Purification, bio-chemical characterization, homology modeling and active site binding mode interactions of thermo-alkali-tolerant β-1,4 endoxylanase from Coprinus cinereus LK-D-NCIM-1369

ABSTRACT CTnDOT integrase (IntDOT) is a member of the tyrosine family of site-specific DNA recombinases. IntDOT is unusual in that it catalyzes recombination between nonidentical sequences. Previous mutational analyses centered on mutants with substitutions of conserved residues in the catalytic (CAT) domain or residues predicted by homology modeling to be close to DNA in the core-binding (CB) domain. That work suggested that a conserved active-site residue (Arg I) of the CAT domain is missing and that some residues in the CB domain are involved in catalysis. Here we used a genetic approach and constructed an Escherichia coli indicator strain to screen for random mutations in IntDOT that disrupt integrative recombination in vivo. Twenty-five IntDOT mutants were isolated and characterized for DNA binding, DNA cleavage, and DNA ligation activities. We found that mutants with substitutions in the amino-terminal (N) domain were catalytically active but defective in forming nucleoprotein complexes, suggesting that they have altered protein-protein interactions or altered interactions with DNA. Replacement of Ala-352 of the CAT domain disrupted DNA cleavage but not DNA ligation, suggesting that Ala-352 may be important for positioning the catalytic tyrosine (Tyr-381) during cleavage. Interestingly, our biochemical data and homology modeling of the CAT domain suggest that Arg-285 is the missing Arg I residue of IntDOT. The predicted position of Arg-285 shows it entering the active site from a position on the polypeptide backbone that is not utilized in other tyrosine recombinases. IntDOT may therefore employ a novel active-site architecture to catalyze recombination.

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Recent insights into lytic polysaccharide monooxygenases (LPMOs)

Biochemical Society Transactions ◽

10.1042/bst20170549 ◽

2018 ◽

Vol 46 (6) ◽

pp. 1431-1447 ◽

Cited By ~ 39

Author(s):

Tobias Tandrup ◽

Kristian E. H. Frandsen ◽

Katja S. Johansen ◽

Jean-Guy Berrin ◽

Leila Lo Leggio

Keyword(s):

Active Site ◽

Room Temperature ◽

Experimental Studies ◽

Binding Mode ◽

Structural Features ◽

Oxygen Species ◽

Animal Kingdom ◽

X Ray ◽

Lytic Polysaccharide Monooxygenases ◽

Polysaccharide Monooxygenases

Lytic polysaccharide monooxygenases (LPMOs) are copper enzymes discovered within the last 10 years. By degrading recalcitrant substrates oxidatively, these enzymes are major contributors to the recycling of carbon in nature and are being used in the biorefinery industry. Recently, two new families of LPMOs have been defined and structurally characterized, AA14 and AA15, sharing many of previously found structural features. However, unlike most LPMOs to date, AA14 degrades xylan in the context of complex substrates, while AA15 is particularly interesting because they expand the presence of LPMOs from the predominantly microbial to the animal kingdom. The first two neutron crystallography structures have been determined, which, together with high-resolution room temperature X-ray structures, have putatively identified oxygen species at or near the active site of LPMOs. Many recent computational and experimental studies have also investigated the mechanism of action and substrate-binding mode of LPMOs. Perhaps, the most significant recent advance is the increasing structural and biochemical evidence, suggesting that LPMOs follow different mechanistic pathways with different substrates, co-substrates and reductants, by behaving as monooxygenases or peroxygenases with molecular oxygen or hydrogen peroxide as a co-substrate, respectively.

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New hypotheses for the binding mode of 4- and 7-substituted indazoles in the active site of neuronal nitric oxide synthase

Bioorganic & Medicinal Chemistry ◽

10.1016/j.bmc.2012.06.025 ◽

2012 ◽

Vol 20 (17) ◽

pp. 5296-5304 ◽

Cited By ~ 9

Author(s):

Elodie Lohou ◽

Jana Sopkova-de Oliveira Santos ◽

Pascale Schumann-Bard ◽

Michel Boulouard ◽

Silvia Stiebing ◽

...

Keyword(s):

Nitric Oxide ◽

Nitric Oxide Synthase ◽

Active Site ◽

Neuronal Nitric Oxide Synthase ◽

Binding Mode ◽

Oxide Synthase

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Molecular mechanism of uncompetitive inhibition of human placental and germ-cell alkaline phosphatase

Biochemical Journal ◽

10.1042/bj2860023 ◽

1992 ◽

Vol 286 (1) ◽

pp. 23-30 ◽

Cited By ~ 55

Author(s):

M F Hoylaerts ◽

T Manes ◽

J L Millán

Keyword(s):

Germ Cell ◽

Active Site ◽

Molecular Model ◽

Inhibition Mechanism ◽

Side Group ◽

Alkaline Phosphatases ◽

Uncompetitive Inhibition ◽

Guanidinium Group ◽

Hydrolysis Of ◽

Site Binding

Placental (PLAP) and germ-cell (GCAP) alkaline phosphatases are inhibited uncompetitively by L-Leu and L-Phe. Whereas L-Phe inhibits PLAP and GCAP to the same extent, L-Leu inhibits GCAP 17-fold more strongly than it does PLAP. This difference has been attributed [Hummer & Millán (1991) Biochem. J 274, 91-95] to a Glu----Gly substitution at position 429 in GCAP. The D-Phe and D-Leu enantiomorphs are also inhibitory through an uncompetitive mechanism but with greatly decreased efficiencies. Replacement of the active-site residue Arg-166 by Ala-166 changes the inhibition mechanism of the resulting PLAP mutant to a more complex mixed-type inhibition, with decreased affinities for L-Leu and L-Phe. The uncompetitive mechanism is restored on the simultaneous introduction of Gly-429 in the Ala-166 mutant, but the inhibitions of [Ala166,Gly429]PLAP and even [Lys166,Gly429]PLAP by L-Leu and L-Phe are considerably decreased compared with that of [Gly429]PLAP. These findings point to the importance of Arg-166 during inhibition. Active-site binding of L-Leu requires the presence of covalently bound phosphate in the active-site pocket, and the inhibition of PLAP by L-Leu is pH-sensitive, gradually disappearing when the pH is decreased from 10.5 to 7.5. Our data are compatible with the following molecular model for the uncompetitive inhibition of PLAP and GCAP by L-Phe and L-Leu: after binding of a phosphorylated substrate to the active site, the guanidinium group of Arg-166 (normally involved in positioning phosphate) is redirected to the carboxy group of L-Leu (or L-Phe), thus stabilizing the inhibitor in the active site. Therefore leucinamide and leucinol are weaker inhibitors of [Gly429]PLAP than is L-Leu. During this Arg-166-regulated event, the amino acid side group is positioned in the loop containing Glu-429 or Gly-429, leading to further stabilization. Replacement of Glu-429 by Gly-429 eliminates steric constraints experienced by the bulky L-Leu side group during its positioning and also increases the active-site accessibility for the inhibitor, providing the basis for the 17-fold difference in inhibition efficiency between PLAP and GCAP. Finally, the inhibitor's unprotonated amino group co-ordinates with the active-site Zn2+ ion 1, interfering with the hydrolysis of the phosphoenzyme intermediate, a phenomenon that determines the uncompetitive nature of the inhibition.

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