Human Cytosolic Enzyme Research Articles

The crystal structure of yeast mitochondrial type pyrophosphatase provides a model to study pathological mutations in its human ortholog

Mutations in human ppa2 gene encoding mitochondrial inorganic pyrophosphatase (PPA2) result in the mitochondria malfunction in heart and brain and lead to early death. In comparison with its cytosolic counterpart, PPA2 of any species is a poorly characterized enzyme with a previously unknown 3D structure. We report here the crystal structure of PPA2 from yeast Ogataea parapolymorpha (OpPPA2), as well as its biochemical characterization. OpPPA2 is a dimer, demonstrating the fold typical for other eukaryotic Family I pyrophosphatases, including the human cytosolic enzyme. Cofactor Mg2+ ions found in OpPPA2 structure have similar coordination to most known Family I pyrophosphatases. Most of the residues associated with the pathological mutations in human PPA2 are conserved in OpPPA2, and their structural context suggests possible explanations for the effects of the mutations on the human enzyme. In this work, the mutant variant of OpPPA2, Met52Val, corresponding to the natural pathogenic variant Met94Val of human PPA2, is characterized. The obtained structural and biochemical data provide a step to understanding the structural basis of PPA2-associated pathologies.

1770-P: Further Evidence Supporting a Potential Role for ADH1B in Obesity

We previously showed that expression of the human cytosolic enzyme, alcohol dehydrogenase 1B (ADH1B), was inversely correlated with measures of obesity and insulin resistance in subcutaneous adipose tissue in Mexican Americans. Here, we sought to determine the relationship between ADH1B RNA, protein expression, adipogenesis and insulin action in human subcutaneous primary adipocytes in culture, derived from lean (BMI < 28 kg.m-2) and obese (BMI ≥ 30 kg.m-2) donors. ADH1B expression increased ∼160-fold during adipogenesis as preadipocytes differentiated into mature adipocytes, reaching maximal levels at 14 days whereas the expression of two homologous genes, ADH1A and ADH1C was relatively unchanged. This indicates that ADH1B may play a role in adipocyte differentiation and proliferation. Adipocytes from lean donors expressed ADH1B at higher levels compared with those from obese donors. Given the multiple passaging of these cells, this indicates likely epigenetic regulation of expression. We performed a meta-analysis of the relationship between ADH1B expression and obesity using data from four populations (Mexican Americans, African Americans, Europeans and Pima Indians). The results showed that BMI was increased by 0.56 standard deviations for each standard deviation of decreased ADH1B expression. This finding illustrates the global relevance of the inverse correlation of ADH1B expression with obesity. Insulin stimulated ADH1B expression dose dependently, with higher levels in the “lean adipocytes.” Phosphorylation of AKT, an important mediator of insulin signaling, was lower in “obese adipocytes” compared with “lean adipocytes” (p<0.01) demonstrating that the “obese adipocytes” were relatively insulin resistant. Knockdown of ADH1B expression resulted in a decrease in insulin-stimulated glucose uptake of 35% (p<0.05). Taken together, these findings show that ADH1B is involved in insulin signaling and action in human subcutaneous adipocytes and that this activity is suppressed in obesity. Disclosure C. Jenkinson: None. L.D. Morales: None. D. Tripathy: None. S.L. Hu: None. J.C. Lopez-Alvarenga: None. R. Duggirala: None. R.A. DeFronzo: Advisory Panel; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Elcelyx Therapeutics, Inc., Intarcia Therapeutics, Inc., Janssen Pharmaceuticals, Inc., Novo Nordisk Inc. Research Support; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Janssen Pharmaceuticals, Inc., Merck & Co., Inc. Speaker's Bureau; Self; AstraZeneca, Novo Nordisk Inc. S. Mummidi: None. R. Arya: None. L. Norton: None. J. Blangero: None. A. Diaz-Badillo: None. Funding National Institutes of Health

Cloning, expression, purification, and characterization of zebrafish cytosolic serine hydroxymethyltransferase

A cDNA which encodes for zebrafish serine hydroxymethyltransferase (SHMT) has been cloned into a pET43.1a vector as a NdeI– EcoRI insert and transformed into HMS174(DE3) cells. After induction with isopropyl thiogalactoside, the enzyme was purified with a three-step purification protocol and about 15 mg of pure enzyme was obtained per liter of culture. Spectral and structural characteristics of the recombinant zebrafish SHMT are similar to the rabbit and human cytosolic SHMT. Kinetic constants for the natural substrates l-serine and tetrahydrofolate are also comparable to the values obtained previously for the rabbit and human cytosolic enzyme.

Location of the Coenzyme Binding Site in the Porcine Mitochondrial NADP-dependent Isocitrate Dehydrogenase

The structure of crystalline porcine mitochondrial NADP-dependent isocitrate dehydrogenase (IDH) has been determined in complex with Mn2+-isocitrate. Based on structural alignment between this porcine enzyme and seven determined crystal structures of complexes of NADP with bacterial IDHs, Arg83, Thr311, and Asn328 were chosen as targets for site-directed mutagenesis of porcine IDH. The circular dichroism spectra of purified wild-type and mutant enzymes are similar. The mutant enzymes exhibit little change in Km for isocitrate or Mn2+, showing that these residues are not involved in substrate binding. In contrast, the Arg83 mutants, Asn328 mutants, and T311A exhibit 3-20-fold increase in the Km(NADP). We propose that Arg83 enhances NADP affinity by hydrogen bonding with the 3'-OH of the nicotinamide ribose, whereas Asn328 hydrogen bonds with N1 of adenine. The pH dependence of Vmax for Arg83 and Asn328 mutants is similar to that of wild-type enzyme, but for all the Thr311 mutants, pK(es) is increased from 5.2 in the wild type to approximately 6.0. We have previously attributed the pH dependence of Vmax to the deprotonation of the metal-bound hydroxyl of isocitrate in the enzyme-substrate complex, prior to the transfer of a hydride from isocitrate to NADP's nicotinamide moiety. Thr311 interacts with the nicotinamide ribose and is the closest of the target amino acids to the nicotinamide ring. Distortion of the nicotinamide by Thr311 mutation will likely be transmitted to Mn2+-isocitrate resulting in an altered pK(es). Because porcine and human mitochondrial NADP-IDH have 95% sequence identity, these results should be applicable to the human enzyme.

Identification of a genotoxic mechanism for 2-nitroanisole carcinogenicity and of its carcinogenic potential for humans.

2-Nitroanisole (2-NA) is an important industrial pollutant and a potent bladder carcinogen for rodents. The mechanism of its carcinogenicity was investigated in this study. Here we have used two independent methods, (32)P-post-labeling and (3)H-labeled 2-NA, to show that 2-NA binds covalently to DNA in vitro after reductive activation by human hepatic cytosol and xanthine oxidase (XO). We also investigated the capacity of 2-NA to form DNA adducts in vivo. Male Wistar rats were treated i.p. with 2-NA (0.15 mg/kg body wt daily for 5 days) and DNA from several organs was analyzed by (32)P-post-labeling. Two 2-NA-specific DNA adducts, identical to those found in DNA incubated with 2-NA and human hepatic cytosol or XO in vitro, were detected in the urinary bladder (3.4 adducts/10(7) nt), the target organ, and, to a lesser extent, in liver, kidney and spleen. The two DNA adducts found in rat tissues in vivo were identified as deoxyguanosine adducts derived from a 2-NA reductive metabolite, N-(2-methoxyphenyl)hydroxylamine. This reactive metabolite of 2-NA was identified in incubations with human hepatic cytosol, besides 2-methoxyaniline (o-anisidine). The results of our study, the first report on the potential of human cytosolic enzymes to contribute to the activation of 2-NA by nitroreduction, strongly suggest a carcinogenic potency of this rodent carcinogen for humans.

Overexpression and Characterization of the Human Mitochondrial and Cytosolic Branched-chain Aminotransferases

We have developed overexpression systems for the human branched-chain aminotransferase isoenzymes. The enzymes function as dimers and have substrate specificity comparable with the rat enzymes. The human cytosolic enzyme appears to turn over 2-5 times faster than the mitochondrial enzyme, and there may be anion and cation effects on the kinetics of both enzymes. The two proteins demonstrate similar absorption profiles, and the far UV circular dichroism spectra show that no global structural changes occur when the proteins are converted from the pyridoxal to pyridoxamine form. On the other hand, the near UV circular dichroism spectra suggest differences in the local environment surrounding tyrosines within these proteins. Both enzymes require a reducing environment for maximal activity, but the mitochondrial enzyme can be inhibited by nickel ions in the presence of reducing agents, while the cytosolic enzyme is unaffected. Chemical denaturation profiles of the proteins show that there are differences in structural stability. Titration of -SH groups with 5,5'-dithiobis(2-nitrobenzoic acid) suggests that no disulfide bonds are present in the mitochondrial enzyme and that at least two disulfide bonds are present in the cytosolic enzyme. Two -SH groups are titrated in the native form of the mitochondrial enzyme, leading to complete inhibition of activity, while only one -SH group is titrated in the cytosolic enzyme with no effect on activity. Although these proteins share 58% identity in primary amino acid sequence, the local environment surrounding the active site appears unique for each isoenzyme.

Cloning of the rat and human mitochondrial branched chain aminotransferases (BCAT m)

The rat and human mitochondrial branched chain aminotransferase (BCAT m) cDNAs have been isolated and shown to encode mature proteins of 41.2 and 41.3 kDa with presequences of 27 amino acids. When rat BCAT m is overexpressed in COS-1 cells, the protein exhibits BCAT activity and correct processing of the mitochondrial targeting sequence. Southern blot analysis of genomic DNA from a panel of rodent-human somatic cell hybrids revealed that the human BCAT m gene resides on chromosome 19 and the human cytosolic enzyme (BCAT c) gene on chromosome 12. Finally, the nomenclature BCAT1 for the cytosolic gene and BCAT2 for the mitochondrial BCAT gene is proposed.

Delivery of a Secretable Adenosine Deaminase Through Microcapsules–A Novel Approach to Somatic Gene Therapy

Many current gene therapy protocols require genetic modification of autologous cells. An alternate approach is to use universal recombinant cell lines engineered to secrete in vivo the desired gene products. Enclosing these cells within immunoprotective devices before implantation would prevent rejection of the nonautologous donor cells. To overcome the limitation that not all therapeutic gene products are secreted, we now propose to fuse a signal sequence to the amino terminus of a nonsecreted protein such as human adenosine deaminase (ADA), thus directing the product into a secretory pathway for release from the cells. A fusion gene constructed between the cDNA of the beta-lactamase signal sequence and human ADA expressed a product after in vitro transcription and translation that was immunologically similar to the human protein. Mouse fibroblasts transfected with the fusion gene demonstrated secreted ADA activity that resembled the human cytosolic enzyme in its heat stability, pH optimum, KM, electrophoretic mobility, and immunologic reactivity. Hence, the secreted enzyme expressed from the fusion gene is antigenically and enzymatically similar to the authentic human form. When transfected mouse fibroblasts or myoblasts were enclosed in permselective alginate-poly-L-lysine alginate microcapsules, ADA activity was secreted from the microcapsules and the cells remained viable for over 5 months. Hence, a secretable and functional human ADA has been constructed that can be delivered from recombinant cells within immunoprotective capsules. The success of this strategy provides the prototype for engineering nonsecreted gene products for therapy via this novel method of somatic gene therapy.