Molecular structure of flavocytochrome b2 at 24 Å resolution

Xia, Fei; Mathews, F.S.

doi:10.1016/0022-2836(90)90240-m

Cited by 330 publications

(405 citation statements)

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“…For example, the distance from the iron to the center of the flavin ring in flavocytochrome b2 is about 16 A (Xia & Mathews, 1990), and in p-cresol methylhydroxylase it is about 18 A . In the photosynthetic center (Deisenhofer et al, 1984) the closest heme iron to bacteriochlorophyll Mg distance is about 21 A.…”

Section: Discussionmentioning

confidence: 99%

Preliminary crystal structure studies of a ternary electron transfer complex between a quinoprotein, a blue copper protein, and a c‐type cytochrome

et al. 1993

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A ternary electron transfer protein complex has been crystallized and a preliminary structure investigation has been carried out. The complex is composed of a quinoprotein, methylamine dehydrogenase (MADH), a blue copper protein, amicyanin, and a C-type cytochrome All three proteins were isolated from Paracoccus denitrificans. The crystals of the complex are orthorhombic, space group C2221 with cell dimensions a = 148.81 A , b = 68.85 A, and c = 187.18 A . Two types of isomorphous crystals were prepared: one using native amicyanin and the other copper-free apo-amicyanin. The diffraction data were collected at 2.75 A resolution from the former and at 2.4 A resolution from the latter. The location of the MADH portion was determined by molecular replacement. The copper site of the amicyanin molecule was located in an isomorphous difference Fourier while the iron site of the cytochrome was found in an anomalous difference Fourier. The MADH from P. denitrificans (PD-MADH) is an H,L, hetero-tetramer with the H subunit containing 373 residues and the L subunit 131 residues, the latter containing a novel redox cofactor, tryptophan tryptophylquinone (TTQ). The amicyanin of P. denitrificans contains 105 residues and the cytochrome cg5li contains 155 residues. The ternary complex consists of one MADH tetramer with two molecules of amicyanin and two of Cssli, forming a hetero-octamer; the octamer is located on a crystallographic diad. The relative positions of the three redox centers-i.e., the TTQ of MADH, the copper of amicyanin, and the heme group of are presented. ., 1983). The crystal structures of MADH from both Thiobucillus versutus (Vellieux et al., 1989) and P. denitrifcuns (Chen et al., 1992b) have been determined, based on an X-ray sequence for the former. The structure of the latter has recently been rebuilt using the DNA-derived amino acid sequence (Chistoserdov et al., 1992) and refined at 1.75 A resolution (Chen et al., in prep.).The nature of the redox cofactor of MADH has been a challenging topic over the last decade. Recently a cofactor model was proposed for MADH based on biophysical studies and on the gene sequence of the L subunit of MADH. This new cofactor, 147

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Section: Discussionmentioning

confidence: 99%

Preliminary crystal structure studies of a ternary electron transfer complex between a quinoprotein, a blue copper protein, and a c‐type cytochrome

et al. 1993

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“…The interaction of the carboxylic acid moiety with these residues has also been observed in homologous enzymes of Hao2. 18,19 The NH group of the pyrazole moiety forms putative hydrogen bonds with the catalytic H247 and nearby Y129 residues. The biphenyl moiety resides in the hydrophobic pocket and is surrounded by residues such as F79, A185, L172, L174, E188, F23, and L161.…”

Section: Acs Medicinal Chemistry Lettersmentioning

confidence: 99%

Potent and Selective Inhibitors of Long Chain l-2-Hydroxy Acid Oxidase Reduced Blood Pressure in DOCA Salt-Treated Rats

Barawkar¹,

Meru²,

Bandyopadhyay³

et al. 2011

ACS Med. Chem. Lett.

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L-2-Hydroxy acid oxidase (Hao2) is a peroxisomal enzyme with predominant expression in the liver and kidney. Hao2 was recently identified as a candidate gene for blood pressure quantitative trait locus in rats. To investigate a pharmacological role of Hao2 in the management of blood pressure, selective Hao2 inhibitors were developed. Optimization of screening hits 1 and 2 led to the discovery of compounds 3 and 4 as potent and selective rat Hao2 inhibitors with pharmacokinetic properties suitable for in vivo studies in rats. Treatment with compound 3 or 4 resulted in a significant reduction or attenuation of blood pressure in an established or developing model of hypertension, deoxycorticosterone acetatetreated rats. This is the first report demonstrating a pharmacological benefit of selective Hao2 inhibitors in a relevant model of hypertension. KEYWORDS: Hao2, hypertension, pyrazolecarboxylic acid, DOCA rat H uman hypertension is a complex, multifactorial disorder resulting from the interplay of multiple environmental and genetic factors, and this common disorder can lead to an increased risk of heart attack, stroke, and renal failure. The mechanisms underlying the initiation and maintenance of the hypertensive process remain unclear. 1 Almost one-third of the U.S. adult population has high blood pressure (BP), which increases the risk of cardiovascular and renal disease and shortened life expectancy. 2−4 Various antihypertensive drugs have been developed, including diuretics, beta blockers, calcium channel blockers (CCBs), renin inhibitors, 5 angiotensinconverting enzyme (ACE) inhibitors, and angiotensin II receptor blockers (ARBs). 6,7 However, these drugs either lack sufficient efficacy or are associated with significant adverse effects. In a search for a novel antihypertensive target, we have identified L-2-hydroxy acid oxidase (Hao2) 8 as a potential target for pharmacological intervention.L-2-Hydroxy acid oxidases are flavin mononucleotide (FMN)-dependent peroxisomal enzymes, which are members of the flavoenzyme family that are responsible for the oxidation of a number of L-2-hydroxy acids to ketoacids at the expense of molecular oxygen, resulting in the formation of hydrogen peroxide. Several examples of such enzymes have been identified in different organisms, e.g., glycolate oxidase from plants, lactate oxidase from Mycobacterium (L-lactate 2-monooxygenase), flavocytochrome b 2 from yeasts (L-lactate cytochrome c oxidoreductase), and mandelate dehydrogenase from Pseudomonas putida. 9 In mammals, this family of enzymes was first identified as an L-amino acid oxidase in the kidney and the liver of rats and later found to have activity similar to that of L-2-hydroxy acid. 10,11 Two α-hydroxy acid oxidases were also reported from hog renal cortex, named long chain L-α-hydroxy acid oxidase (Hao2) and short chain L-α-hydroxy acid oxidase (Hao1), because of their substrate specificity toward long and short carbon chain L-α-hydroxy acids, respectively. In both prokaryotes and eukaryotes, all the memb...

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“…This can probably be explained on steric grounds. The flavocytochrome b2 crystal structure at the active site is shown in Figure 3 (Xia & Mathews, 1990), together with the postulated lactate binding mode, derived from the binding of pyruvate in subunit S2 (Lederer & Mathews, 1987;Dubois et al, 1990;Lederer, 1991a). In subunit S 1, a water network is observed at the active site instead of the ligand.…”

Section: Discussionmentioning

confidence: 99%

“…In subunit S 1, a water network is observed at the active site instead of the ligand. WAT662 is hydrogen bonded to both H373 and Y254, as well as to other waters (Xia & Mathews, 1990). Unless large local movements take place in solution, it can be surmised that this chelate-type binding to the two residues might be favor-…”

Section: Discussionmentioning

confidence: 99%

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About the pK_a of the active‐site histidine in flavocytochrome b₂ (yeast L‐lactate dehydrogenase)

Rao

Lederer

1998

Protein Science

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Flavocytochrome b2 or L-lactate dehydrogenase from yeasts catalyzes the oxidation of L-lactate at the expense of monoelectronic acceptors such as cytochrome c, its physiological partner. When incubated in the presence of both L-lactate and a keto acid, the enzyme catalyzes a transhydrogenation reaction wherein only the flavin is involved. During this reaction, the substrate a-hydrogen is transferred not only to the solvent but also in part to the keto acid, which acts as reverse substrate. Thus, when bound to the reduced enzyme, this hydrogen is sticky. In the context of a carbanion mechanism, it resides on NE of His373, the active site base. We have shown before that a correlation between the amount of intermolecular hydrogen transfer from [2-'H] lactate and the keto acid reverse substrate concentration enables the determination of the first-order rate constant, k:, for exchange of the substrate-derived protein-bound hydrogen with bulk solvent (Urban P, Lederer F, 1985, J BioZ Chem 260: 1 1 1 15-1 1 122). In this work, we show that the exchange with the solvent appears to be independent of the phosphate buffer concentration in the range from 40 to 500 mM. It is thus probable that exchange occurs directly with water molecules. The second-order rate constant for exchange is then 0.16 (kO.03) M" s". Using the Eigen equation, this figure yields a pK, of 9.1 f 0.1 for His373 in the reduced enzyme, compared to a probable value of 6.0 or less in the oxidized enzyme (Suzuki H, Ogura YC, 1970, JBiochem 67:291-295). The mechanistic significance of these results is discussed.

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Molecular structure of flavocytochrome b2 at 24 Å resolution

Cited by 330 publications

References 91 publications

Preliminary crystal structure studies of a ternary electron transfer complex between a quinoprotein, a blue copper protein, and a c‐type cytochrome

Preliminary crystal structure studies of a ternary electron transfer complex between a quinoprotein, a blue copper protein, and a c‐type cytochrome

Potent and Selective Inhibitors of Long Chain l-2-Hydroxy Acid Oxidase Reduced Blood Pressure in DOCA Salt-Treated Rats

About the pK_a of the active‐site histidine in flavocytochrome b₂ (yeast L‐lactate dehydrogenase)

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Molecular structure of flavocytochrome b2 at 24 Å resolution

Cited by 330 publications

References 91 publications

Preliminary crystal structure studies of a ternary electron transfer complex between a quinoprotein, a blue copper protein, and a c‐type cytochrome

Preliminary crystal structure studies of a ternary electron transfer complex between a quinoprotein, a blue copper protein, and a c‐type cytochrome

Potent and Selective Inhibitors of Long Chain l-2-Hydroxy Acid Oxidase Reduced Blood Pressure in DOCA Salt-Treated Rats

About the pKa of the active‐site histidine in flavocytochrome b2 (yeast L‐lactate dehydrogenase)

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About the pK_a of the active‐site histidine in flavocytochrome b₂ (yeast L‐lactate dehydrogenase)