scholarly journals Determination of the Catalytic Base in Family 48 Glycosyl Hydrolases

2011 ◽  
Vol 77 (17) ◽  
pp. 6274-6276 ◽  
Author(s):  
Maxim Kostylev ◽  
David B. Wilson
Keyword(s):  
Aspartic Acid ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Thermobifida Fusca ◽  
Glycosyl Hydrolases ◽  
Content Type ◽  
Modeling Data ◽  
Partial Activity

ABSTRACTThe catalytic base in family 48 glycosyl hydrolases has not been previously established experimentally. Based on structural and modeling data published to date, we used site-directed mutagenesis and azide rescue activity assays to show definitively that the catalytic base inThermobifida fuscaCel48A is aspartic acid 225. Of the tested mutants, only Cel48A with the D225E mutation retained partial activity on soluble and insoluble substrates. In azide rescue experiments, only the D225G mutation, in the smallest residue tested, showed an increase in activity with added azide.

scholarly journals Hydrolysis of Clavulanate by Mycobacterium tuberculosis β-Lactamase BlaC Harboring a Canonical SDN Motif

2015 ◽  
Vol 59 (9) ◽  
pp. 5714-5720 ◽  
Author(s):  
Daria Soroka ◽  
Inès Li de la Sierra-Gallay ◽  
Vincent Dubée ◽  
Sébastien Triboulet ◽  
Herman van Tilbeurgh ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Fold Increase ◽  
Mycobacterium Abscessus ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Content Type ◽  
Emergence Of Resistance ◽  
Hydrolysis Of ◽  
Sequential Formation

ABSTRACTCombinations of β-lactams with clavulanate are currently being investigated for tuberculosis treatment. SinceMycobacterium tuberculosisproduces a broad spectrum β-lactamase, BlaC, the success of this approach could be compromised by the emergence of clavulanate-resistant variants, as observed for inhibitor-resistant TEM variants in enterobacteria. Previous analyses based on site-directed mutagenesis of BlaC have led to the conclusion that this risk was limited. Here, we used a different approach based on determination of the crystal structure of β-lactamase BlaMAbofMycobacterium abscessus, which efficiently hydrolyzes clavulanate. Comparison of BlaMAband BlaC allowed for structure-assisted site-directed mutagenesis of BlaC and identification of the G132N substitution that was sufficient to switch the interaction of BlaC with clavulanate from irreversible inactivation to efficient hydrolysis. The substitution, which restored the canonical SDN motif (SDG→SDN), allowed for efficient hydrolysis of clavulanate, with a more than 104-fold increase inkcat(0.41 s−1), without affecting the hydrolysis of other β-lactams. Mass spectrometry revealed that acylation of BlaC and of its G132N variant by clavulanate follows similar paths, involving sequential formation of two acylenzymes. Decarboxylation of the first acylenzyme results in a stable secondary acylenzyme in BlaC, whereas hydrolysis occurs in the G132N variant. The SDN/SDG polymorphism defines two mycobacterial lineages comprising rapidly and slowly growing species, respectively. Together, these results suggest that the efficacy of β-lactam–clavulanate combinations may be limited by the emergence of resistance. β-Lactams active without clavulanate, such as faropenem, should be prioritized for the development of new therapies.

scholarly journals Site-Directed Mutagenesis of the Heterotrimeric Killer Toxin Zymocin Identifies Residues Required for Early Steps in Toxin Action

2014 ◽  
Vol 80 (20) ◽  
pp. 6549-6559 ◽  
Author(s):  
Sabrina Wemhoff ◽  
Roland Klassen ◽  
Friedhelm Meinhardt
Keyword(s):  
Kluyveromyces Lactis ◽  
Killer Toxin ◽  
Cysteine Residue ◽  
Site Directed Mutagenesis ◽  
Target Cells ◽  
Directed Mutagenesis ◽  
Content Type ◽  
Protein Toxin ◽  
Assisted Delivery

ABSTRACTZymocin is aKluyveromyces lactisprotein toxin composed of αβγ subunits encoded by the cytoplasmic virus-like element k1 and functions by αβ-assisted delivery of the anticodon nuclease (ACNase) γ into target cells. The toxin binds to cells' chitin and exhibits chitinase activityin vitrothat might be important during γ import.Saccharomyces cerevisiaestrains carrying k1-derived hybrid elements deficient in either αβ (k1ORF2) or γ (k1ORF4) were generated. Loss of either gene abrogates toxicity, and unexpectedly, Orf2 secretion depends on Orf4 cosecretion. Functional zymocin assembly can be restored by nuclear expression of k1ORF2 or k1ORF4, providing an opportunity to conduct site-directed mutagenesis of holozymocin. Complementation required active site residues of α's chitinase domain and the sole cysteine residue of β (Cys250). Since βγ are reportedly disulfide linked, the requirement for the conserved γ C231 was probed. Toxicity of intracellularly expressed γ C231A indicated no major defect in ACNase activity, while complementation of k1ΔORF4 by γ C231A was lost, consistent with a role of β C250 and γ C231 in zymocin assembly. To test the capability of αβ to carry alternative cargos, the heterologous ACNase fromPichia acaciae(P. acaciaeOrf2 [PaOrf2]) was expressed, along with its immunity gene, in k1ΔORF4. While efficient secretion of PaOrf2 was detected, suppression of the k1ΔORF4-derived k1Orf2 secretion defect was not observed. Thus, the dependency of k1Orf2 on k1Orf4 cosecretion needs to be overcome prior to studying αβ's capability to deliver other cargo proteins into target cells.

scholarly journals Versatile Fungal Polyphenol Oxidase with Chlorophenol Bioremediation Potential: Characterization and Protein Engineering

2018 ◽  
Vol 84 (23) ◽  
Author(s):  
Efstratios Nikolaivits ◽  
Maria Dimarogona ◽  
Ioanna Karagiannaki ◽  
Angelina Chalima ◽  
Ayelet Fishman ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Fruits And Vegetables ◽  
Homology Model ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Gene Encoding ◽  
Content Type ◽  
Polyphenol Oxidases ◽  
Increased Activity ◽  
Work Knowledge

ABSTRACTPolyphenol oxidases (PPOs) have been mostly associated with the undesirable postharvest browning in fruits and vegetables and have implications in human melanogenesis. Nonetheless, they are considered useful biocatalysts in the food, pharmaceutical, and cosmetic industries. The aim of the present work was to characterize a novel PPO and explore its potential as a bioremediation agent. A gene encoding an extracellular tyrosinase-like enzyme was amplified from the genome ofThermothelomyces thermophilaand expressed inPichia pastoris. The recombinant enzyme (TtPPO) was purified and biochemically characterized. Its production reached 40 mg/liter, and it appeared to be a glycosylated and N-terminally processed protein.TtPPO showed broad substrate specificity, as it could oxidize 28/30 compounds tested, including polyphenols, substituted phenols, catechols, and methoxyphenols. Its optimum temperature was 65°C, with a half-life of 18.3 h at 50°C, while its optimum pH was 7.5. The homology model ofTtPPO was constructed, and site-directed mutagenesis was performed in order to increase its activity on mono- and dichlorophenols (di-CPs). The G292N/Y296V variant ofTtPPO 5.3-fold increased activity on 3,5-dichlorophenol (3,5-diCP) compared to the wild type.IMPORTANCEA novel fungal PPO was heterologously expressed and biochemically characterized. Construction of single and double mutants led to the generation of variants with altered specificity against CPs. Through this work, knowledge is gained regarding the effect of mutations on the substrate specificity of PPOs. This work also demonstrates that more potent biocatalysts for the bioremediation of harmful CPs can be developed by applying site-directed mutagenesis.

scholarly journals TheTreponema denticolaPAS Domain-Containing Histidine Kinase Hpk2 Is a Heme Binding Sensor of Oxygen Levels

2018 ◽  
Vol 200 (18) ◽  
Author(s):  
Juni Sarkar ◽  
Daniel P. Miller ◽  
Lee D. Oliver ◽  
Richard T. Marconi
Keyword(s):  
Microbial Community ◽  
Periodontal Disease ◽  
Histidine Kinase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Treponema Denticola ◽  
Pas Domain ◽  
Adaptive Responses ◽  
Content Type

ABSTRACTPeriodontal disease (PD) results from a shift in the composition of the microbial community of the subgingival crevice. As the bacterial population transitions from Gram-positive bacteria to predominantly Gram-negative anaerobes and spirochetes, dramatic changes occur in the physiological and immunological environment at diseased sites.Treponema denticolathrives in periodontal pockets, indicating that it has a unique ability to adapt to changing environmental conditions. Hpk2 (tde1970), a Per-Arnt-Sim motif (PAS) domain-containing histidine kinase (HK), is part of theT. denticolaHpk2-Rrp2 (tde1969) two-component regulatory (TCR) system. This TCR system is growth phase regulated and has been postulated to play a key role in adaptive responses. In this study, we employ predictive structural analyses and site-directed mutagenesis to investigate the functional role of specific amino acid residues located within the Hpk2 PAS domain. Specific substitutions impacted autophosphorylation (AP), phosphotransfer (PT), oligomerization, and hemin binding. The AP, PT, hemin binding, and oligomerization potential of some mutated Hpk2 proteins differed under aerobic versus anaerobic reaction conditions. The data presented here suggest that the regulatory activity of Hpk2 is linked to diatomic gas levels. In a broader sense, this study highlights the importance of studying proteins produced by anaerobes under conditions that approximate the environment in which they thrive.IMPORTANCEPeriodontal disease affects nearly 60% of the global adult population. Its costs to individuals, and to society as a whole, are enormous. As periodontal disease develops, there is a shift in the composition of the oral microbial community. The bacteria that become dominant are able to cause significant damage to the tissues that support the teeth, leading to tooth loss.Treponema denticolais one of the keystone pathogens associated with periodontal disease. An earlier study demonstrated that the Hpk2 and Rrp2 proteins play an important role in adaptive responses. Here, we explore the role of specific Hpk2 amino acids in environmental sensing and function, using structural analyses and site-directed mutagenesis.

scholarly journals Site-directed mutagenesis of dicarboxylic acids near the active site of Bacillus cereus 5/B/6 β-lactamase II

1991 ◽  
Vol 276 (2) ◽  
pp. 401-404 ◽  
Author(s):  
H M Lim ◽  
R K Iyer ◽  
J J Pène
Keyword(s):  
Bacillus Cereus ◽  
Aspartic Acid ◽  
Carboxy Group ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Aspartic Acid Residue ◽  
Beta Lactamase Ii

An amino acid residue functioning as a general base has been proposed to assist in the hydrolysis of beta-lactam antibiotics by the zinc-containing Bacillus cereus beta-lactamase II [Bicknell & Waley (1985) Biochemistry 24, 6876-6887]. Oligonucleotide-directed mutagenesis of cloned Bacillus cereus 5/B/6 beta-lactamase II was used in an ‘in vivo’ study to investigate the role of carboxy-group-containing amino acids near the active site of the enzyme. Substitution of asparagine for the wild-type aspartic acid residue at position 81 resulted in fully functional enzyme. An aspartic acid residue at position 90 is essential for beta-lactamase II to confer any detectable ampicillin and cephalosporin C resistance to Escherichia coli. Conversion of Asp90 into Asn90 or Glu90 lead to the synthesis of inactive enzyme, suggesting that the spatial position of the beta-carboxy group of Asp90 is critical for enzyme function.

scholarly journals Production of l-Ribose from l-Ribulose by a Triple-Site Variant of Mannose-6-Phosphate Isomerase from Geobacillus thermodenitrificans

2012 ◽  
Vol 78 (11) ◽  
pp. 3880-3884 ◽  
Author(s):  
Yu-Ri Lim ◽  
Soo-Jin Yeom ◽  
Deok-Kun Oh
Keyword(s):  
Specific Activity ◽  
Thermus Thermophilus ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Geobacillus Thermodenitrificans ◽  
Wild Type Enzyme ◽  
Content Type ◽  
Mannose 6 Phosphate

ABSTRACTA triple-site variant (W17Q N90A L129F) of mannose-6-phosphate isomerase fromGeobacillus thermodenitrificanswas obtained by combining variants with residue substitutions at different positions after random and site-directed mutagenesis. The specific activity and catalytic efficiency (kcat/Km) forl-ribulose isomerization of this variant were 3.1- and 7.1-fold higher, respectively, than those of the wild-type enzyme at pH 7.0 and 70°C in the presence of 1 mM Co2+. The triple-site variant produced 213 g/literl-ribose from 300 g/literl-ribulose for 60 min, with a volumetric productivity of 213 g liter−1h−1, which was 4.5-fold higher than that of the wild-type enzyme. Thekcat/Kmand productivity of the triple-site variant were approximately 2-fold higher than those of theThermus thermophilusR142N variant of mannose-6-phosphate isomerase, which exhibited the highest values previously reported.

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