scholarly journals Characterization of Glycerol Trinitrate Reductase (NerA) and the Catalytic Role of Active-Site Residues

2004 ◽  
Vol 186 (6) ◽  
pp. 1802-1810 ◽  
Author(s):  
Samantha J. Marshall ◽  
Doreen Krause ◽  
Dayle K. Blencowe ◽  
Graham F. White
Keyword(s):  
Active Site ◽  
Structural Model ◽  
Flavin Mononucleotide ◽  
Active Site Residues ◽  
Old Yellow Enzyme ◽  
Ping Pong ◽  
Glycerol Trinitrate ◽  
Catalytic Role

ABSTRACT Glycerol trinitrate reductase (NerA) from Agrobacterium radiobacter, a member of the old yellow enzyme (OYE) family of oxidoreductases, was expressed in and purified from Escherichia coli. Denaturation of pure enzyme liberated flavin mononucleotide (FMN), and spectra of NerA during reduction and reoxidation confirmed its catalytic involvement. Binding of FMN to apoenzyme to form the holoenzyme occurred with a dissociation constant of ca. 10−7 M and with restoration of activity. The NerA-dependent reduction of glycerol trinitrate (GTN; nitroglycerin) by NADH followed ping-pong kinetics. A structural model of NerA based on the known coordinates of OYE showed that His-178, Asn-181, and Tyr-183 were close to FMN in the active site. The NerA mutation H178A produced mutant protein with bound FMN but no activity toward GTN. The N181A mutation produced protein that did not bind FMN and was isolated in partly degraded form. The mutation Y183F produced active protein with the same k cat as that of wild-type enzyme but with altered Km values for GTN and NADH, indicating a role for this residue in substrate binding. Correlation of the ratio of Km GTN to Km NAD(P)H, with sequence differences for NerA and several other members of the OYE family of oxidoreductases that reduce GTN, indicated that Asn-181 and a second Asn-238 that lies close to Tyr-183 in the NerA model structure may influence substrate specificity.

scholarly journals FMN-dependent oligomerization of putative lactate oxidase from Pediococcus acidilactici

2019 ◽  
Author(s):  
Yashwanth Ashok ◽  
Mirko M. Maksimainen ◽  
Tuija Kallio ◽  
Pekka Kilpeläinen ◽  
Lari Lehtiö
Keyword(s):  
Hydrogen Peroxide ◽  
Active Site ◽  
Pediococcus Acidilactici ◽  
Active Site Residues ◽  
Lactate Oxidase ◽  
Monooxygenase Activity ◽  
Aerococcus Viridans ◽  
Lactate Levels

AbstractLactate oxidases belong to a group of FMN-dependent enzymes and they catalyze a conversion of lactate to pyruvate with a release of hydrogen peroxide. Hydrogen peroxide is also utilized as a read out in biosensors to quantitate lactate levels in biological samples. Aerococcus viridans lactate oxidase is the best characterized lactate oxidase and our knowledge of lactate oxidases relies largely to studies conducted with that particular enzyme. Pediococcus acidilactici lactate oxidase is also commercially available for e.g. lactate measurements, but this enzyme has not been characterized before in detail. Here we report structural characterization of the recombinant enzyme and its co-factor dependent oligomerization. The crystal structures revealed two distinct conformations in the loop closing the active site, consistent with previous biochemical studies implicating the role of loop in catalysis. Despite the structural conservation of active site residues when compared to Aerococcus viridans lactate oxidase we were not able to detect either oxidase or monooxygenase activity when L-lactate or other potential alpha hydroxyl acids were used as a substrate. Pediococcus acidilactici lactate oxidase is therefore an example of a misannotation of an FMN-dependent enzyme, which catalyzes likely a so far unknown oxidation reaction.

scholarly journals Analyzing the catalytic role of active site residues in the Fe-type nitrile hydratase from Comamonas testosteroni Ni1

2015 ◽  
Vol 20 (5) ◽  
pp. 885-894 ◽  
Author(s):  
Salette Martinez ◽  
Rui Wu ◽  
Karoline Krzywda ◽  
Veronika Opalka ◽  
Hei Chan ◽  
...  
Keyword(s):  
Active Site ◽  
Nitrile Hydratase ◽  
Comamonas Testosteroni ◽  
Active Site Residues ◽  
Catalytic Role

scholarly journals Clarification of the role of key active site residues of glutathione transferase Zeta/maleylacetoacetate isomerase by a new spectrophotometric technique

2003 ◽  
Vol 374 (3) ◽  
pp. 731-737 ◽  
Author(s):  
Philip G. BOARD ◽  
Matthew C. TAYLOR ◽  
Marjorie COGGAN ◽  
Michael W. PARKER ◽  
Hoffman B. LANTUM ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Glutathione Transferase ◽  
Salt Bridge ◽  
Side Chain ◽  
Active Site Residues ◽  
Conserved Residues ◽  
Isomerase Activity ◽  
Catalytic Role

hGSTZ1-1 (human glutathione transferase Zeta 1-1) catalyses a range of glutathione-dependent reactions and plays an important role in the metabolism of tyrosine via its maleylacetoacetate isomerase activity. The crystal structure and sequence alignment of hGSTZ1 with other GSTs (glutathione transferases) focused attention on three highly conserved residues (Ser-14, Ser-15, Cys-16) as candidates for an important role in catalysis. Progress in the investigation of these residues has been limited by the absence of a convenient assay for kinetic analysis. In this study we have developed a new spectrophotometric assay with a novel substrate [(±)-2-bromo-3-(4-nitrophenyl)propionic acid]. The assay has been used to rapidly assess the potential catalytic role of several residues in the active site. Despite its less favourable orientation in the crystal structure, Ser-14 was the only residue found to be essential for catalysis. It is proposed that a conformational change may favourably reposition the hydroxyl of Ser-14 during the catalytic cycle. The Cys16→Ala (Cys-16 mutated to Ala) mutation caused a dramatic increase in the Km for glutathione, indicating that Cys-16 plays an important role in the binding and orientation of glutathione in the active site. Previous structural studies implicated Arg-175 in the orientation of α-halo acid substrates in the active site of hGSTZ1-1. Mutation of Arg-175 to Lys or Ala resulted in a significant lowering of the kcat in the Ala-175 variant. This result is consistent with the proposal that the charged side chain of Arg-175 forms a salt bridge with the carboxylate of the α-halo acid substrates.

Characterization of anEscherichia coliSulfite Oxidase Homologue Reveals the Role of a Conserved Active Site Cysteine in Assembly and Function†

Biochemistry ◽  
2005 ◽  
Vol 44 (30) ◽  
pp. 10339-10348 ◽  
Author(s):  
Stephen J. Brokx ◽  
Richard A. Rothery ◽  
Guijin Zhang ◽  
Derek P. Ng ◽  
Joel H. Weiner
Keyword(s):  
Active Site ◽  
Active Site Cysteine ◽  
And Function

scholarly journals Characterization of active-site residues in diadenosine tetraphosphate hydrolase from Lupinus angustifolius

2001 ◽  
Vol 357 (2) ◽  
pp. 399 ◽  
Author(s):  
Danuta MAKSEL ◽  
Paul R. GOOLEY ◽  
James D. SWARBRICK ◽  
Andrzej GURANOWSKI ◽  
Christine GANGE ◽  
...  
Keyword(s):  
Active Site ◽  
Lupinus Angustifolius ◽  
Active Site Residues ◽  
Diadenosine Tetraphosphate

scholarly journals The mechanistic role of active site residues in non-stereo haloacid dehalogenase E (DehE)

2019 ◽  
Vol 90 ◽  
pp. 219-225 ◽  
Author(s):  
Muhammad Hasanuddin Zainal Abidin ◽  
Khairul Bariyyah Abd Halim ◽  
Fahrul Huyop ◽  
Tengku Haziyamin Tengku Abdul Hamid ◽  
Roswanira Abdul Wahab ◽  
...  
Keyword(s):  
Active Site ◽  
Active Site Residues ◽  
Haloacid Dehalogenase

scholarly journals Mutations in the Predicted Active Site of Xanthomonas oryzae pv. oryzae XopQ Differentially Affect Virulence, Suppression of Host Innate Immunity, and Induction of the HR in a Nonhost Plant

2015 ◽  
Vol 28 (2) ◽  
pp. 195-206 ◽  
Author(s):  
Mahesh Kumar Gupta ◽  
Rajkanwar Nathawat ◽  
Dipanwita Sinha ◽  
Asfarul S. Haque ◽  
Rajan Sankaranarayanan ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Active Site ◽  
Plant Cell Wall ◽  
Structural Model ◽  
Bioinformatic Analysis ◽  
Xanthomonas Oryzae ◽  
Active Site Residues ◽  
Callose Deposition ◽  
Nonhost Plant ◽  
Bacterial Blight Pathogen

Xanthomonas oryzae pv. oryzae, the bacterial blight pathogen of rice, secretes a number of effectors through a type 3 secretion system. One of these effectors, called XopQ, is required for virulence and suppression of rice innate immune responses induced by the plant cell-wall-degrading enzyme lipase/esterase A (LipA). Bioinformatic analysis suggested that XopQ is homologous to inosine-uridine nucleoside hydrolases (NH). A structural model of XopQ with the protozoan Crithidia fasciculata purine NH suggested that D116 and Y279 are potential active site residues. X. oryzae pv. oryzae xopQ mutants (xopQ−/pHM1::xopQD116A and xopQ−/pHM1::xopQY279A) show reduced virulence on rice compared with xopQ−/pHM1::xopQ. The two predicted XopQ active site mutants (xopQ−/pHM1::xopQD116A and xopQ−/pHM1::xopQY279A) exhibit a reduced hypersensitive response (HR) on Nicotiana benthamiana, a nonhost. However, Arabidopsis lines expressing either xopQ or xopQY279A are equally proficient at suppression of LipA-induced callose deposition. Purified XopQ does not show NH activity on standard nucleoside substrates but exhibits ribose hydrolase activity on the nucleoside substrate analogue 4-nitrophenyl β-D-ribofuranoside. The D116A and Y279A mutations cause a reduction in biochemical activity. These results indicate that mutations in the predicted active site of XopQ affect virulence and induction of the HR but do not affect suppression of innate immunity.

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