Wild Type and Mutant Human Heart (<i>R</i>)-3-Hydroxybutyrate Dehydrogenase Expressed in Insect Cells

D, Green; Marks, Adam; Fleischer, Sidney; Jo, McIntyre

doi:10.1021/bi952807n

Cited by 15 publications

(34 citation statements)

References 32 publications

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“…In contrast, one member (DHRS6) of the large family of short chain dehydrogenases has been characterized as a novel human cytosolic hydroxybutyrate dehydrogenase (BDH2) (23), which catalyzes another important reaction in ketone body metabolism. This assignment was offered even though the available functional characterization indicates specific activity and substrate K m values that differ by an order of magnitude from comparable parameters for the well established mitochondrial enzyme (BDH1) (24). This cytosolic dehydrogenase may possess adequate BDH function to support extramitochondrial ketone body metabolism.…”

Section: Discussionmentioning

confidence: 99%

Identification and Characterization of an Extramitochondrial Human 3-Hydroxy-3-methylglutaryl-CoA Lyase

Montgomery

Pei

Watkins

et al. 2012

Journal of Biological Chemistry

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Background: Ketone bodies have been implicated not only in energy metabolism, but also in lipogenesis. Results: Discovery and characterization of human extramitochondrial HMG-CoA lyase-like protein (HMGCLL1) has been accomplished. Conclusion: Catalytically active HMGCLL1 is myristoylated and vesicle associated. Significance: Extramitochondrial HMG-CoA lyase may be crucial to lipid biosynthesis or to energy metabolism in certain tissues and cancer cells.

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Section: Discussionmentioning

confidence: 99%

Identification and Characterization of an Extramitochondrial Human 3-Hydroxy-3-methylglutaryl-CoA Lyase

Montgomery

Pei

Watkins

et al. 2012

Journal of Biological Chemistry

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“…To study the effects of diabetes and diets, both of which are generally correlated with high concentrations of ketone bodies in the blood, the previously published mitochondrial metabolic network was expanded to include ketone body degradation. We added one enzymatic reaction ((R)-3-hydroxybutanoate:NAD ϩ oxidoreductase, EC 1.1.1.30, (38,39)) and six transport reactions (supplemental data Table S2a) to the previous reconstruction. These reactions added five more metabolites to the network (supplemental data Table S2b).…”

Section: Content Of the Reconstructed Mitochondrial Networkmentioning

confidence: 99%

Candidate Metabolic Network States in Human Mitochondria

Thiele

Price

et al. 2005

Journal of Biological Chemistry

136

123

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The human mitochondrial metabolic network was recently reconstructed based on proteomic and biochemical data. Linear programming and uniform random sampling were applied herein to identify candidate steady states of the metabolic network that were consistent with the imposed physico-chemical constraints and available experimental data. The activity of the mitochondrion was studied under four metabolic conditions: normal physiologic, diabetic, ischemic, and dietetic. Pairwise correlations between steady-state reaction fluxes were calculated in each condition to evaluate the dependence among the reactions in the network. Applying constraints on exchange fluxes resulted in predictions for intracellular fluxes that agreed with experimental data. Analyses of the steady-state flux distributions showed that the experimentally observed reduced activity of pyruvate dehydrogenase in vivo could be a result of stoichiometric constraints and therefore would not necessarily require enzymatic inhibition. The observed changes in the energy metabolism of the mitochondrion under diabetic conditions were used to evaluate the impact of previously suggested treatments. The results showed that neither normalized glucose uptake nor decreased ketone body uptake have a positive effect on the mitochondrial energy metabolism or network flexibility. Taken together, this study showed that sampling of the steady-state flux space is a powerful method to investigate network properties under different conditions and provides a basis for in silico evaluations of effects of potential disease treatments.The emergence of genomic, metabolic, and proteomic data has facilitated the reconstruction of genome-scale metabolic networks. Various network reconstructions have been carried out in recent years for microorganisms such as Escherichia coli (1), Saccharomyces cerevisiae (2), Helicobacter pylori (3), Haemophilus influenzae (4), and Geobacter sulferreducans.1 Most recently, proteomics data (6, 7) were utilized to reconstruct the metabolic network of the human cardiac mitochondrion (8).Constraint-based analyses have proven to be valuable for studying genome-scale metabolic networks. The constraintbased approach is based on the fact that cellular networks are constrained to operate within boundaries set by physico-chemical constraints (mass conservation, directional flow, enzymatic capacity, etc). In this study, uniform random sampling was used to calculate candidate steady-state flux distributions in the human cardiac mitochondrion under different sets of constraints representing various physiological conditions. Experimental data from many literature sources were integrated as physico-chemical constraints of the mitochondrial metabolic network. Constraints based on these experimental data were applied to segment the steady-state flux space defined by mass-balance constraints. This segmentation resulted in a characterization of all feasible steadystate flux distributions, termed candidate steady-state flux distributions, that occurred under each speci...

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“…Each predicted membraneanchoring region has been deleted to produce the mutant cDNA that encodes the enzyme lacking one of the two segments. These cDNA molecules have been introduced into recombinant baculovirus for expression in eukaryotic Sf9 cells, which have subcellular organelle structures that are identical to mammalian cells (Green et al 1996, Gough et al 1998. After differential ultracentrifugation, the distribution of the two mutant enzymes and the wild-type enzyme among the endoplasmic reticulum (microsomes), mitochondria and cytosol has been determined.…”

Section: Introductionmentioning

confidence: 99%

Creation of a fully active, cytosolic form of human type I 3beta-hydroxysteroid dehydrogenase/isomerase by the deletion of a membrane-spanning domain

Jl¹,

Evans²,

Blanco³

et al. 1999

Journal of Molecular Endocrinology

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Human 3 -hydroxysteroid dehydrogenase/steroid 5 -4 -isomerase (3 -HSD/isomerase) is a bifunctional, single enzyme protein that is membranebound in the endoplasmic reticulum (microsomes) and mitochondria of cells in the placenta (type I) and in the adrenals and gonads (type II). Two membrane-binding domains (residues 72-89 and 283-310) have been predicted by analyses of hydrophobicity in the type I and II isoenzymes (90% regional homology). These putative membrane domains were deleted in the cDNA by PCR-based mutagenesis, and the two mutant enzymes were expressed by baculovirus in insect Sf9 cells. Differential centrifugation of the Sf9 cell homogenate containing the 283-310 deletion mutant revealed that 94% of the 3 -HSD and isomerase activities were in the cell cytosol, 6% of the activities were in the microsomes, and no activity was in the mitochondria. This is the opposite of the subcellular distribution of the wild-type enzyme with 94% of the activities in the microsomes and mitochondria and only 6% activity in the cytosol. The organelle distribution of the 72-89 deletion mutant lies between these two extremes with 72% of the enzyme activity in the cytosol and 28% in the microsomes/ mitochondria. The integrity of the subcellular organelle preparations was confirmed by electron microscopy. Western immunoblots confirmed the presence of the 283-310 deletion mutant enzyme and the absence of the wild-type enzyme in the insect cell cytosol. The unpurified, cytosolic 383-310 deletion mutant exhibited 3 -HSD (22 nmol/min per mg) and isomerase (33 nmol/min per mg) specific activities that were comparable with those of the membrane-bound, wild-type enzyme. The isomerase reaction of the cytosolic 283-311 deletion mutant requires activation by NADH just like the isomerase of the microsomal or mitochondrial wild-type enzyme. In contrast, the 72-89 deletion mutant had low 3 -HSD and isomerase specific activities that were only 12% of the wild-type levels. This innovative study identifies the 283-310 region as the critical membrane domain of 3 -HSD/isomerase that can be deleted without compromising enzyme function. The shorter 72-89 region is also a membrane domain, but deletion of this NH 2 -terminal region markedly diminishes the enzyme activities. Purification of the active, cytosolic 283-310 deletion mutant will produce a valuable tool for crystallographic studies that may ultimately determine the tertiary/ quaternary structure of this key steroidogenic enzyme.

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Wild Type and Mutant Human Heart (R)-3-Hydroxybutyrate Dehydrogenase Expressed in Insect Cells

Cited by 15 publications

References 32 publications

Identification and Characterization of an Extramitochondrial Human 3-Hydroxy-3-methylglutaryl-CoA Lyase

Identification and Characterization of an Extramitochondrial Human 3-Hydroxy-3-methylglutaryl-CoA Lyase

Candidate Metabolic Network States in Human Mitochondria

Creation of a fully active, cytosolic form of human type I 3beta-hydroxysteroid dehydrogenase/isomerase by the deletion of a membrane-spanning domain

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