Stability

ABSTRACT A prolipase from Rhizopus oryzae (proROL) was engineered in order to increase its stability toward lipid oxidation products such as aldehydes with the aim of improving its performance in oleochemical industries. Out of 22 amino acid residues (15 Lys and 7 His) prone to react with aldehydes, 6 Lys and all His residues (except for the catalytic histidine) were chosen and subjected to saturation mutagenesis. In order to quickly and reliably identify stability mutants within the resulting libraries, active variants were prescreened by an activity staining method on agar plates. Active mutants were expressed in Escherichia coli Origami in a 96-well microtiterplate format, and a stability test using octanal as a model deactivating agent was performed. The most stable histidine mutant (H201S) conferred a stability increase of 60%, which was further enhanced to 100% by combination with a lysine mutant (H201S/K168I). This increase in stability was also confirmed for other aldehydes. Interestingly, the mutations did not affect specific activity, as this was still similar to the wild-type enzyme.

scholarly journals ISOLATION AND CHARACTERIZATION OF NEUROSPORA MUTANTS AFFECTED IN INVERTASE SYNTHESIS

Genetics ◽  
1984 ◽  
Vol 106 (4) ◽  
pp. 591-599
Author(s):  
Deborah B Lee ◽  
Stephen J Free
Keyword(s):  
Neurospora Crassa ◽  
Large Scale ◽  
Invertase Activity ◽  
Free Medium ◽  
Wild Type ◽  
Isolation And Characterization ◽  
Normal Enzyme ◽  
Enzyme Molecules ◽  
Large Scale Screening

ABSTRACT We have outlined a procedure that allows the large-scale screening of mutagenized Neurospora crassa populations for invertaseless mutants. We have isolated and characterized three mutations, inv(DBL1), inv(DBL9) and inv(DBL14), which have been mapped at or near the invertase structural gene. One of these, inv(DBL1), is particularly interesting. Our experiments indicate that the reduced level of invertase activity in the inv(DBL1)-containing cell can be explained as the result of a reduced number of normal enzyme molecules. We also show that wild-type Neurospora is able to respond rapidly to a change of medium and can dramatically increase its production of invertase within 20 min after a transfer to a carbon-free medium.

scholarly journals Site-directed mutagenesis and substrate-induced inactivation of β-lactamase I

1992 ◽  
Vol 288 (3) ◽  
pp. 1045-1051 ◽  
Author(s):  
S J Thornewell ◽  
S G Waley
Keyword(s):  
Alpha Helix ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Beta Lactamase ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Extracellular Medium ◽  
The Stability ◽  
Hydrolysis Of ◽  
Reduced Activity

The substrate-induced inactivation of beta-lactamase I from Bacillus cereus 569/H has been studied. Both the wild-type enzyme and mutants have been used. The kinetics follow a branched pathway of the type recently analysed [Waley (1991) Biochem. J. 279, 87-94]. The substrate cloxacillin (a penicillin) formed an acyl-enzyme (characterized by m.s.), and it was probably the instability of this intermediate that brought about inactivation. A disulphide bond was introduced into beta-lactamase I (the wild-type enzyme lacks this bond) by site-directed mutagenesis: Ala-77 and Ala-123 were replaced by cysteine. Spontaneous oxidation yielded the disulphide. The activity of this newly cross-linked enzyme was a little diminished, but the stability towards inactivation by cloxacillin was not increased. A second mutant of beta-lactamase I was studied: this mutant lacked the first 17 residues, i.e. the first alpha-helix. The mutant had reduced activity towards ordinary (non-inactivating) substrates and no hydrolysis of cloxacillin could be detected. These mutant enzymes were expressed in Bacillus subtilis, and were purified from the extracellular medium.

A survey of proteins encoded by non-synonymous single nucleotide polymorphisms reveals a significant fraction with altered stability and activity

2009 ◽  
Vol 424 (1) ◽  
pp. 15-26 ◽  
Author(s):  
Abdellah Allali-Hassani ◽  
Gregory A. Wasney ◽  
Irene Chau ◽  
Bum Soo Hong ◽  
Guillermo Senisterra ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphisms ◽  
Three Dimensional ◽  
Catalytic Efficiency ◽  
Nucleotide Polymorphisms ◽  
Wild Type ◽  
Coding Region ◽  
Wild Type Enzyme ◽  
Single Nucleotide ◽  
The Stability ◽  
And Function

On average, each human gene has approximately four SNPs (single nucleotide polymorphisms) in the coding region, half of which are nsSNPs (non-synonymous SNPs) or missense SNPs. Current attention is focused on those that are known to perturb function and are strongly linked to disease. However, the vast majority of SNPs have not been investigated for the possibility of causing disease. We set out to assess the fraction of nsSNPs that encode proteins that have altered stability and activity, for this class of variants would be candidates to perturb cellular function. We tested the thermostability and, where possible, the catalytic activity for the most common variant (wild-type) and minor variants (total of 46 SNPs) for 16 human enzymes for which the three-dimensional structures were known. There were significant differences in the stability of almost half of the variants (48%) compared with their wild-type counterparts. The catalytic efficiency of approx. 14 variants was significantly altered, including several variants of human PKM2 (pyruvate kinase muscle 2). Two PKM2 variants, S437Y and E28K, also exhibited changes in their allosteric regulation compared with the wild-type enzyme. The high proportion of nsSNPs that affect protein stability and function, albeit subtly, underscores the need for experimental analysis of the diverse human proteome.

scholarly journals Complementation relationships of Neurospora am mutants in relation to their formation of abnormal varieties of glutamate dehydrogenase

1965 ◽  
Vol 6 (1) ◽  
pp. 121-129 ◽  
Author(s):  
J. R. S. Fincham ◽  
D. R. Stadler
Keyword(s):  
Enzyme Activity ◽  
Glutamate Dehydrogenase ◽  
Nitrous Acid ◽  
Active Enzyme ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Inactive Form ◽  
Normal Enzyme ◽  
Altered Form

Six new am mutants of Neurospora, am14–am19, with defective formation of NADP-linked glutamate dehydrogenase, were isolated following treatment of conidia with nitrous acid. This brings the number of genetically distinct and viable am mutants to sixteen. Heterocaryon-compatible isolates of each of the new mutants were tested for complementation with each of the mutants previously known. A number of new complementary combinations were found which add to the complexity of the complementation map which, however, remains linear.The mutants which show complementation are am1, am2, am3, am7, am14 and am19. It has proved possible to isolate an altered form of glutamate dehydrogenase from each of these except am14, which appears to produce no protein fractionating like the normal enzyme. Among the non-complementing mutants, am4 produces an easily-isolated inactive form of glutamate dehydrogenase. These results agree with the immunochemical findings of Roberts & Pateman (1964).Of the mutationally altered forms of the enzyme, the am1, am2, am3 and am7varieties are electrophoretically identical or closely similar to the normal protein, while the am4 and am19 varieties move faster towards the anode at pH 8·5, am19being the faster of the two. The am19 protein, like the am2 and am3 proteins previously reported on, can be activated to give some enzyme activity, but the activity obtained is much lower for this mutant than for am2 or am3.All the complementary pairs of mutants, including those involving am19, form active enzyme varieties which have very close to normal electrophoretic properties. In several cases the complementation enzyme is less stable to heat than the wild-type enzyme.

scholarly journals Simultaneous Enhancement of Thermostability and Catalytic Activity of Phospholipase A1 by Evolutionary Molecular Engineering

2000 ◽  
Vol 66 (3) ◽  
pp. 890-894 ◽  
Author(s):  
Jae Kwang Song ◽  
Joon Shick Rhee
Keyword(s):  
Catalytic Activity ◽  
Molecular Engineering ◽  
Effect Of Temperature ◽  
Mutant Library ◽  
Wild Type ◽  
Phospholipase A1 ◽  
Wild Type Enzyme ◽  
Melting Temperatures ◽  
Different Types ◽  
The Stability

ABSTRACT The thermal stability and catalytic activity of phospholipase A1 from Serratia sp. strain MK1 were improved by evolutionary molecular engineering. Two thermostable mutants were isolated after sequential rounds of error-prone PCR performed to introduce random mutations and filter-based screening of the resultant mutant library; we determined that these mutants had six (mutant TA3) and seven (mutant TA13) amino acid substitutions. Different types of substitutions were found in the two mutants, and these substitutions resulted in an increase in nonploar residues (mutant TA3) or in differences between side chains for polar or charged residues (mutant TA13). The wild-type and mutant enzymes were purified, and the effect of temperature on the stability and catalytic activity of the enzymes was investigated. The melting temperatures of the TA3 and TA13 enzymes were increased by 7 and 11°C, respectively, compared with the melting temperature of the wild-type enzyme. Thus, we found that evolutionary molecular engineering was an effective and efficient approach for increasing thermostability without compromising enzyme activity.

scholarly journals InsP7is a small-molecule regulator of NUDT3-mediated mRNA decapping and processing-body dynamics

2020 ◽  
Vol 117 (32) ◽  
pp. 19245-19253 ◽  
Author(s):  
Soumyadip Sahu ◽  
Zhenzhen Wang ◽  
Xinfu Jiao ◽  
Chunfang Gu ◽  
Nikolaus Jork ◽  
...  
Keyword(s):  
Stress Responses ◽  
Messenger Rna ◽  
Control Process ◽  
Stem Cell Differentiation ◽  
Wild Type ◽  
Inositol Pyrophosphate ◽  
Body Dynamics ◽  
Processing Body ◽  
The Stability

Regulation of enzymatic 5′ decapping of messenger RNA (mRNA), which normally commits transcripts to their destruction, has the capacity to dynamically reshape the transcriptome. For example, protection from 5′ decapping promotes accumulation of mRNAs into processing (P) bodies—membraneless, biomolecular condensates. Such compartmentalization of mRNAs temporarily removes them from the translatable pool; these repressed transcripts are stabilized and stored until P-body dissolution permits transcript reentry into the cytosol. Here, we describe regulation of mRNA stability and P-body dynamics by the inositol pyrophosphate signaling molecule 5-InsP7(5-diphosphoinositol pentakisphosphate). First, we demonstrate 5-InsP7inhibits decapping by recombinant NUDT3 (Nudix [nucleoside diphosphate linked moiety X]-type hydrolase 3) in vitro. Next, in intact HEK293 and HCT116 cells, we monitored the stability of a cadre of NUDT3 mRNA substrates following CRISPR-Cas9 knockout ofPPIP5Ks(diphosphoinositol pentakisphosphate 5-kinases type 1 and 2, i.e.,PPIP5KKO), which elevates cellular 5-InsP7levels by two- to threefold (i.e., within the physiological rheostatic range). ThePPIP5KKO cells exhibited elevated levels of NUDT3 mRNA substrates and increased P-body abundance. Pharmacological and genetic attenuation of 5-InsP7synthesis in the KO background reverted both NUDT3 mRNA substrate levels and P-body counts to those of wild-type cells. Furthermore, liposomal delivery of a metabolically resistant 5-InsP7analog into wild-type cells elevated levels of NUDT3 mRNA substrates and raised P-body abundance. In the context that cellular 5-InsP7levels normally fluctuate in response to changes in the bioenergetic environment, regulation of mRNA structure by this inositol pyrophosphate represents an epitranscriptomic control process. The associated impact on P-body dynamics has relevance to regulation of stem cell differentiation, stress responses, and, potentially, amelioration of neurodegenerative diseases and aging.

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 An MDM2 inhibitor achieves synergistic cytotoxic effects with adenoviruses lacking E1B55kDa gene on mesothelioma with the wild-type p53 through augmenting NFI expression

2021 ◽  
Vol 12 (7) ◽  
Author(s):  
Thao Thi Thanh Nguyen ◽  
Masato Shingyoji ◽  
Michiko Hanazono ◽  
Boya Zhong ◽  
Takao Morinaga ◽  
...  
Keyword(s):  
Wild Type ◽  
Inhibitory Effects ◽  
Cytotoxic Effects ◽  
P53 Expression ◽  
Nuclear Factor I ◽  
Infected Cells ◽  
Wild Type P53 ◽  
Mdm2 Inhibitor ◽  
P16 Expression

AbstractA majority of mesothelioma specimens were defective of p14 and p16 expression due to deletion of the INK4A/ARF region, and the p53 pathway was consequently inactivated by elevated MDM2 functions which facilitated p53 degradaton. We investigated a role of p53 elevation by MDM2 inhibitors, nutlin-3a and RG7112, in cytotoxicity of replication-competent adenoviruses (Ad) lacking the p53-binding E1B55kDa gene (Ad-delE1B). We found that a growth inhibition by p53-activating Ad-delE1B was irrelevant to p53 expression in the infected cells, but combination of Ad-delE1B and the MDM2 inhibitor produced synergistic inhibitory effects on mesothelioma with the wild-type but not mutated p53 genotype. The combination augmented p53 phosphorylation, activated apoptotic but not autophagic pathway, and enhanced DNA damage signals through ATM-Chk2 phosphorylation. The MDM2 inhibitors facilitated production of the Ad progenies through augmented expression of nuclear factor I (NFI), one of the transcriptional factors involved in Ad replications. Knocking down of p53 with siRNA did not increase the progeny production or the NFI expression. We also demonstrated anti-tumor effects by the combination of Ad-delE1B and the MDM2 inhibitors in an orthotopic animal model. These data collectively indicated that upregulation of wild-type p53 expression contributed to cytotoxicity by E1B55kDa-defective replicative Ad through NFI induction and suggested that replication-competent Ad together with augmented p53 levels was a therapeutic strategy for p53 wild-type mesothelioma.

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.

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