scholarly journals Catalytic Efficiency Diversification of Duplicate β-1,3-1,4-Glucanases from Neocallimastix patriciarum J11

2012 ◽  
Vol 78 (12) ◽  
pp. 4294-4300 ◽  
Author(s):  
Yu-Lung Hung ◽  
Hui-Jye Chen ◽  
Jeng-Chen Liu ◽  
Yo-Chia Chen
Keyword(s):  
Substrate Specificity ◽  
Catalytic Efficiency ◽  
Substrate Affinity ◽  
Transfer Event ◽  
Content Type ◽  
Close Phylogenetic Relationship ◽  
Neocallimastix Patriciarum ◽  
Rumen Fungus ◽  
Specific Activities ◽  
Streptococcus Equinus

ABSTRACTFour types of β-1,3-1,4 glucanase (β-glucanase, EC 3.2.1.73) genes, designatedbglA13,bglA16,bglA51, andbglM2, were found in the cDNA library ofNeocallimastix patriciarumJ11. All were highly homologous with each other and demonstrated a close phylogenetic relationship with and a similar codon bias toStreptococcus equinus. The presence of expansion and several predicted secondary structures in the 3′ untranslated regions (3′UTRs) ofbglA16andbglM2suggest that these two genes were duplicated recently, whereasbglA13andbglA16, which contain very short 3′UTRs, were replicated earlier. These findings indicate that the β-glucanase genes fromN. patriciarumJ11 may have arisen by horizontal transfer from the bacterium and subsequent duplication in the rumen fungus. β-Glucanase genes ofStreptococcus equinus,Ruminococcus albus7, andN. patriciarumJ11 were cloned and expressed byEscherichia coli. The recombinant β-glucanases cloned fromS. equinus,R. albus7, andN. patriciarumJ11 were endo-acting and had similar substrate specificity, but they demonstrated different properties in other tests. The specific activities and catalytic efficiency of the bacterial β-glucanases were also significantly lower than those of the fungal β-glucanases. Our results also revealed that the activities and some characteristics of enzymes were changed during the horizontal gene transfer event. The specific activities of the fungal β-glucanases ranged from 26,529 to 41,209 U/mg of protein when barley-derived β-glucan was used as the substrate. They also demonstrated similar pH and temperature optima, substrate specificity, substrate affinity, and hydrolysis patterns. Nevertheless, BglA16 and BglM2, two recently duplicated β-glucanases, showed much higherkcatvalues than others. These results support the notion that duplicated β-glucanase genes, namely,bglA16andbglM2, increase the reaction efficiency of β-glucanases and suggest that the catalytic efficiency of β-glucanase is likely to be a criterion determining the evolutionary fate of duplicate forms inN. patriciarumJ11.

scholarly journals Substrate specificity and structure of human aminoadipate aminotransferase/kynurenine aminotransferase II

2008 ◽  
Vol 28 (4) ◽  
pp. 205-215 ◽  
Author(s):  
Qian Han ◽  
Tao Cai ◽  
Danilo A. Tagle ◽  
Howard Robinson ◽  
Jianyong Li
Keyword(s):  
Substrate Specificity ◽  
Catalytic Efficiency ◽  
Structural Basis ◽  
Substrate Affinity ◽  
Kinetic Properties ◽  
Amino Group ◽  
Kynurenine Aminotransferase ◽  
Broad Substrate Specificity ◽  
Systematic Assessment ◽  
The Brain

KAT (kynurenine aminotransferase) II is a primary enzyme in the brain for catalysing the transamination of kynurenine to KYNA (kynurenic acid). KYNA is the only known endogenous antagonist of the N-methyl-D-aspartate receptor. The enzyme also catalyses the transamination of aminoadipate to α-oxoadipate; therefore it was initially named AADAT (aminoadipate aminotransferase). As an endotoxin, aminoadipate influences various elements of glutamatergic neurotransmission and kills primary astrocytes in the brain. A number of studies dealing with the biochemical and functional characteristics of this enzyme exist in the literature, but a systematic assessment of KAT II addressing its substrate profile and kinetic properties has not been performed. The present study examines the biochemical and structural characterization of a human KAT II/AADAT. Substrate screening of human KAT II revealed that the enzyme has a very broad substrate specificity, is capable of catalysing the transamination of 16 out of 24 tested amino acids and could utilize all 16 tested α-oxo acids as amino-group acceptors. Kinetic analysis of human KAT II demonstrated its catalytic efficiency for individual amino-group donors and acceptors, providing information as to its preferred substrate affinity. Structural analysis of the human KAT II complex with α-oxoglutaric acid revealed a conformational change of an N-terminal fraction, residues 15–33, that is able to adapt to different substrate sizes, which provides a structural basis for its broad substrate specificity.

scholarly journals Cloning, Baeyer-Villiger Biooxidations, and Structures of the Camphor Pathway 2-Oxo-Δ3-4,5,5-Trimethylcyclopentenylacetyl-Coenzyme A Monooxygenase of Pseudomonas putida ATCC 17453

2012 ◽  
Vol 78 (7) ◽  
pp. 2200-2212 ◽  
Author(s):  
Hannes Leisch ◽  
Rong Shi ◽  
Stephan Grosse ◽  
Krista Morley ◽  
Hélène Bergeron ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Pseudomonas Putida ◽  
Coenzyme A ◽  
Free Acid ◽  
Three Dimensional ◽  
Catalytic Efficiency ◽  
Dimensional Structure ◽  
Content Type ◽  
Crystal Forms

ABSTRACTA dimeric Baeyer-Villiger monooxygenase (BVMO) catalyzing the lactonization of 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetyl-coenzyme A (CoA), a key intermediate in the metabolism of camphor byPseudomonas putidaATCC 17453, had been initially characterized in 1983 by Ougham and coworkers (H. J. Ougham, D. G. Taylor, and P. W. Trudgill, J. Bacteriol. 153:140–152, 1983). Here we cloned and overexpressed the 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase (OTEMO) inEscherichia coliand determined its three-dimensional structure with bound flavin adenine dinucleotide (FAD) at a 1.95-Å resolution as well as with bound FAD and NADP+at a 2.0-Å resolution. OTEMO represents the first homodimeric type 1 BVMO structure bound to FAD/NADP+. A comparison of several crystal forms of OTEMO bound to FAD and NADP+revealed a conformational plasticity of several loop regions, some of which have been implicated in contributing to the substrate specificity profile of structurally related BVMOs. Substrate specificity studies confirmed that the 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetic acid coenzyme A ester is preferred over the free acid. However, the catalytic efficiency (kcat/Km) favors 2-n-hexyl cyclopentanone (4.3 × 105M−1s−1) as a substrate, although its affinity (Km= 32 μM) was lower than that of the CoA-activated substrate (Km= 18 μM). In whole-cell biotransformation experiments, OTEMO showed a unique enantiocomplementarity to the action of the prototypical cyclohexanone monooxygenase (CHMO) and appeared to be particularly useful for the oxidation of 4-substituted cyclohexanones. Overall, this work extends our understanding of the molecular structure and mechanistic complexity of the type 1 family of BVMOs and expands the catalytic repertoire of one of its original members.

scholarly journals Loop 3 of Fungal Endoglucanases of Glycoside Hydrolase Family 12 Modulates Catalytic Efficiency

2016 ◽  
Vol 83 (6) ◽  
Author(s):  
Hong Yang ◽  
Pengjun Shi ◽  
Yun Liu ◽  
Wei Xia ◽  
Xiaoyu Wang ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Glycoside Hydrolase ◽  
Turnover Rate ◽  
Catalytic Efficiency ◽  
Glycoside Hydrolase Family ◽  
Multiple Sequence ◽  
Content Type ◽  
Hydrogen Bond Interactions ◽  
Wide Range ◽  
Hydrogen Network

ABSTRACT Glycoside hydrolase (GH) family 12 comprises enzymes with a wide range of activities critical for the degradation of lignocellulose. However, the important roles of the loop regions of GH12 enzymes in substrate specificity and catalytic efficiency remain poorly understood. This study examined how the loop 3 region affects the enzymatic properties of GH12 glucanases using NfEG12A from Neosartorya fischeri P1 and EG (PDB 1KS4 ) from Aspergillus niger. Acidophilic and thermophilic NfEG12A had the highest catalytic efficiency (k cat/Km , 3,001 and 263 ml/mg/s toward lichenin and carboxymethyl cellulose sodium [CMC-Na], respectively) known so far. Based on the multiple-sequence alignment and homology modeling, two specific sequences (FN and STTQA) were identified in the loop 3 region of GH12 endoglucanases from fungi. To determine their functions, these sequences were introduced into NfEG12A, or the counterpart sequence STTQA was removed from EG. These modifications had no effects on the optimal pH and temperature or substrate specificity but changed the catalytic efficiency (k cat/Km ) of these enzymes (in descending order, NfEG12A [100%], NfEG12A-FN [140%], and NfEG12A-STTQA [190%]; EG [100%] and EGΔSTTQA [41%]). Molecular docking and dynamic simulation analyses revealed that the longer loop 3 in GH12 may strengthen the hydrogen-bond interactions between the substrate and protein, thereby increasing the turnover rate (k cat). This study provides a new insight to understand the vital roles of loop 3 for GH12 endoglucanases in catalysis. IMPORTANCE Loop structures play critical roles in the substrate specificity and catalytic hydrolysis of GH12 enzymes. Three typical loops exist in these enzymes. Loops 1 and 2 are recognized as the catalytic loops and are closely related to the substrate specificity and catalytic efficiency. Loop 3 locates in the −1 or +1 subsite and varies a lot in amino acid composition, which may play a role in catalysis. In this study, two GH12 glucanases, NfEG12A and EG, which were mutated by introducing or deleting partial loop 3 sequences FN and/or STTQA, were selected to identify the function of loop 3. It revealed that the longer loop 3 of GH12 glucanases may strengthen the hydrogen network interactions between the substrate and protein, consequently increasing the turnover rate (k cat). This study proposes a strategy to increase the catalytic efficiency of GH12 glucanases by improving the hydrogen network between substrates and catalytic loops.

scholarly journals Heterologous Production and Characterization of Two Glyoxal Oxidases from Pycnoporus cinnabarinus

2016 ◽  
Vol 82 (16) ◽  
pp. 4867-4875 ◽  
Author(s):  
Marianne Daou ◽  
François Piumi ◽  
Daniel Cullen ◽  
Eric Record ◽  
Craig B. Faulds
Keyword(s):  
Substrate Specificity ◽  
Metal Ion ◽  
Inorganic Compounds ◽  
Glyoxylic Acid ◽  
Catalytic Efficiency ◽  
White Rot ◽  
White Rot Fungus ◽  
Pycnoporus Cinnabarinus ◽  
Content Type ◽  
Glyoxal Oxidase

ABSTRACTThe genome of the white rot fungusPycnoporus cinnabarinusincludes a large number of genes encoding enzymes implicated in lignin degradation. Among these, three genes are predicted to encode glyoxal oxidase, an enzyme previously isolated fromPhanerochaete chrysosporium. The glyoxal oxidase ofP. chrysosporiumis physiologically coupled to lignin-oxidizing peroxidases via generation of extracellular H2O2and utilizes an array of aldehydes and α-hydroxycarbonyls as the substrates. Two of the predicted glyoxal oxidases ofP. cinnabarinus, GLOX1 (PciGLOX1) and GLOX2 (PciGLOX2), were heterologously produced inAspergillus nigerstrain D15#26 (pyrGnegative) and purified using immobilized metal ion affinity chromatography, yielding 59 and 5 mg of protein forPciGLOX1 andPciGLOX2, respectively. Both proteins were approximately 60 kDa in size and N-glycosylated. The optimum temperature for the activity of these enzymes was 50°C, and the optimum pH was 6. The enzymes retained most of their activity after incubation at 50°C for 4 h. The highest relative activity and the highest catalytic efficiency of both enzymes occurred with glyoxylic acid as the substrate. The twoP. cinnabarinusenzymes generally exhibited similar substrate preferences, butPciGLOX2 showed a broader substrate specificity and was significantly more active on 3-phenylpropionaldehyde.IMPORTANCEThis study addresses the poorly understood role of how fungal peroxidases obtain anin situsupply of hydrogen peroxide to enable them to oxidize a variety of organic and inorganic compounds. This cooperative activity is intrinsic in the living organism to control the amount of toxic H2O2in its environment, thus providing a feed-on-demand scenario, and can be used biotechnologically to supply a cheap source of peroxide for the peroxidase reaction. The secretion of multiple glyoxal oxidases by filamentous fungi as part of a lignocellulolytic mechanism suggests a controlled system, especially as these enzymes utilize fungal metabolites as the substrates. Two glyoxal oxidases have been isolated and characterized to date, and the differentiation of the substrate specificity of the two enzymes produced byPycnoporus cinnabarinusillustrates the alternative mechanisms existing in a single fungus, together with the utilization of these enzymes to prepare platform chemicals for industry.

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 Improving the Substrate Affinity and Catalytic Efficiency of β-Glucosidase Bgl3A from Talaromyces leycettanus JCM12802 by Rational Design

Biomolecules ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1882
Author(s):  
Wei Xia ◽  
Yingguo Bai ◽  
Pengjun Shi
Keyword(s):  
Hydrogen Bonding ◽  
Rational Design ◽  
Substrate Binding ◽  
Enzymatic Saccharification ◽  
Catalytic Efficiency ◽  
Binding Pocket ◽  
Glycoside Hydrolases ◽  
Cellulosic Biomass ◽  
Substrate Affinity ◽  
Hydrogen Bonding Networks

Improving the substrate affinity and catalytic efficiency of β-glucosidase is necessary for better performance in the enzymatic saccharification of cellulosic biomass because of its ability to prevent cellobiose inhibition on cellulases. Bgl3A from Talaromyces leycettanus JCM12802, identified in our previous work, was considered a suitable candidate enzyme for efficient cellulose saccharification with higher catalytic efficiency on the natural substrate cellobiose compared with other β-glucosidase but showed insufficient substrate affinity. In this work, hydrophobic stacking interaction and hydrogen-bonding networks in the active center of Bgl3A were analyzed and rationally designed to strengthen substrate binding. Three vital residues, Met36, Phe66, and Glu168, which were supposed to influence substrate binding by stabilizing adjacent binding site, were chosen for mutagenesis. The results indicated that strengthening the hydrophobic interaction between stacking aromatic residue and the substrate, and stabilizing the hydrogen-bonding networks in the binding pocket could contribute to the stabilized substrate combination. Four dominant mutants, M36E, M36N, F66Y, and E168Q with significantly lower Km values and 1.4–2.3-fold catalytic efficiencies, were obtained. These findings may provide a valuable reference for the design of other β-glucosidases and even glycoside hydrolases.

scholarly journals Characterization of the Efflux Capability and Substrate Specificity of Aspergillus fumigatus PDR5-like ABC Transporters Expressed in Saccharomyces cerevisiae

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Brooke D. Esquivel ◽  
Jeffrey M. Rybak ◽  
Katherine S. Barker ◽  
Jarrod R. Fortwendel ◽  
P. David Rogers ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Drug Resistance ◽  
Substrate Specificity ◽  
Abc Transporter ◽  
Drug Susceptibility ◽  
Antifungal Drugs ◽  
Efflux Transporters ◽  
Content Type ◽  
Background Strain

ABSTRACT This research analyzed six Aspergillus fumigatus genes encoding putative efflux proteins for their roles as transporters. The A. fumigatus genes abcA, abcC, abcF, abcG, abcH, and abcI were cloned into plasmids and overexpressed in a Saccharomyces cerevisiae strain in which the highly active endogenous ABC transporter gene PDR5 was deleted. The activity of each transporter was measured by efflux of rhodamine 6G and accumulation of alanine β-naphthylamide. The transporters AbcA, AbcC, and AbcF had the strongest efflux activities of these compounds. All of the strains with plasmid-expressed transporters had more efflux activity than did the PDR5-deleted background strain. We performed broth microdilution drug susceptibility testing and agar spot assays using an array of compounds and antifungal drugs to determine the transporter specificity and drug susceptibility of the strains. The transporters AbcC and AbcF showed the broadest range of substrate specificity, while AbcG and AbcH had the narrowest range of substrates. Strains expressing the AbcA, AbcC, AbcF, or AbcI transporter were more resistant to fluconazole than was the PDR5-deleted background strain. Strains expressing AbcC and AbcF were additionally more resistant to clotrimazole, itraconazole, ketoconazole, and posaconazole than was the background strain. Finally, we analyzed the expression levels of the genes by reverse transcription-quantitative PCR (RT-qPCR) in triazole-susceptible and -resistant A. fumigatus clinical isolates. All of these transporters are expressed at a measurable level, and transporter expression varied significantly between strains, demonstrating the high degree of phenotypic variation, plasticity, and divergence of which this species is capable. IMPORTANCE One mechanism behind drug resistance is altered export out of the cell. This work is a multifaceted analysis of membrane efflux transporters in the human fungal pathogen A. fumigatus. Bioinformatics evidence infers that there is a relatively large number of genes in A. fumigatus that encode ABC efflux transporters. However, very few of these transporters have been directly characterized and analyzed for their potential role in drug resistance. Our objective was to determine if these undercharacterized proteins function as efflux transporters and then to better define whether their efflux substrates include antifungal drugs used to treat fungal infections. We chose six A. fumigatus potential plasma membrane ABC transporter genes for analysis and found that all six genes produced functional transporter proteins. We used two fungal systems to look for correlations between transporter function and drug resistance. These transporters have the potential to produce drug-resistant phenotypes in A. fumigatus. Continued characterization of these and other transporters may assist in the development of efflux inhibitor drugs.

scholarly journals Molecular Basis of Substrate Recognition of Endonuclease Q from the Euryarchaeon Pyrococcus furiosus

2019 ◽  
Vol 202 (2) ◽  
Author(s):  
Miyako Shiraishi ◽  
Shigenori Iwai
Keyword(s):  
Dna Repair ◽  
Bacillus Subtilis ◽  
Substrate Specificity ◽  
Molecular Basis ◽  
Pyrococcus Furiosus ◽  
Substrate Recognition ◽  
Base Flipping ◽  
Content Type ◽  
Cleavage Activity

ABSTRACT Endonuclease Q (EndoQ), a DNA repair endonuclease, was originally identified in the hyperthermophilic euryarchaeon Pyrococcus furiosus in 2015. EndoQ initiates DNA repair by generating a nick on DNA strands containing deaminated bases and an abasic site. Although EndoQ is thought to be important for maintaining genome integrity in certain bacteria and archaea, the underlying mechanism catalyzed by EndoQ remains unclear. Here, we provide insights into the molecular basis of substrate recognition by EndoQ from P. furiosus (PfuEndoQ) using biochemical approaches. Our results of the substrate specificity range and the kinetic properties of PfuEndoQ demonstrate that PfuEndoQ prefers the imide structure in nucleobases along with the discovery of its cleavage activity toward 5,6-dihydrouracil, 5-hydroxyuracil, 5-hydroxycytosine, and uridine in DNA. The combined results for EndoQ substrate binding and cleavage activity analyses indicated that PfuEndoQ flips the target base from the DNA duplex, and the cleavage activity is highly dependent on spontaneous base flipping of the target base. Furthermore, we find that PfuEndoQ has a relatively relaxed substrate specificity; therefore, the role of EndoQ in restriction modification systems was explored. The activity of the EndoQ homolog from Bacillus subtilis was found not to be inhibited by the uracil glycosylase inhibitor from B. subtilis bacteriophage PBS1, whose genome is completely replaced by uracil instead of thymine. Our findings suggest that EndoQ not only has additional functions in DNA repair but also could act as an antiviral enzyme in organisms with EndoQ. IMPORTANCE Endonuclease Q (EndoQ) is a lesion-specific DNA repair enzyme present in certain bacteria and archaea. To date, it remains unclear how EndoQ recognizes damaged bases. Understanding the mechanism of substrate recognition by EndoQ is important to grasp genome maintenance systems in organisms with EndoQ. Here, we find that EndoQ from the euryarchaeon Pyrococcus furiosus recognizes the imide structure in nucleobases by base flipping, and the cleavage activity is enhanced by the base pair instability of the target base, along with the discovery of its cleavage activity toward 5,6-dihydrouracil, 5-hydroxyuracil, 5-hydroxycytosine, and uridine in DNA. Furthermore, a potential role of EndoQ in Bacillus subtilis as an antiviral enzyme by digesting viral genome is demonstrated.

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