scholarly journals Fusion of Glutamate Dehydrogenase and Formate Dehydrogenase Yields a Bifunctional Efficient Biocatalyst for the Continuous Removal of Ammonia

2021 ◽  
Vol 1 ◽  
Author(s):  
Valentina Marchini ◽  
Ana I. Benítez-Mateos ◽  
David Roura Padrosa ◽  
Francesca Paradisi
Keyword(s):  
Fusion Protein ◽  
Glutamate Dehydrogenase ◽  
Formate Dehydrogenase ◽  
Reductive Amination ◽  
Candida Boidinii ◽  
Initial Activity ◽  
Wild Type ◽  
Immobilized Biocatalyst ◽  
Clostridium Symbiosum ◽  
The Individual

A novel fusion protein has been rationally designed, combining the hexameric glutamate dehydrogenase from Clostridium symbiosum with the dimeric formate dehydrogenase from Candida boidinii. The former enzyme consumes ammonia for the reductive amination of α-ketoglutarate using NADH, while the latter biocatalyst regenerates continuously the cofactor. This enzymes fusion opens new perspectives for the detection and the removal of ammonia. The bifunctional biocatalyst has been successfully created, expressed, and then characterized. The two fused protein domains retained identical properties and catalytic activity of the individual enzymes. Additionally, the immobilization on a methacrylate resin optimized the assembly providing a reusable and stable biocatalyst. This is an example of immobilization of a fusion protein, so that efficiency and sustainability of the process are enhanced. The immobilized biocatalyst could be recycled 10 times retaining still half of the initial activity. Such preparation outperforms the co-immobilized wild-type enzymes in the conversion of 300 mM of ammonia, which could be carried out also in continuous mode.

Allosteric behaviour of 1:5 hybrids of mutant subunits of Clostridium symbiosum glutamate dehydrogenase differing in their amino acid specificity

2001 ◽  
Vol 360 (3) ◽  
pp. 651-656 ◽  
Author(s):  
Arun GOYAL ◽  
Xing-Guo WANG ◽  
Paul C. ENGEL
Keyword(s):  
Amino Acid ◽  
Glutamate Dehydrogenase ◽  
Cysteine Residue ◽  
The Other ◽  
Wild Type Enzyme ◽  
Triple Mutant ◽  
Subunit Stoichiometry ◽  
Clostridium Symbiosum ◽  
Mutant Forms ◽  
Fold Activation

Hybrid hexamers were made by refolding mixtures of two mutant forms of clostridial glutamate dehydrogenase. Mutant Cys320Ser (C320S) has a similar activity to the wild-type enzyme, but is unreactive with Ellman's reagent, 5,5′-dithiobis(2-nitrobenzoate) (DTNB). The triple mutant Lys89Leu/Ala163Gly/Ser380Ala (K89L/A163G/S380A), active with norleucine but not glutamate, is inactivated by DTNB, since the amino acid residue at position 320 is a cysteine residue. The chosen ratio favoured 1:5 hybrids of the triple mutant and C320S. The renatured mixture was treated with DTNB and separated on an NAD+–agarose column to which only C320S subunits bind tightly. Fractions were monitored for glutamate and norleucine activity and for releasable thionitrobenzoate to establish subunit stoichiometry. A fraction highly enriched in the 1:5 hybrid was identified. Homohexamers (C320S with 40mM glutamate and 1mM NAD+ at pH8.8, or K89L/A163G/S380A with 70mM norleucine and 1mM NAD+ at pH8.5) showed allosteric activation; succinate activated C320S approx. 50-fold (EC50 = 70mM, h = 2.4), and glutarate gave approx. 30-fold activation (EC50 = 35mM, h = 2.3). For the triple mutant, corresponding values were 80mM and 2.2 for succinate, and 75mM and 1.7 for glutarate, but maximal activation was only about 2-fold. In the 1:5 hybrid, with only one norleucine-active subunit per hexamer, responses to glutarate and succinate were still co-operative, and activation was more extensive than in the triple mutant homohexamer. A single norleucine-active subunit can thus respond co-operatively to a substrate analogue at the other five inactive sites. On the other hand, similar hyperbolic dependence on the norleucine concentration for the hybrid and the triple mutant homohexamer reflected the inability of C320S subunits to bind norleucine. With glutamate at pH8.8, an h value of 3.6 was obtained for the 1:5 hybrid, in contrast with an h value of 5.2 for the C320S homohexamer. The ‘foreign’ subunit evidently impedes inter-subunit communication to some extent.

scholarly journals Synthesis of Trypsin-Resistant Variants of the Listeria-Active Bacteriocin Salivaricin P

2010 ◽  
Vol 76 (16) ◽  
pp. 5356-5362 ◽  
Author(s):  
Eileen F. O'Shea ◽  
Paula M. O'Connor ◽  
Paul D. Cotter ◽  
R. Paul Ross ◽  
Colin Hill
Keyword(s):  
Antimicrobial Activity ◽  
Gastrointestinal Tract ◽  
Cleavage Sites ◽  
Wild Type ◽  
Enhanced Resistance ◽  
Fold Reduction ◽  
Two Component ◽  
Specific Protease ◽  
The Individual ◽  
Protease Action

ABSTRACT Two-component salivaricin P-like bacteriocins have demonstrated potential as antimicrobials capable of controlling infections in the gastrointestinal tract (GIT). The anti-Listeria activity of salivaricin P is optimal when the individual peptides Sln1 and Sln2 are added in succession at a 1:1 ratio. However, as degradation by digestive proteases may compromise the functionality of these peptides within the GIT, we investigated the potential to create salivaricin variants with enhanced resistance to the intestinal protease trypsin. A total of 11 variants of the salivaricin P components, in which conservative modifications at the trypsin-specific cleavage sites were explored in order to protect the peptides from trypsin degradation while maintaining their potent antimicrobial activity, were generated. Analysis of these variants revealed that eight were resistant to trypsin digestion while retaining antimicrobial activity. Combining the complementary trypsin-resistant variants Sln1-5 and Sln2-3 resulted in a MIC50 of 300 nM against Listeria monocytogenes, a 3.75-fold reduction in activity compared to the level for wild-type salivaricin P. This study demonstrates the potential of engineering bacteriocin variants which are resistant to specific protease action but which retain significant antimicrobial activity.

Properties of NAD-dependent glutamate dehydrogenase from the tylosin producer Streptomyces fradiae

1997 ◽  
Vol 43 (11) ◽  
pp. 1005-1010 ◽  
Author(s):  
Kien Trung Nguyen ◽  
Lieu Thi Nguyen ◽  
Jan Kopecký ◽  
Vladislav Běhal
Keyword(s):  
Key Words ◽  
Glutamate Dehydrogenase ◽  
Reductive Amination ◽  
Divalent Cations ◽  
Ammonium Assimilation ◽  
Alkaline Ph ◽  
Streptomyces Fradiae ◽  
Oxidative Deamination

Glutamate dehydrogenase is an enzyme responsible for ammonium assimilation and glutamate catabolism in organisms. The tylosin producer Streptomyces fradiae possesses both NADP- and NAD-dependent glutamate dehydrogenases. The latter enzyme was purified 498-fold with a 7.5% recovery by a six-step protocol. The enzyme is composed of two subunits, each of Mr 47 000, and could form active aggregates of four or eight subunits. Its activity was inactivated by alkaline pH or temperatures of −20 °C or above 40 °C. Activities assayed in the direction of oxidative deamination and reductive amination were optimal at pH 9.2 and 8.8, respectively, and at temperatures of 30–35 °C. No activity was found when NAD(H) was replaced with NADP(H). The Km values were 32.2 mM for L-glutamate, 0.3 mM for NAD+, 3.4 mM for 2-ketoglutarate, 14.2 mM for NH4+, and 0.05 mM for NADH. Deamination activity was partially inhibited by adenyl nucleotides and several divalent cations; amination activity was not affected by the nucleotides but significantly inhibited by Cu2+ or Ni2+.Key words: Streptomyces fradiae, NAD-dependent glutamate dehydrogenase, purification, properties.

Structural insights into the efficient CO2-reducing activity of an NAD-dependent formate dehydrogenase fromThiobacillussp. KNK65MA

2015 ◽  
Vol 71 (2) ◽  
pp. 313-323 ◽  
Author(s):  
Hyunjun Choe ◽  
Jung Min Ha ◽  
Jeong Chan Joo ◽  
Hyunook Kim ◽  
Hye-Jin Yoon ◽  
...  
Keyword(s):  
Formate Dehydrogenase ◽  
Computational Simulation ◽  
Candida Boidinii ◽  
Free Energy Barrier ◽  
Asymmetric Unit ◽  
Key Factors ◽  
Biological Unit ◽  
Practical Applications ◽  
Reducing Activity ◽  
Structural Insights

CO2fixation is thought to be one of the key factors in mitigating global warming. Of the various methods for removing CO2, the NAD-dependent formate dehydrogenase fromCandida boidinii(CbFDH) has been widely used in various biological CO2-reduction systems; however, practical applications of CbFDH have often been impeded owing to its low CO2-reducing activity. It has recently been demonstrated that the NAD-dependent formate dehydrogenase fromThiobacillussp. KNK65MA (TsFDH) has a higher CO2-reducing activity compared with CbFDH. The crystal structure of TsFDH revealed that the biological unit in the asymmetric unit has two conformations,i.e.open (NAD+-unbound) and closed (NAD+-bound) forms. Three major differences are observed in the crystal structures of TsFDH and CbFDH. Firstly, hole 2 in TsFDH is blocked by helix α20, whereas it is not blocked in CbFDH. Secondly, the sizes of holes 1 and 2 are larger in TsFDH than in CbFDH. Thirdly, Lys287 in TsFDH, which is crucial for the capture of formate and its subsequent delivery to the active site, is an alanine in CbFDH. A computational simulation suggested that the higher CO2-reducing activity of TsFDH is owing to its lower free-energy barrier to CO2reduction than in CbFDH.

scholarly journals GAGA Factor Isoforms Have Distinct but Overlapping Functions In Vivo

2001 ◽  
Vol 21 (24) ◽  
pp. 8565-8574 ◽  
Author(s):  
Anthony J. Greenberg ◽  
Paul Schedl
Keyword(s):  
Fusion Protein ◽  
Transcriptional Activation ◽  
Functional Difference ◽  
Wild Type ◽  
Gaga Factor ◽  
Phenotypic Analysis ◽  
Alternatively Spliced

ABSTRACT The Drosophila melanogaster GAGA factor (encoded by the Trithorax-like [Trl] gene) is required for correct chromatin architecture at diverse chromosomal sites. The Trl gene encodes two alternatively spliced isoforms of the GAGA factor (GAGA-519 and GAGA-581) that are identical except for the length and sequence of the C-terminal glutamine-rich (Q) domain. In vitro and tissue culture experiments failed to find any functional difference between the two isoforms. We made a set of transgenes that constitutively express cDNAs coding for either of the isoforms with the goal of elucidating their roles in vivo. Phenotypic analysis of the transgenes in Trl mutant background led us to the conclusion that GAGA-519 and GAGA-581 perform different, albeit largely overlapping, functions. We also expressed a fusion protein with LacZ disrupting the Q domain of GAGA-519. This LacZ fusion protein compensated for the loss of wild-type GAGA factor to a surprisingly large extent. This suggests that the Q domain either is not required for the essential functions performed by the GAGA protein or is exclusively used for tetramer formation. These results are inconsistent with a major role of the Q domain in chromatin remodeling or transcriptional activation. We also found that GAGA-LacZ was able to associate with sites not normally occupied by the GAGA factor, pointing to a role of the Q domain in binding site choice in vivo.

scholarly journals Multienzymatic in situ hydrogen peroxide generation cascade for peroxygenase-catalysed oxyfunctionalisation reactions

2019 ◽  
Vol 74 (3-4) ◽  
pp. 101-104 ◽  
Author(s):  
Milja Pesic ◽  
Sébastien Jean-Paul Willot ◽  
Elena Fernández-Fueyo ◽  
Florian Tieves ◽  
Miguel Alcalde ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Formate Dehydrogenase ◽  
Flavin Mononucleotide ◽  
Candida Boidinii ◽  
Reaction Parameters ◽  
Old Yellow Enzyme ◽  
Hydroxylation Reaction ◽  
In Situ Generation ◽  
Hydrogen Peroxide Generation

Abstract There is an increasing interest in the application of peroxygenases in biocatalysis, because of their ability to catalyse the oxyfunctionalisation reaction in a stereoselective fashion and with high catalytic efficiencies, while using hydrogen peroxide or organic peroxides as oxidant. However, enzymes belonging to this class exhibit a very low stability in the presence of peroxides. With the aim of bypassing this fast and irreversible inactivation, we study the use of a gradual supply of hydrogen peroxide to maintain its concentration at stoichiometric levels. In this contribution, we report a multienzymatic cascade for in situ generation of hydrogen peroxide. In the first step, in the presence of NAD+ cofactor, formate dehydrogenase from Candida boidinii (FDH) catalysed the oxidation of formate yielding CO2. Reduced NADH was reoxidised by the reduction of the flavin mononucleotide cofactor bound to an old yellow enzyme homologue from Bacillus subtilis (YqjM), which subsequently reacts with molecular oxygen yielding hydrogen peroxide. Finally, this system was coupled to the hydroxylation of ethylbenzene reaction catalysed by an evolved peroxygenase from Agrocybe aegerita (rAaeUPO). Additionally, we studied the influence of different reaction parameters on the performance of the cascade with the aim of improving the turnover of the hydroxylation reaction.

scholarly journals Differential expression of five tRNA(UAGTrp) amber suppressors in Caenorhabditis elegans.

1988 ◽  
Vol 8 (9) ◽  
pp. 3627-3635 ◽  
Author(s):  
K Kondo ◽  
J Hodgkin ◽  
R H Waterston
Keyword(s):  
Nervous System ◽  
Caenorhabditis Elegans ◽  
Gene Family ◽  
Development Stage ◽  
Base Change ◽  
Wild Type ◽  
Specific Manner ◽  
Flanking Sequences ◽  
Single Base Change ◽  
The Individual

Caenorhabditis elegans has 12 tRNA(UGGTrp) genes as defined by Southern analysis. In order to evaluate the function of the individual members of this multigene family, we sought to recover amber (UAG)-suppressing mutations from reversion experiments with animals carrying amber mutations in a nervous system-affecting gene (unc-13) or a sex-determining gene (tra-3). Revertants were analyzed by Southern blot, exploiting the fact that the CCA to CTA change at the anticodon creates a new XbaI site. Five different members of the tRNATrp gene family were identified as suppressors: sup-7 X, sup-5 III, sup-24 IV, sup-28 X, and sup-29 IV. All five suppressor genes were sequenced and found to encode identical tRNA(UAGTrp) molecules with a single base change (CCA to CTA) at the anticodon compared with their wild-type counterparts. The flanking sequences had only limited homology. The relative expression of these five genes was determined by measuring the efficiencies of suppressers against amber mutations in genes affecting the nervous system, hypodermis, muscle, and sex determination. The results of these cross-suppression tests showed that the five members of the tRNA(Trp) gene family were differentially regulated in a tissue- or development stage-specific manner.

scholarly journals Sequence Analysis and Expression of the Attachment and Fusion Proteins of Canine Distemper Virus Wild-Type Strain A75/17

1999 ◽  
Vol 73 (3) ◽  
pp. 2263-2269 ◽  
Author(s):  
Pascal Cherpillod ◽  
Karin Beck ◽  
Andreas Zurbriggen ◽  
Riccardo Wittek
Keyword(s):  
Amino Acid ◽  
Fusion Protein ◽  
Translation Initiation ◽  
Fusion Proteins ◽  
Canine Distemper Virus ◽  
Protein A ◽  
Biological Properties ◽  
Canine Distemper ◽  
Wild Type ◽  
Attachment Protein

ABSTRACT The biological properties of wild-type A75/17 and cell culture-adapted Onderstepoort canine distemper virus differ markedly. To learn more about the molecular basis for these differences, we have isolated and sequenced the protein-coding regions of the attachment and fusion proteins of wild-type canine distemper virus strain A75/17. In the attachment protein, a total of 57 amino acid differences were observed between the Onderstepoort strain and strain A75/17, and these were distributed evenly over the entire protein. Interestingly, the attachment protein of strain A75/17 contained an extension of three amino acids at the C terminus. Expression studies showed that the attachment protein of strain A75/17 had a higher apparent molecular mass than the attachment protein of the Onderstepoort strain, in both the presence and absence of tunicamycin. In the fusion protein, 60 amino acid differences were observed between the two strains, of which 44 were clustered in the much smaller F2 portion of the molecule. Significantly, the AUG that has been proposed as a translation initiation codon in the Onderstepoort strain is an AUA codon in strain A75/17. Detailed mutation analyses showed that both the first and second AUGs of strain A75/17 are the major translation initiation sites of the fusion protein. Similar analyses demonstrated that, also in the Onderstepoort strain, the first two AUGs are the translation initiation codons which contribute most to the generation of precursor molecules yielding the mature form of the fusion protein.

Sign in / Sign up

Export Citation Format

Share Document