Role of tryptophan-208 residue in cytochrome c oxidation by ascorbate peroxidase from Leishmania major-kinetic studies on Trp208Phe mutant and wild type enzyme

The two distinct domains of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase) are connected by a typical hinge peptide. The amino acid sequence of this interdomain hinge is dramatically different in flavocytochromes b2 from Saccharomyces cerevisiae and Hansenula anomala. This difference in the hinge is believed to contribute to the difference in kinetic properties between the two enzymes. To probe the importance of the hinge, an interspecies hybrid enzyme has been constructed comprising the bulk of the S. cerevisiae enzyme but containing the H. anomala flavocytochrome b2 hinge. The kinetic properties of this ‘hinge-swap’ enzyme have been investigated by steady-state and stopped-flow methods. The hinge-swap enzyme remains a good lactate dehydrogenase as is evident from steady-state experiments with ferricyanide as acceptor (only 3-fold less active than wild-type enzyme) and stopped-flow experiments monitoring flavin reduction (2.5-fold slower than in wild-type enzyme). The major effect of the hinge-swap mutation is to lower dramatically the enzyme's effectiveness as a cytochrome c reductase; kcat. for cytochrome c reduction falls by more than 100-fold, from 207 +/- 10 s-1 (25 degrees C, pH 7.5) in the wild-type enzyme to 1.62 +/- 0.41 s-1 in the mutant enzyme. This fall in cytochrome c reductase activity results from poor interdomain electron transfer between the FMN and haem groups. This can be demonstrated by the fact that the kcat. for haem reduction in the hinge-swap enzyme (measured by the stopped-flow method) has a value of 1.61 +/- 0.42 s-1, identical with the value for cytochrome c reduction and some 300-fold lower than the value for the wild-type enzyme. From these and other kinetic parameters, including kinetic isotope effects with [2-2H]lactate, we conclude that the hinge plays a crucial role in allowing efficient electron transfer between the two domains of flavocytochrome b2.

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The Role of Vitamin D and Vitamin D Receptor in Immunity toLeishmania majorInfection

Journal of Parasitology Research ◽

10.1155/2012/134645 ◽

2012 ◽

Vol 2012 ◽

pp. 1-10 ◽

Cited By ~ 18

Author(s):

James P. Whitcomb ◽

Mary DeAgostino ◽

Mark Ballentine ◽

Jun Fu ◽

Martin Tenniswood ◽

...

Keyword(s):

Vitamin D ◽

Immune Responses ◽

Vitamin D Receptor ◽

Leishmania Major ◽

Active Form ◽

Wild Type ◽

Vitamin D Signaling ◽

Deficient Mice ◽

Th 1

Vitamin D signaling modulates a variety of immune responses. Here, we assessed the role of vitamin D in immunity to experimental leishmaniasis infection in vitamin D receptor-deficient mice (VDRKO). We observed that VDRKO mice on a genetically resistant background have decreasedLeishmania major-induced lesion development compared to wild-type (WT) mice; additionally, parasite loads in infected dermis were significantly lower at the height of infection. Enzymatic depletion of the active form of vitamin D mimics the ablation of VDR resulting in an increased resistance toL. major. Conversely, VDRKO or vitamin D-deficient mice on the susceptible Th2-biased background had no change in susceptibility. These studies indicate vitamin D deficiency, either through the ablation of VDR or elimination of its ligand, 1,25D3, leads to an increase resistance toL. majorinfection but only in a host that is predisposed for Th-1 immune responses.

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Leishmania-Derived Trimannose Modulates the Inflammatory Response To Significantly Reduce Leishmania major-Induced Lesions

Infection and Immunity ◽

10.1128/iai.00672-17 ◽

2017 ◽

Vol 86 (1) ◽

Cited By ~ 2

Author(s):

Tara L. Grinnage-Pulley ◽

Daniel E. K. Kabotso ◽

Chelsea L. Rintelmann ◽

Rajarshi Roychoudhury ◽

Robert G. Schaut ◽

...

Keyword(s):

Leishmania Major ◽

Parasite Load ◽

Mannose Receptor ◽

Lesion Size ◽

Wild Type ◽

Cutaneous Lesions ◽

Content Type ◽

Latex Beads

ABSTRACTLeishmanialipophosphoglycan (LPG) is a key virulence factor, initiating inflammation resulting in cutaneous lesions. LPG is capped by various oligosaccharides. How these glycans are recognized and how they alter the course ofLeishmaniainfection are poorly understood. Previous studies synthesized α-1,2-trimannose cap sugars on latex beads and demonstrated that C57BL/6 mice coinoculated withLeishmania majorand trimannose-coated beads produced significantly higher levels of interleukin-12p40 (IL-12p40) and other proinflammatory, type 1 cytokines than mice inoculated withL. majoralone within the first 48 h of infection. However, asL. majorinfection typically progress over weeks to months, the role of trimannose in altering disease progression over the course of infection was unknown. Wild-type mice were inoculated with either trimannose-coated or carrier (uncoated) beads, infected withL. majoralone, coinoculated with carrier beads andL. major, or coinoculated with trimannose-coated beads andL. major. Trimannose treatment ofL. major-infected mice decreased the parasite load and significantly decreased the lesion size at 14 days postinfection (p.i.) compared to results for nontreated, infected mice. Infected, trimannose-treated mice had decreased IL-12p40 and IL-10 secretion and increased interferon gamma secretion at 14 days p.i. Mannose receptor knockout (MR−/−) mice lack the ability to detect trimannose. When MR−/−mice were infected withL. majorand treated with trimannose beads, they did not have decreased lesion size.Leishmania-derived trimannose represents a novel immunomodulator that provides early type 1-skewed cytokine production to control the parasite load and alter the course of cutaneous leishmaniasis.

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Structural and Biochemical Studies on the Regulation of Biotin Carboxylase by Substrate Inhibition and Dimerization

Journal of Biological Chemistry ◽

10.1074/jbc.m111.220517 ◽

2011 ◽

Vol 286 (27) ◽

pp. 24417-24425 ◽

Cited By ~ 14

Author(s):

Chi-Yuan Chou ◽

Liang Tong

Keyword(s):

Binding Sites ◽

Analytical Ultracentrifugation ◽

Substrate Inhibition ◽

Kinetic Studies ◽

The Other ◽

Biotin Carboxylase ◽

Binding Modes ◽

Wild Type ◽

Wild Type Enzyme ◽

Dimer Interface

Biotin carboxylase (BC) activity is shared among biotin-dependent carboxylases and catalyzes the Mg-ATP-dependent carboxylation of biotin using bicarbonate as the CO2 donor. BC has been studied extensively over the years by structural, kinetic, and mutagenesis analyses. Here we report three new crystal structures of Escherichia coli BC at up to 1.9 Å resolution, complexed with different ligands. Two structures are wild-type BC in complex with two ADP molecules and two Ca2+ ions or two ADP molecules and one Mg2+ ion. One ADP molecule is in the position normally taken by the ATP substrate, whereas the other ADP molecule occupies the binding sites of bicarbonate and biotin. One Ca2+ ion and the Mg2+ ion are associated with the ADP molecule in the active site, and the other Ca2+ ion is coordinated by Glu-87, Glu-288, and Asn-290. Our kinetic studies confirm that ATP shows substrate inhibition and that this inhibition is competitive against bicarbonate. The third structure is on the R16E mutant in complex with bicarbonate and Mg-ADP. Arg-16 is located near the dimer interface. The R16E mutant has only a 2-fold loss in catalytic activity compared with the wild-type enzyme. Analytical ultracentrifugation experiments showed that the mutation significantly destabilized the dimer, although the presence of substrates can induce dimer formation. The binding modes of bicarbonate and Mg-ADP are essentially the same as those to the wild-type enzyme. However, the mutation greatly disrupted the dimer interface and caused a large re-organization of the dimer. The structures of these new complexes have implications for the catalysis by BC.

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The role of distal tryptophan in the bifunctional activity of catalase-peroxidases

Biochemical Society Transactions ◽

10.1042/bst0290099 ◽

2001 ◽

Vol 29 (2) ◽

pp. 99-105 ◽

Cited By ~ 36

Author(s):

G. Regelsberger ◽

C. Jakopitsch ◽

P. G. Furtmüller ◽

F. Rueker ◽

J. Switala ◽

...

Keyword(s):

Catalase Activity ◽

Intermediate Compound ◽

Stopped Flow ◽

Wild Type ◽

Compound I ◽

Wild Type Enzyme ◽

Catalase Peroxidase ◽

Electron Reduction ◽

The One

Catalase-peroxidases are bifunctional peroxidases exhibiting an overwhelming catalase activity and a substantial peroxidase activity. Here we present a kinetic study of the formation and reduction of the key intermediate compound I by probing the role of the conserved tryptophan at the distal haem cavity site. Two wild-type proteins and three mutants of Synechocystis catalase-peroxidase (W122A and W122F) and Escherichia coli catalase-peroxidase (W105F) have been investigated by steady-state and stopped-flow spectroscopy. W122F and W122A completely lost their catalase activity whereas in W105F the catalase activity was reduced by a factor of about 5000. However, the mutations did not influence both formation of compound I and its reduction by peroxidase substrates. It was demonstrated unequivocally that the rate of compound I reduction by pyrogallol or o-dianisidine sometimes even exceeded that of the wild-type enzyme. This study demonstrates that the indole ring of distal Trp in catalase-peroxidases is essential for the two-electron reduction of compound I by hydrogen peroxide but not for compound I formation or for peroxidase reactivity (i.e. the one-electron reduction of compound I).

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Role of Arg-401 of cytosolic serine hydroxymethyltransferase in subunit assembly and interaction with the substrate carboxy group

Biochemical Journal ◽

10.1042/bj3270877 ◽

1997 ◽

Vol 327 (3) ◽

pp. 877-882 ◽

Cited By ~ 9

Author(s):

Junutula Reddy JAGATH ◽

Naropantul APPAJI RAO ◽

Handanahal SubbaRao SAVITHRI

Keyword(s):

Carboxy Group ◽

Gel Filtration ◽

Serine Hydroxymethyltransferase ◽

Site Directed Mutagenesis ◽

Mutant Enzyme ◽

Wild Type ◽

Wild Type Enzyme ◽

Partial Dissociation ◽

Tetrameric Form

In an attempt to identify the arginine residue involved in binding of the carboxylate group of serine to mammalian serine hydroxymethyltransferase, a highly conserved Arg-401 was mutated to Ala by site-directed mutagenesis. The mutant enzyme had a characteristic visible absorbance at 425 nm indicative of the presence of bound pyridoxal 5ʹ-phosphate as an internal aldimine with a lysine residue. However, it had only 0.003% of the catalytic activity of the wild-type enzyme. It was also unable to perform reactions with glycine, β-phenylserine or D-alanine, suggesting that the binding of these substrates to the mutant enzyme was affected. This was also evident from the interaction of amino-oxyacetic acid, which was very slow (8.4×10-4 s-1 at 50 μM) for the R401A mutant enzyme compared with the wild-type enzyme (44.6 s-1 at 50 μM). In contrast, methoxyamine (which lacks the carboxy group) reacted with the mutant enzyme (1.72 s-1 at 250 μM) more rapidly than the wild-type enzyme (0.2 s-1 at 250 μM). Further, both wild-type and the mutant enzymes were capable of forming unique quinonoid intermediates absorbing at 440 and 464 nm on interaction with thiosemicarbazide, which also does not have a carboxy group. These results implicate Arg-401 in the binding of the substrate carboxy group. In addition, gel-filtration profiles of the apoenzyme and the reconstituted holoenzyme of R401A and the wild-type enzyme showed that the mutant enzyme remained in a tetrameric form even when the cofactor had been removed. However, the wild-type enzyme underwent partial dissociation to a dimer, suggesting that the oligomeric structure was rendered more stable by the mutation of Arg-401. The increased stability of the mutant enzyme was also reflected in the higher apparent melting temperature (Tm) (61 °C) than that of the wild-type enzyme (56 °C). The addition of serine or serinamide did not change the apparent Tm of R401A mutant enzyme. These results suggest that the mutant enzyme might be in a permanently ‘open’ form and the increased apparent Tm could be due to enhanced subunit interactions.

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The role of tyrosine-9 and the C-terminal helix in the catalytic mechanism of Alpha-class glutathione S-transferases

Biochemical Journal ◽

10.1042/bj3430525 ◽

1999 ◽

Vol 343 (3) ◽

pp. 525-531 ◽

Cited By ~ 17

Author(s):

Claire S. ALLARDYCE ◽

Paul D. MCDONAGH ◽

Lu-Yun LIAN ◽

C. Roland WOLF ◽

Gordon C. K. ROBERTS

Keyword(s):

Conformational Change ◽

Catalytic Mechanism ◽

Ethacrynic Acid ◽

Marked Decrease ◽

Wild Type ◽

Wild Type Enzyme ◽

Turnover Number ◽

Hydrophobic Substrate ◽

Glutathione S Transferases

Glutathione S-transferases (GSTs) play a key role in the metabolism of drugs and xenobiotics. To investigate the catalytic mechanism, substrate binding and catalysis by the wild-type and two mutants of GST A1-1 have been studied. Substitution of the ‘essential’ Tyr9 by phenylalanine leads to a marked decrease in the kcat for 1-chloro-2,4-dinitrobenzene (CDNB), but has no affect on kcat for ethacrynic acid. Similarly, removal of the C-terminal helix by truncation of the enzyme at residue 209 leads to a decrease in kcat for CDNB, but an increase in kcat for ethacrynic acid. The binding of a GSH analogue increases the affinity of the wild-type enzyme for CDNB, and increases the rate of the enzyme-catalysed conjugation of this substrate with the small thiols 2-mercaptoethanol and dithiothreitol. This suggests that GSH binding produces a conformational change which is transmitted to the binding site for the hydrophobic substrate, where it alters both the affinity for the substrate and the catalytic-centre activity (‘turnover number‘) for conjugation, perhaps by increasing the proportion of the substrate bound productively. Neither of these two effects of GSH analogues are seen in the C-terminally truncated enzyme, indicating a role for the C-terminal helix in the GSH-induced conformational change.

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Selectivity of Fungal Sesquiterpene Synthases: Role of the Active Site's H-1α Loop in Catalysis

Applied and Environmental Microbiology ◽

10.1128/aem.01811-10 ◽

2010 ◽

Vol 76 (23) ◽

pp. 7723-7733 ◽

Cited By ~ 39

Author(s):

Fernando LÃ¯Â¿Â½pez-Gallego ◽

GraysonT. Wawrzyn ◽

Claudia Schmidt-Dannert

Keyword(s):

Marked Effect ◽

Biological Activities ◽

Site Directed Mutagenesis ◽

Gas Chromatography Mass Spectrometry ◽

Farnesyl Pyrophosphate ◽

Wild Type ◽

Wild Type Enzyme ◽

Terpene Synthases ◽

Product Profile

ABSTRACT Sesquiterpene synthases are responsible for the cyclization of farnesyl pyrophosphate into a myriad of structurally diverse compounds with various biological activities. We examine here the role of the conserved active site H-α1 loop in catalysis in three previously characterized fungal sesquiterpene synthases. The H-α1 loops of Cop3, Cop4, and Cop6 from Coprinus cinereus were altered by site-directed mutagenesis and the resultant product profiles were analyzed by gas chromatography-mass spectrometry and compared to the wild-type enzymes. In addition, we examine the effect of swapping the H-α1 loop from the promiscuous enzyme Cop4 with the more selective Cop6 and the effect of acidic or basic conditions on loop mutations in Cop4. Directed mutations of the H-α1 loop had a marked effect on the product profile of Cop3 and Cop4, while little to no change was shown in Cop6. Swapping of the Cop4 and Cop6 loops with one another was again shown to influence the product profile of Cop4, while the product profile of Cop6 remained identical to the wild-type enzyme. The loop mutations in Cop4 also implicate specific residues responsible for the pH sensitivity of the enzyme. These results affirm the role of the H-α1 loop in catalysis and provide a potential target to increase the product diversity of terpene synthases.

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Kinetic Studies on CphA Mutants Reveal the Role of the P158-P172 Loop in Activity versus Carbapenems

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01703-15 ◽

2016 ◽

Vol 60 (5) ◽

pp. 3123-3126 ◽

Cited By ~ 5

Author(s):

Carlo Bottoni ◽

Mariagrazia Perilli ◽

Francesca Marcoccia ◽

Alessandra Piccirilli ◽

Cristina Pellegrini ◽

...

Keyword(s):

Catalytic Activity ◽

Catalytic Efficiency ◽

Kinetic Studies ◽

Zinc Ion ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Wild Type ◽

The Stability

ABSTRACTSite-directed mutagenesis of CphA indicated that prolines in the P158-P172 loop are essential for the stability and the catalytic activity of subclass B2 metallo-β-lactamases against carbapenems. The sequential substitution of proline led to a decrease of the catalytic efficiency of the variant compared to the wild-type (WT) enzyme but also to a higher affinity for the binding of the second zinc ion.

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Investigation of putative active-site lysine residues in hydroxymethylbilane synthase. Preparation and characterization of mutants in which (a) Lys-55, (b) Lys-59 and (c) both Lys-55 and Lys-59 have been replaced by glutamine

Biochemical Journal ◽

10.1042/bj2710487 ◽

1990 ◽

Vol 271 (2) ◽

pp. 487-491 ◽

Cited By ~ 10

Author(s):

A Hädener ◽

P R Alefounder ◽

G J Hart ◽

C Abell ◽

A R Battersby

Keyword(s):

Escherichia Coli ◽

Active Site ◽

Kinetic Studies ◽

Enzyme Kinetic ◽

Wild Type ◽

Wild Type Enzyme ◽

Over Expression ◽

Putative Active Site ◽

Lysine Residues

A new construct carrying the hemC gene was transformed into Escherichia coli, resulting in approx. 1000-fold over-expression of hydroxymethylbilane synthase (HMBS). This construct was used to generate HMBS in which (a) Lys-55, (b) Lys-59 and (c) both Lys-55 and Lys-59 were replaced by glutamine (K55Q, K59Q and K55Q-K59Q respectively). All three modified enzymes are chromatographically separable from wild-type enzyme. Kinetic studies showed that the substitution K55Q has little effect whereas K59Q causes a 25-fold decrease in Kapp. cat./Kapp. m. Treatment of K55Q, K59Q and K55Q-K59Q separately with pyridoxal 5′-phosphate and NaBH4 resulted in incomplete and non-specific reaction with the remaining lysine residues. Pyridoxal modification of Lys-59 in the K55Q mutant caused greater enzymic inactivation than similar modification of Lys-55 in K59Q. The results in sum show that, though Lys-55 and Lys-59 may be at or near the active site, neither is indispensable for the catalytic activity of HMBS.

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