scholarly journals Crystal Structures of Pyrophosphatase from Acinetobacter baumannii: Snapshots of Pyrophosphate Binding and Identification of a Phosphorylated Enzyme Intermediate

2019 ◽  
Vol 20 (18) ◽  
pp. 4394
Author(s):  
Yunlong Si ◽  
Xing Wang ◽  
Guosong Yang ◽  
Tong Yang ◽  
Yuying Li ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Water Molecule ◽  
Acinetobacter Baumannii ◽  
Divalent Cations ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Catalytic Center ◽  
Favorable Reaction ◽  
Enzyme Intermediate

All living things have pyrophosphatases that hydrolyze pyrophosphate and release energy. This energetically favorable reaction drives many energetically unfavorable reactions. An accepted catalytic model of pyrophosphatase shows that a water molecule activated by two divalent cations (M1 and M2) within the catalytic center can attack pyrophosphate in an SN2 mechanism and thus hydrolyze the molecule. However, our co-crystal structure of Acinetobacter baumannii pyrophosphatase with pyrophosphate shows that a water molecule from the solvent may, in fact, be the actual catalytic water. In the co-crystal structure of the wild-type pyrophosphatase with pyrophosphate, the electron density of the catalytic centers of each monomer are different from one another. This indicates that pyrophosphates in the catalytic center are dynamic. Our mass spectroscopy results have identified a highly conserved lysine residue (Lys30) in the catalytic center that is phosphorylated, indicating that the enzyme could form a phosphoryl enzyme intermediate during hydrolysis. Mutation of Lys30 to Arg abolished the activity of the enzyme. In the structure of the apo wild type enzyme, we observed that a Na+ ion is coordinated by residues within a loop proximal to the catalytic center. Therefore, we mutated three key residues within the loop (K143R, P147G, and K149R) and determined Na+ and K+-induced inhibition on their activities. Compared to the wild type enzyme, P147G is most sensitive to these cations, whereas K143R was inactive and K149R showed no change in activity. These data indicate that monovalent cations could play a role in down-regulating pyrophosphatase activity in vivo. Overall, our results reveal new aspects of pyrophosphatase catalysis and could assist in the design of specific inhibitors of Acinetobacter baumannii growth.

scholarly journals Effects of Saline, an Ambient Acidic Environment, and Sodium Salicylate on OXA-Mediated Carbapenem Resistance in Acinetobacter baumannii: TABLE 1

2016 ◽  
Vol 60 (6) ◽  
pp. 3415-3418 ◽  
Author(s):  
Esther Zander ◽  
Harald Seifert ◽  
Paul G. Higgins
Keyword(s):  
Gene Expression ◽  
Acinetobacter Baumannii ◽  
Sodium Salicylate ◽  
Carbapenem Resistance ◽  
Low Ph ◽  
Wild Type ◽  
Experimental Conditions ◽  
Content Type ◽  
Reference Strains

Different physiological conditions, such as NaCl, low pH, and sodium salicylate, have been shown to affect antibiotic resistance determinants inAcinetobacter baumanniiisolates. Therefore, the aim of this study was to investigate the effects of NaCl, sodium salicylate, and low pH on the susceptibility ofA. baumanniito carbapenem. We cloned genes encoding oxacillinases (OXA) of different subclasses, with their associated promoters, from carbapenem-resistantA. baumanniiisolates into the same vector and transferred them to theA. baumanniireference strains ATCC 19606 and ATCC 17978. Carbapenem MICs were determined at least in triplicate by agar dilution under standard conditions, as well as in the presence of 200 mM NaCl or 16 mM sodium salicylate, or at pH 5.8. OXA-58-like gene expression was determined by reverse transcription-quantitative PCR (qRT-PCR). Under some experimental conditions, significant MIC reductions were shown for some transformants but not for others. Only in one instance were all transformants harboring the same OXA affected by the same condition: at pH 5.8, the imipenem and meropenem MICs for strains expressing OXA-58-like enzymes decreased from a resistant level (32 to 64 mg/liter) to an intermediate-susceptible level (8 mg/liter). However,blaOXA-58-likegene expression remained the same. MICs for both wild-type reference strains were not affected by the conditions tested. Our results indicate that the effects of the experimental conditions tested on OXAin vivoare mostly strain dependent. MICs were not reduced to wild-type levels, suggesting that the conditions tested do not lead to complete OXA inhibition in the bacterial cell.

scholarly journals Active-Site Residues of Escherichia coli DNA Gyrase Required in Coupling ATP Hydrolysis to DNA Supercoiling and Amino Acid Substitutions Leading to Novobiocin Resistance

2003 ◽  
Vol 47 (3) ◽  
pp. 1037-1046 ◽  
Author(s):  
Christian H. Gross ◽  
Jonathan D. Parsons ◽  
Trudy H. Grossman ◽  
Paul S. Charifson ◽  
Steven Bellon ◽  
...  
Keyword(s):  
Amino Acid ◽  
Atp Hydrolysis ◽  
Dna Gyrase ◽  
Dna Supercoiling ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Mutant Proteins ◽  
E Coli ◽  
Novobiocin Resistance

ABSTRACT DNA gyrase is a bacterial type II topoisomerase which couples the free energy of ATP hydrolysis to the introduction of negative supercoils into DNA. Amino acids in proximity to bound nonhydrolyzable ATP analog (AMP · PNP) or novobiocin in the gyrase B (GyrB) subunit crystal structures were examined for their roles in enzyme function and novobiocin resistance by site-directed mutagenesis. Purified Escherichia coli GyrB mutant proteins were complexed with the gyrase A subunit to form the functional A2B2 gyrase enzyme. Mutant proteins with alanine substitutions at residues E42, N46, E50, D73, R76, G77, and I78 had reduced or no detectable ATPase activity, indicating a role for these residues in ATP hydrolysis. Interestingly, GyrB proteins with P79A and K103A substitutions retained significant levels of ATPase activity yet demonstrated no DNA supercoiling activity, even with 40-fold more enzyme than the wild-type enzyme, suggesting that these amino acid side chains have a role in the coupling of the two activities. All enzymes relaxed supercoiled DNA to the same extent as the wild-type enzyme did, implying that only ATP-dependent reactions were affected. Mutant genes were examined in vivo for their abilities to complement a temperature-sensitive E. coli gyrB mutant, and the activities correlated well with the in vitro activities. We show that the known R136 novobiocin resistance mutations bestow a significant loss of inhibitor potency in the ATPase assay. Four new residues (D73, G77, I78, and T165) that, when changed to the appropriate amino acid, result in both significant levels of novobiocin resistance and maintain in vivo function were identified in E. coli.

scholarly journals Mutagenesis of solvent-exposed amino acids in Photinus pyralis luciferase improves thermostability and pH-tolerance

2006 ◽  
Vol 397 (2) ◽  
pp. 305-312 ◽  
Author(s):  
G. H. Erica Law ◽  
Olga A. Gandelman ◽  
Laurence C. Tisi ◽  
Christopher R. Lowe ◽  
James A. H. Murray
Keyword(s):  
Amino Acids ◽  
Specific Activity ◽  
Green Light ◽  
Firefly Luciferase ◽  
Wild Type ◽  
Photinus Pyralis ◽  
Wild Type Enzyme ◽  
Ph Tolerance

Firefly luciferase catalyses a two-step reaction, using ATP-Mg2+, firefly luciferin and molecular oxygen as substrates, leading to the efficient emission of yellow–green light. We report the identification of novel luciferase mutants which combine improved pH-tolerance and thermostability and that retain the specific activity of the wild-type enzyme. These were identified by the mutagenesis of solvent-exposed non-conserved hydrophobic amino acids to hydrophilic residues in Photinus pyralis firefly luciferase followed by in vivo activity screening. Mutants F14R, L35Q, V182K, I232K and F465R were found to be the preferred substitutions at the respective positions. The effects of these amino acid replacements are additive, since combination of the five substitutions produced an enzyme with greatly improved pH-tolerance and stability up to 45 °C. All mutants, including the mutant with all five substitutions, showed neither a decrease in specific activity relative to the recombinant wild-type enzyme, nor any substantial differences in kinetic constants. It is envisaged that the combined mutant will be superior to wild-type luciferase for many in vitro and in vivo applications.

Structural and functional consequences of the replacement of proximal residues Cys172 and Cys192 in the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from Chlamydomonas reinhardtii

2008 ◽  
Vol 411 (2) ◽  
pp. 241-247 ◽  
Author(s):  
María-Jesús García-Murria ◽  
Saeid Karkehabadi ◽  
Julia Marín-Navarro ◽  
Sriram Satagopan ◽  
Inger Andersson ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Higher Plants ◽  
Large Subunit ◽  
Dimensional Structure ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Bisphosphate Carboxylase ◽  
Specificity Factor

Proximal Cys172 and Cys192 in the large subunit of the photosynthetic enzyme Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase; EC 4.1.1.39) are evolutionarily conserved among cyanobacteria, algae and higher plants. Mutation of Cys172 has been shown to affect the redox properties of Rubisco in vitro and to delay the degradation of the enzyme in vivo under stress conditions. Here, we report the effect of the replacement of Cys172 and Cys192 by serine on the catalytic properties, thermostability and three-dimensional structure of Chlamydomonas reinhardtii Rubisco. The most striking effect of the C172S substitution was an 11% increase in the specificity factor when compared with the wild-type enzyme. The specificity factor of C192S Rubisco was not altered. The Vc (Vmax for carboxylation) was similar to that of wild-type Rubisco in the case of the C172S enzyme, but approx. 30% lower for the C192S Rubisco. In contrast, the Km for CO2 and O2 was similar for C192S and wild-type enzymes, but distinctly higher (approximately double) for the C172S enzyme. C172S Rubisco showed a critical denaturation temperature approx. 2 °C lower than wild-type Rubisco and a distinctly higher denaturation rate at 55 °C, whereas C192S Rubisco was only slightly more sensitive to temperature denaturation than the wild-type enzyme. X-ray crystal structures reveal that the C172S mutation causes a shift of the main-chain backbone atoms of β-strand 1 of the α/β-barrel affecting a number of amino acid side chains. This may cause the exceptional catalytic features of C172S. In contrast, the C192S mutation does not produce similar structural perturbations.

scholarly journals Cloning of hexokinase structural genes from Saccharomyces cerevisiae mutants with regulatory mutations responsible for glucose repression.

1985 ◽  
Vol 5 (11) ◽  
pp. 3035-3040 ◽  
Author(s):  
K D Entian ◽  
F Hilberg ◽  
H Opitz ◽  
D Mecke
Keyword(s):  
Structural Gene ◽  
Glucose Repression ◽  
Regulatory System ◽  
Structural Genes ◽  
Wild Type ◽  
Tetrad Analysis ◽  
Catalytic Center ◽  
Regulatory Mutations ◽  
Pii Proteins

The regulatory hexokinase PII mutants isolated previously (K.-D. Entian and K.-U. Fröhlich, J. Bacteriol. 158:29-35, 1984) were characterized further. These mutants were defective in glucose repression. The mutation was thought to be in the hexokinase PII structural gene, but it did not affect the catalytic activity of the enzyme. Hence, a regulatory domain for glucose repression was postulated. For further understanding of this regulatory system, the mutationally altered hexokinase PII proteins were isolated from five mutants obtained independently and characterized by their catalytic constants and bisubstrate kinetics. None of these characteristics differed from those of the wild type, so the catalytic center of the mutant enzymes remained unchanged. The only noticeable difference observed was that the in vivo modified form of hexokinase PII, PIIM, which has been described recently (K.-D. Entian and E. Kopetzki, Eur. J. Biochem. 146:657-662, 1985), was absent from one of these mutants. It is possible that the PIIM modification is directly connected with the triggering of glucose repression. To establish with certainty that the mutation is located in the hexokinase PII structural gene, the genes of these mutants were isolated after transforming a hexokinaseless mutant strain and selecting for concomitant complementation of the nuclear function. Unlike hexokinase PII wild-type transformants, glucose repression was not restored in the hexokinase PII mutant transformants. In addition mating experiments with these transformants followed by tetrad analysis of sporulated diploids gave clear evidence of allelism to the hexokinase PII structural gene.

scholarly journals Altering Toluene 4-Monooxygenase by Active-Site Engineering for the Synthesis of 3-Methoxycatechol, Methoxyhydroquinone, and Methylhydroquinone

2004 ◽  
Vol 186 (14) ◽  
pp. 4705-4713 ◽  
Author(s):  
Ying Tao ◽  
Ayelet Fishman ◽  
William E. Bentley ◽  
Thomas K. Wood
Keyword(s):  
Burkholderia Cepacia ◽  
Saturation Mutagenesis ◽  
Hydroxyl Groups ◽  
Pseudomonas Mendocina ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Protein Variant ◽  
Catalytic Center ◽  
Hydrogen Bonding Interactions ◽  
Formed Structure

ABSTRACT Wild-type toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 oxidizes toluene to p-cresol (96%) and oxidizes benzene sequentially to phenol, to catechol, and to 1,2,3-trihydroxybenzene. In this study T4MO was found to oxidize o-cresol to 3-methylcatechol (91%) and methylhydroquinone (9%), to oxidize m-cresol and p-cresol to 4-methylcatechol (100%), and to oxidize o-methoxyphenol to 4-methoxyresorcinol (87%), 3-methoxycatechol (11%), and methoxyhydroquinone (2%). Apparent V max values of 6.6 ± 0.9 to 10.7 ± 0.1 nmol/min/ mg of protein were obtained for o-, m-, and p-cresol oxidation by wild-type T4MO, which are comparable to the toluene oxidation rate (15.1 ± 0.8 nmol/min/mg of protein). After these new reactions were discovered, saturation mutagenesis was performed near the diiron catalytic center at positions I100, G103, and A107 of the alpha subunit of the hydroxylase (TmoA) based on directed evolution of the related toluene o-monooxygenase of Burkholderia cepacia G4 (K. A. Canada, S. Iwashita, H. Shim, and T. K. Wood, J. Bacteriol. 184 :344-349, 2002) and a previously reported T4MO G103L regiospecific mutant (K. H. Mitchell, J. M. Studts, and B. G. Fox, Biochemistry 41 :3176-3188, 2002). By using o-cresol and o-methoxyphenol as model substrates, regiospecific mutants of T4MO were created; for example, TmoA variant G103A/A107S produced 3-methylcatechol (98%) from o-cresol twofold faster and produced 3-methoxycatechol (82%) from 1 mM o-methoxyphenol seven times faster than the wild-type T4MO (1.5 ± 0.2 versus 0.21 ± 0.01 nmol/min/mg of protein). Variant I100L produced 3-methoxycatechol from o-methoxyphenol four times faster than wild-type T4MO, and G103S/A107T produced methylhydroquinone (92%) from o-cresol fourfold faster than wild-type T4MO and there was 10 times more in terms of the percentage of the product. Variant G103S produced 40-fold more methoxyhydroquinone from o-methoxyphenol than the wild-type enzyme produced (80 versus 2%) and produced methylhydroquinone (80%) from o-cresol. Hence, the regiospecific oxidation of o-methoxyphenol and o-cresol was changed for significant synthesis of 3-methoxycatechol, methoxyhydroquinone, 3-methylcatechol, and methylhydroquinone. The enzyme variants also demonstrated altered monohydroxylation regiospecificity for toluene; for example, G103S/A107G formed 82% o-cresol, so saturation mutagenesis converted T4MO into an ortho-hydroxylating enzyme. Furthermore, G103S/A107T formed 100% p-cresol from toluene; hence, a better para-hydroxylating enzyme than wild-type T4MO was formed. Structure homology modeling suggested that hydrogen bonding interactions of the hydroxyl groups of altered residues S103, S107, and T107 influence the regiospecificity of the oxygenase reaction.

scholarly journals Scalable continuous evolution of genes at mutation rates above genomic error thresholds

2018 ◽  
Author(s):  
Arjun Ravikumar ◽  
Garri A. Arzumanyan ◽  
Muaeen K.A. Obadi ◽  
Alex A. Javanpour ◽  
Chang C. Liu
Keyword(s):  
Directed Evolution ◽  
Fitness Landscape ◽  
Drug Resistant ◽  
Wild Type ◽  
New Paradigm ◽  
Wild Type Enzyme ◽  
Epistatic Interactions ◽  
Powerful Approach ◽  
Experimental Strategies

Directed evolution is a powerful approach for engineering biomolecules and understanding adaptation1-3. However, experimental strategies for directed evolution are notoriously low-throughput, limiting access to demanding functions, multiple functions in parallel, and the study of molecular evolution in replicate. Here, we report OrthoRep, a yeast orthogonal DNA polymerase-plasmid pair that stably mutates ~100,000-fold faster than the host genome in vivo, exceeding error thresholds of genomic replication that lead to single-generation extinction4. User-defined genes in OrthoRep continuously and rapidly evolve through serial passaging, a highly scalable process. Using OrthoRep, we evolved drug resistant malarial DHFRs 90 times and uncovered a more complex fitness landscape than previously realized5-9. We find rare fitness peaks that resist the maximum soluble concentration of the antimalarial pyrimethamine – these resistant variants support growth at pyrimethamine concentrations >40,000-fold higher than the wild-type enzyme can tolerate – and also find that epistatic interactions direct adaptive trajectories to convergent outcomes. OrthoRep enables a new paradigm of routine, high-throughput evolution of biomolecular and cellular function.

A glycosynthase derived from an inverting GH19 chitinase from the moss Bryum coronatum

2012 ◽  
Vol 444 (3) ◽  
pp. 437-443 ◽  
Author(s):  
Takayuki Ohnuma ◽  
Tatsuya Fukuda ◽  
Satoshi Dozen ◽  
Yuji Honda ◽  
Motomitsu Kitaoka ◽  
...  
Keyword(s):  
Water Molecule ◽  
Hydrogen Fluoride ◽  
Amino Sugar ◽  
Hydrolytic Activity ◽  
Major Product ◽  
Polymerization Degree ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Glycosidic Bonds ◽  
Inverting Mechanism

BcChi-A, a GH19 chitinase from the moss Bryum coronatum, is an endo-acting enzyme that hydrolyses the glycosidic bonds of chitin, (GlcNAc)n [a β-1,4-linked polysaccharide of GlcNAc (N-acetylglucosamine) with a polymerization degree of n], through an inverting mechanism. When the wild-type enzyme was incubated with α-(GlcNAc)2-F [α-(GlcNAc)2 fluoride] in the absence or presence of (GlcNAc)2, (GlcNAc)2 and hydrogen fluoride were found to be produced through the Hehre resynthesis–hydrolysis mechanism. To convert BcChi-A into a glycosynthase, we employed the strategy reported by Honda et al. [(2006) J. Biol. Chem. 281, 1426–1431; (2008) Glycobiology 18, 325–330] of mutating Ser102, which holds a nucleophilic water molecule, and Glu70, which acts as a catalytic base, producing S102A, S102C, S102D, S102G, S102H, S102T, E70G and E70Q. In all of the mutated enzymes, except S102T, hydrolytic activity towards (GlcNAc)6 was not detected under the conditions we used. Among the inactive BcChi-A mutants, S102A, S102C, S102G and E70G were found to successfully synthesize (GlcNAc)4 as a major product from α-(GlcNAc)2-F in the presence of (GlcNAc)2. The S102A mutant showed the greatest glycosynthase activity owing to its enhanced F− releasing activity and its suppressed hydrolytic activity. This is the first report on a glycosynthase that employs amino sugar fluoride as a donor substrate.

scholarly journals Characterization of a New DyP-Peroxidase from the Alkaliphilic Cellulomonad, Cellulomonas bogoriensis

Molecules ◽  
2019 ◽  
Vol 24 (7) ◽  
pp. 1208 ◽  
Author(s):  
Mohamed H. Habib ◽  
Henriëtte J. Rozeboom ◽  
Marco W. Fraaije
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Active Site ◽  
Biochemical Characterization ◽  
Sequence Motif ◽  
Acidic Ph ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Ph Values

DyP-type peroxidases are heme-containing enzymes that have received increasing attention over recent years with regards to their potential as biocatalysts. A novel DyP-type peroxidase (CboDyP) was discovered from the alkaliphilic cellulomonad, Cellulomonas bogoriensis, which could be overexpressed in Escherichia coli. The biochemical characterization of the recombinant enzyme showed that it is a heme-containing enzyme capable to act as a peroxidase on several dyes. With the tested substrates, the enzyme is most active at acidic pH values and is quite tolerant towards solvents. The crystal structure of CboDyP was solved which revealed atomic details of the dimeric heme-containing enzyme. A peculiar feature of CboDyP is the presence of a glutamate in the active site which in most other DyPs is an aspartate, being part of the DyP-typifying sequence motif GXXDG. The E201D CboDyP mutant was prepared and analyzed which revealed that the mutant enzyme shows a significantly higher activity on several dyes when compared with the wild-type enzyme.

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