scholarly journals A Single Mutation Increases the Thermostability and Activity of Aspergillus terreus Amine Transaminase

Molecules ◽  
2019 ◽  
Vol 24 (7) ◽  
pp. 1194 ◽  
Author(s):  
Wan-Li Zhu ◽  
Sheng Hu ◽  
Chang-Jiang Lv ◽  
Wei-Rui Zhao ◽  
Hong-Peng Wang ◽  
...  
Keyword(s):  
Mutual Information ◽  
Catalytic Efficiency ◽  
Saturation Mutagenesis ◽  
Single Mutation ◽  
Wild Type ◽  
Information Server ◽  
Differential Scanning Fluorimetry ◽  
Stable Mutant ◽  
The Stability ◽  
And Function

Enhancing the thermostability of (R)-selective amine transaminases (AT-ATA) will expand its application in the asymmetric synthesis of chiral amines. In this study, mutual information and coevolution networks of ATAs were analyzed by the Mutual Information Server to Infer Coevolution (MISTIC). Subsequently, the amino acids most likely to influence the stability and function of the protein were investigated by alanine scanning and saturation mutagenesis. Four stabilized mutants (L118T, L118A, L118I, and L118V) were successfully obtained. The best mutant, L118T, exhibited an improved thermal stability with a 3.7-fold enhancement in its half-life (t1/2) at 40 °C and a 5.3 °C increase in T5010 compared to the values for the wild-type protein. By the differential scanning fluorimetry (DSF) analysis, the best mutant, L118T, showed a melting temperature (Tm) of 46.4 °C, which corresponded to a 5.0 °C increase relative to the wild-type AT-ATA (41.4 °C). Furthermore, the most stable mutant L118T displayed the highest catalytic efficiency among the four stabilized mutants.

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 Rational thermostabilisation of four-helix bundle dimeric de novo proteins

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shin Irumagawa ◽  
Kaito Kobayashi ◽  
Yutaka Saito ◽  
Takeshi Miyata ◽  
Mitsuo Umetsu ◽  
...  
Keyword(s):  
Protein Design ◽  
De Novo ◽  
Protein Complexes ◽  
Salt Bridge ◽  
Dynamics Simulation ◽  
Saturation Mutagenesis ◽  
Helix Bundle ◽  
Stable Mutant ◽  
Four Helix Bundle ◽  
The Stability

AbstractThe stability of proteins is an important factor for industrial and medical applications. Improving protein stability is one of the main subjects in protein engineering. In a previous study, we improved the stability of a four-helix bundle dimeric de novo protein (WA20) by five mutations. The stabilised mutant (H26L/G28S/N34L/V71L/E78L, SUWA) showed an extremely high denaturation midpoint temperature (Tm). Although SUWA is a remarkably hyperstable protein, in protein design and engineering, it is an attractive challenge to rationally explore more stable mutants. In this study, we predicted stabilising mutations of WA20 by in silico saturation mutagenesis and molecular dynamics simulation, and experimentally confirmed three stabilising mutations of WA20 (N22A, N22E, and H86K). The stability of a double mutant (N22A/H86K, rationally optimised WA20, ROWA) was greatly improved compared with WA20 (ΔTm = 10.6 °C). The model structures suggested that N22A enhances the stability of the α-helices and N22E and H86K contribute to salt-bridge formation for protein stabilisation. These mutations were also added to SUWA and improved its Tm. Remarkably, the most stable mutant of SUWA (N22E/H86K, rationally optimised SUWA, ROSA) showed the highest Tm (129.0 °C). These new thermostable mutants will be useful as a component of protein nanobuilding blocks to construct supramolecular protein complexes.

scholarly journals Elimination of 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid 9-phosphate synthase activity from human N-acetylneuraminic acid 9-phosphate synthase by a single mutation

2006 ◽  
Vol 397 (1) ◽  
pp. 195-201 ◽  
Author(s):  
Jijun Hao ◽  
Willie F. Vann ◽  
Stephan Hinderlich ◽  
Munirathinam Sundaramoorthy
Keyword(s):  
Aldol Condensation ◽  
Saturation Mutagenesis ◽  
Single Mutation ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
High Sequence Identity ◽  
Acetylneuraminic Acid ◽  
Nonulosonic Acid ◽  
The Impact ◽  
Phosphate Synthase

The most commonly occurring sialic acid Neu5Ac (N-acetylneuraminic acid) and its deaminated form, KDN (2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid), participate in many biological functions. The human Neu5Ac-9-P (Neu5Ac 9-phosphate) synthase has the unique ability to catalyse the synthesis of not only Neu5Ac-9-P but also KDN-9-P (KDN 9-phosphate). Both reactions are catalysed by the mechanism of aldol condensation of PEP (phosphoenolpyruvate) with sugar substrates, ManNAc-6-P (N-acetylmannosamine 6-phosphate) or Man-6-P (mannose 6-phosphate). Mouse and putative rat Neu5Ac-9-P synthases, however, do not show KDN-9-P synthase activity, despite sharing high sequence identity (>95%) with the human enzyme. Here, we demonstrate that a single mutation, M42T, in human Neu5Ac-9-P synthase can abolish the KDN-9-P synthase activity completely without compromising the Neu5Ac-9-P synthase activity. Saturation mutagenesis of Met42 of the human Neu5Ac-9-P synthase showed that the substitution with all amino acids except leucine retains only the Neu5Ac-9-P synthase activity at levels comparable with the wild-type enzyme. The M42L mutant, like the wild-type enzyme, showed the additional KDN-9-P synthase activity. In the homology model of human Neu5Ac-9-P synthase, Met42 is located 22 Å (1 Å=0.1 nm) away from the substrate-binding site and the impact of this distant residue on the enzyme functions is discussed.

scholarly journals Systemic effects of missense mutations on SARS-CoV-2 spike glycoprotein stability and receptor-binding affinity

2020 ◽  
Author(s):  
Shaolei Teng ◽  
Adebiyi Sobitan ◽  
Raina Rhoades ◽  
Dongxiao Liu ◽  
Qiyi Tang
Keyword(s):  
Receptor Binding ◽  
Saturation Mutagenesis ◽  
Systemic Effects ◽  
Missense Mutations ◽  
Host Cell Membrane ◽  
S Protein ◽  
Angiotensin Converting Enzyme 2 ◽  
Glycosylation Sites ◽  
The Stability ◽  
And Function

Abstract The spike (S) glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the binding to the permissive cells. The receptor-binding domain (RBD) of SARS-CoV-2 S protein directly interacts with the human angiotensin-converting enzyme 2 (ACE2) on the host cell membrane. In this study, we used computational saturation mutagenesis approaches, including structure-based energy calculations and sequence-based pathogenicity predictions, to quantify the systemic effects of missense mutations on SARS-CoV-2 S protein structure and function. A total of 18 354 mutations in S protein were analyzed, and we discovered that most of these mutations could destabilize the entire S protein and its RBD. Specifically, residues G431 and S514 in SARS-CoV-2 RBD are important for S protein stability. We analyzed 384 experimentally verified S missense variations and revealed that the dominant pandemic form, D614G, can stabilize the entire S protein. Moreover, many mutations in N-linked glycosylation sites can increase the stability of the S protein. In addition, we investigated 3705 mutations in SARS-CoV-2 RBD and 11 324 mutations in human ACE2 and found that SARS-CoV-2 neighbor residues G496 and F497 and ACE2 residues D355 and Y41 are critical for the RBD–ACE2 interaction. The findings comprehensively provide potential target sites in the development of drugs and vaccines against COVID-19.

scholarly journals Natural Mutations Affect Structure and Function of gC1q Domain of Otolin-1

2021 ◽  
Vol 22 (16) ◽  
pp. 9085
Author(s):  
Rafał Hołubowicz ◽  
Andrzej Ożyhar ◽  
Piotr Dobryszycki
Keyword(s):  
Wild Type ◽  
Positional Vertigo ◽  
Natural Variants ◽  
Self Association ◽  
Function Relationship ◽  
The Stability ◽  
And Function ◽  
Relationship Of ◽  
Wild Type Form ◽  
Paroxysmal Positional Vertigo

Otolin-1 is a scaffold protein of otoliths and otoconia, calcium carbonate biominerals from the inner ear. It contains a gC1q domain responsible for trimerization and binding of Ca2+. Knowledge of a structure–function relationship of gC1q domain of otolin-1 is crucial for understanding the biology of balance sensing. Here, we show how natural variants alter the structure of gC1q otolin-1 and how Ca2+ are able to revert some effects of the mutations. We discovered that natural substitutions: R339S, R342W and R402P negatively affect the stability of apo-gC1q otolin-1, and that Q426R has a stabilizing effect. In the presence of Ca2+, R342W and Q426R were stabilized at higher Ca2+ concentrations than the wild-type form, and R402P was completely insensitive to Ca2+. The mutations affected the self-association of gC1q otolin-1 by inducing detrimental aggregation (R342W) or disabling the trimerization (R402P) of the protein. Our results indicate that the natural variants of gC1q otolin-1 may have a potential to cause pathological changes in otoconia and otoconial membrane, which could affect sensing of balance and increase the probability of occurrence of benign paroxysmal positional vertigo (BPPV).

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 Kinetic Studies on CphA Mutants Reveal the Role of the P158-P172 Loop in Activity versus Carbapenems

2016 ◽  
Vol 60 (5) ◽  
pp. 3123-3126 ◽  
Author(s):  
Carlo Bottoni ◽  
Mariagrazia Perilli ◽  
Francesca Marcoccia ◽  
Alessandra Piccirilli ◽  
Cristina Pellegrini ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Zinc Ion ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
The Stability

ABSTRACTSite-directed mutagenesis of CphA indicated that prolines in the P158-P172 loop are essential for the stability and the catalytic activity of subclass B2 metallo-β-lactamases against carbapenems. The sequential substitution of proline led to a decrease of the catalytic efficiency of the variant compared to the wild-type (WT) enzyme but also to a higher affinity for the binding of the second zinc ion.

scholarly journals Improving the Stability and Activity of Arginine Decarboxylase at Alkaline pH for the Production of Agmatine

2021 ◽  
Vol 1 ◽  
Author(s):  
Eun Young Hong ◽  
Sun-Gu Lee ◽  
Hyungdon Yun ◽  
Byung-Gee Kim
Keyword(s):  
Disulfide Bond ◽  
Specific Activity ◽  
Catalytic Efficiency ◽  
Arginine Decarboxylase ◽  
Saturation Mutagenesis ◽  
Alkaline Ph ◽  
Wild Type ◽  
Active Site Residues ◽  
Cellular Mechanisms

Agmatine, involved in various modulatory actions in cellular mechanisms, is produced from arginine (Arg) by decarboxylation reaction using arginine decarboxylase (ADC, EC 4.1.1.19). The major obstacle of using wild-type Escherichia coli ADC (ADCes) in agmatine production is its sharp activity loss and instability at alkaline pH. Here, to overcome this problem, a new disulfide bond was rationally introduced in the decameric interface region of the enzyme. Among the mutants generated, W16C/D43C increased both thermostability and activity. The half-life (T1/2) of W16C/D43C at pH 8.0 and 60°C was 560 min, which was 280-fold longer than that of the wild-type, and the specific activity at pH 8.0 also increased 2.1-fold. Site-saturation mutagenesis was subsequently performed at the active site residues of ADCes using the disulfide-bond mutant (W16C/D43C) as a template. The best variant W16C/D43C/I258A displayed a 4.4-fold increase in the catalytic efficiency when compared with the wild-type. The final mutant (W16C/D43C/I258A) was successfully applied to in vitro synthesis of agmatine with an improved yield and productivity (>89.0% yield based on 100 mM of Arg within 5  h).

scholarly journals Overview and Clinical Significance of Multiple Mutations in Individual Genes in Hepatocellular Carcinoma

2021 ◽  
Author(s):  
Taisuke Imamura ◽  
Yukiyasu Okamura ◽  
Keiichi Ohshima ◽  
Katsuhiko Uesaka ◽  
Teiichi Sugiura ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Clinical Significance ◽  
Independent Predictor ◽  
Single Mutation ◽  
Recurrence Free Survival ◽  
Wild Type ◽  
Free Survival ◽  
Significant Difference ◽  
And Function ◽  
Worse Prognosis

Abstract BackgroundMultiple mutations (MMs) within individual oncogenes have been newly characterized as a mechanism for promotion of carcinogenesis. We investigated the spectra of the MMs and the clinical significance in hepatocellular carcinoma (HCC). MethodsWhole-exome sequencing and gene expression profiling were performed in 223 surgically resected HCCs. ResultsMMs within individual genes was identified in 178 samples (79.8%, MMs tumors). All the remaining samples carried single mutation (20.2%, SM tumors). Mutations identified as MMs show different mutational patterns with higher functional impact compared with mutations identified as SM. Recurrence-free survival was significantly worse in the group with MMs tumors than the group with SM tumors (P = 0.012). MMs tumor was identified as an independent predictor for worse prognosis (hazard ratio, 1.72; 95%, P = 0.045). MMs were observed particularly in MUC16 (15% of samples with at least one mutation in the gene) and CTNNB1 (14%). Although there was no significant difference in MUC16 mRNA expression between MUC16 wild-type and MUC16 SM tumors, the expression in MUC16 MMs tumors was significantly enhanced compared with MUC16 SM tumors (P < 0.001). MMs in MUC16 were associated with viral hepatitis, higher tumor markers and vascular invasion. Recurrence-free survival was significantly worse in the MUC16 MMs group than the MUC16 SM group (P = 0.022); no significant difference was observed between the MUC16 SM group and MUC16 wild-type group (P = 0.324).ConclusionsMMs are relatively common driver events that selectively occur in specific oncogenes and function in tumor-promoting activity.

Role of the N-terminus in human 4-hydroxyphenylpyruvate dioxygenase activity

2019 ◽  
Vol 167 (3) ◽  
pp. 315-322
Author(s):  
An-Ning Feng ◽  
Chih-Wei Huang ◽  
Chi-Huei Lin ◽  
Yung-Lung Chang ◽  
Meng-Yuan Ni ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Efficiency ◽  
Dynamics Simulation ◽  
Wild Type ◽  
Catalytic Function ◽  
C Terminus ◽  
N Terminus ◽  
Key Enzyme ◽  
The Stability

Abstract 4-Hydroxyphenylpyruvate dioxygenase (HPPD) is a key enzyme in tyrosine catabolism, catalysing the oxidation of 4-hydroxyphenylpyruvate to homogentisate. Genetic deficiency of this enzyme causes type III tyrosinaemia. The enzyme comprises two barrel-shaped domains formed by the N- and C-termini, with the active site located in the C-terminus. This study investigated the role of the N-terminus, located at the domain interface, in HPPD activity. We observed that the kcat/Km decreased ∼8-fold compared with wild type upon removal of the 12 N-terminal residues (ΔR13). Interestingly, the wild-type level of activity was retained in a mutant missing the 17 N-terminal residues, with a kcat/Km 11-fold higher than that of the ΔR13 mutant; however, the structural stability of this mutant was lower than that of wild type. A 2-fold decrease in catalytic efficiency was observed for the K10A and E12A mutants, indicating synergism between these residues in the enzyme catalytic function. A molecular dynamics simulation showed large RMS fluctuations in ΔR13 suggesting that conformational flexibility at the domain interface leads to lower activity in this mutant. These results demonstrate that the N-terminus maintains the stability of the domain interface to allow for catalysis at the active site of HPPD.

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