Towards Dissecting the Mechanism of Protein Phosphatase-1 Inhibition by Its C-Terminal Phosphorylation.

Phosphoprotein phosphatase-1 (PP1) is a key player in the regulation of phospho-serine (pSer) and phospho-threonine (pThr) dephosphorylation and is involved in a large fraction of cellular signaling pathways. Aberrant activity of PP1 has been linked to many diseases, including cancer and heart failure. Besides a well-established activity control by regulatory proteins, an inhibitory function for phosphorylation (p) of a Thr residue in the C-terminal intrinsically disordered tail of PP1 has been demonstrated. The associated phenotype of cell-cycle arrest was repeatedly proposed to be due to autoinhibition of PP1 through either conformational changes or substrate competition. Here, we use PP1 variants created by mutations and protein semisynthesis to differentiate between these hypotheses. Our data support the hypothesis that pThr exerts its inhibitory function by mediating protein complex formation rather than by a direct mechanism of structural changes or substrate competition.

Kinetic Investigation of a Presumed Nitronate Monooxygenase from Pseudomonas aeruginosa PAO1 Establishes a New Class of NAD(P)H:Quinone Reductases.

PA0660 from Pseudomonas aeruginosa PAO1 is currently classified as a hypothetical nitronate monooxygenase (NMO), but no evidence at the transcript or protein level has been presented. In this study, PA0660 was purified and its biochemical and kinetic properties were characterized. Absorption spectroscopy and mass spectrometry demonstrated a tightly, noncovalently bound FMN in the active site of the enzyme. Analytical ultracentrifugation showed that the enzyme exists as a dimer in solution. Despite its annotation, PA0660 did not exhibit nitronate monooxygenase activity. The enzyme could be reduced with NADPH or NADH with a marked preference for NADPH, as indicated by ∼30-fold larger kcat/ Km and kred/ Kd values. Turnover could be sustained with NAD(P)H and quinones, DCPIP, and to a lesser extent molecular oxygen. However, PA0660 did not turn over with methyl red, consistent with a lack of azoreductase activity. The enzyme turned over through a ping-pong bi-bi steady-state kinetic mechanism with NADPH and 1,4-benzoquinone showing a kcat value of 90 s-1. The rate constant for flavin reduction with saturating NADPH was 360 s-1, whereas that for flavin oxidation with 1,4-benzoquinone was 270 s-1, consistent with both hydride transfers from the pyridine nucleotide to the flavin and from the flavin to 1,4-benzoquinone being partially rate-limiting for enzyme turnover. A BlastP search and a multiple-sequence alignment analysis of PA0660 highlighted the presence of six conserved motifs in >1000 open reading frames currently annotated as hypothetical NMOs. Our results suggest that PA0660 should be classified as an NAD(P)H:quinone reductase and serve as a paradigm enzyme for a new class of enzymes.

Effects of stably incorporated iron on protein phosphatase-1 structure and activity.

Protein phosphatase-1 (PP1) drives a large amount of phosphoSer/Thr protein dephosphorylations in eukaryotes to counteract multiple kinases in signaling pathways. The phosphatase requires divalent metal cations for catalytic activity and contains iron naturally. Iron has been suggested to have an influence on PP1 activity through Fe2+ and Fe3+ oxidation states. However, much biochemical and all structural data have been obtained with recombinant PP1 containing Mn2+ ions. Purifying iron-containing PP1 from Escherichia coli has thus far not been possible. Here, we present the preparation, characterization, and structure of iron-bound PP1α in inactive and active states. We establish a key role for the electronic/redox properties of iron in PP1 activity and shed light on the difference in substrate specificity between iron- and manganese-containing PP1.

Nonpegylated liposomal doxorubicin combination regimen in patients with diffuse large B-cell lymphoma and cardiac comorbidity. Results of the HEART01 phase II trial conducted by the Fondazione Italiana Linfomi.

The purpose of this phase 2, multicenter study was to determine the activity and safety of nonpegylated liposomal doxorubicin as part of "R-COMP" combination in patients with diffuse large B-cell lymphoma and coexisting cardiac disorders. The study was conducted using a Bayesian continuing assessment method using complete remission rate and rate of cardiac events as study endpoints. Between November 2009 and October 2011, 50 evaluable patients were enrolled (median age, 76 years). Median baseline left ventricular ejection fraction (LVEF) was 60%. Ischemic cardiopathy was the most frequent preexisting cardiac disorder (35%), followed by atrial fibrillation (15%), left ventricular hypertrophy (13%), and baseline LVEF <50% (12%). Based on the intent to treat analysis, overall response rate was 72%, including 28 patients in complete remission (complete remission rate, 56%), and 8 in partial remission (16%). At the end of treatment, grades 3 to 4 cardiac events were observed in 6 patients. No significant modifications from baseline values of LVEF were observed during treatment and follow-up. Nonpegylated liposomal doxorubicin instead of doxorubicin in the R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) regimen is a feasible option for patients with diffuse large B-cell lymphoma presenting with concomitant cardiac disorders.

Synthesis of Highly Selective Submicromolar Microcystin-Based Inhibitors of Protein Phosphatase (PP)2A over PP1.

Research and therapeutic targeting of the phosphoserine/threonine phosphatases PP1 and PP2A is hindered by the lack of selective inhibitors. The microcystin (MC) natural toxins target both phosphatases with equal potency, and their complex synthesis has complicated structure-activity relationship studies in the past. We report herein the synthesis and biochemical evaluation of 11 MC analogues, which was accomplished through an efficient strategy combining solid- and solution-phase approaches. Our approach led to the first MC analogue with submicromolar inhibitory potency that is strongly selective for PP2A over PP1 and does not require the complex lipophilic Adda group. Through mutational and structural analyses, we identified a new key element for binding, as well as reasons for the selectivity. This work gives unprecedented insight into how selectivity between these phosphatases can be achieved with MC analogues.

Functional Annotation of a Presumed Nitronate Monoxygenase Reveals a New Class of NADH:Quinone Reductases.

The protein PA1024 from Pseudomonas aeruginosa PAO1 is currently classified as 2-nitropropane dioxygenase, the previous name for nitronate monooxygenase in the GenBankTM and PDB databases, but the enzyme was not kinetically characterized. In this study, PA1024 was purified to high levels, and the enzymatic activity was investigated by spectroscopic and polarographic techniques. Purified PA1024 did not exhibit nitronate monooxygenase activity; however, it displayed NADH:quinone reductase and a small NADH:oxidase activity. The enzyme preferred NADH to NADPH as a reducing substrate. PA1024 could reduce a broad spectrum of quinone substrates via a Ping Pong Bi Bi steady-state kinetic mechanism, generating the corresponding hydroquinones. The reductive half-reaction with NADH showed a kred value of 24 s-1 and an apparent Kd value estimated in the low micromolar range. The enzyme was not able to reduce the azo dye methyl red, routinely used in the kinetic characterization of azoreductases. Finally, we revisited and modified the existing six conserved motifs of PA1024, which define a new class of NADH:quinone reductases and are present in more than 490 hypothetical proteins in the GenBankTM, the vast majority of which are currently misannotated as nitronate monooxygenase.

Role of F357 as an Oxygen Gate in the Oxidative Half-Reaction of Choline Oxidase.

Choline oxidase from Arthrobacter globiformis catalyzes the oxidation of choline to glycine betaine by using oxygen as an electron acceptor. A partially rate limiting isomerization of the reduced wild-type enzyme during the reaction with oxygen was previously detected using solvent viscosity effects. In this study, we hypothesized that the side chains of M62 and F357, located at the entrance to the active site of choline oxidase, may be related to the slow isomerization detected. We engineered a double-variant enzyme M62A/F357A. The kinetic characterization of the double-variant enzyme showed a lack of the isomerization detected in wild-type choline oxidase, and a lack of saturation with an oxygen concentration as high as 1 mM, while most other kinetic parameters were similar to those of wild-type choline oxidase. The kinetic characterization of the single-variant enzymes established that only the side chain of F357 plays a role in the isomerization of choline oxidase in the oxidative half-reaction. Molecular dynamics studies suggest that the slow isomerization related to F357 is possibly due to the participation of the phenyl ring in a newly proposed gating mechanism for a narrow tunnel, assumed to regulate the access of oxygen to the reduced cofactor.

Pseudomonas aeruginosa LysR PA4203 regulator NmoR acts as a repressor of the PA4202 nmoA gene, encoding a nitronate monooxygenase.

The PA4203 gene encodes a LysR regulator and lies between the ppgL gene (PA4204), which encodes a periplasmic gluconolactonase, and, in the opposite orientation, the PA4202 (nmoA) gene, coding for a nitronate monooxygenase, and ddlA (PA4201), encoding a d-alanine alanine ligase. The intergenic regions between PA4203 and ppgL and between PA4203 and nmoA are very short (79 and 107 nucleotides, respectively). Here we show that PA4203 (nmoR) represses its own transcription and the expression of nmoA. A chromatin immunoprecipitation analysis showed the presence of a single NmoR binding site between nmoA and nmoR, which was confirmed by electrophoretic mobility shift assays (EMSAs) with the purified NmoR protein. Despite this observation, a transcriptome analysis revealed more genes to be affected in an nmoR mutant, including genes known to be part of the MexT LysR activator regulon. The PA1225 gene, encoding a quinone oxidoreductase, was the most highly upregulated gene in the nmoR deletion mutant, independently of MexT. Finally, deletion of the nmoA gene resulted in an increased sensitivity of the cells to 3-nitropropionic acid (3-NPA), confirming the role of the nitronate monooxygenase protein in the detoxification of nitronate.

The combined structural and kinetic characterization of a bacterial nitronate monooxygenase from Pseudomonas aeruginosa PAO1 establishes NMO class I and II.

Nitronate monooxygenase (NMO) oxidizes the mitochondrial toxin propionate 3-nitronate (P3N) to malonate semialdehyde. The enzyme has been previously characterized biochemically in fungi, but no structural information is available. Based on amino acid similarity 4,985 genes are annotated in the GenBank(TM) as NMO. Of these, 4,424 (i.e. 89%) are bacterial genes, including several Pseudomonads that have been shown to use P3N as growth substrate. Here, we have cloned and expressed the gene pa4202 of Pseudomonas aeruginosa PAO1, purified the resulting protein, and characterized it. The enzyme is active on P3N and other alkyl nitronates, but cannot oxidize nitroalkanes. P3N is the best substrate at pH 7.5 and atmospheric oxygen with k(cat)(app)/K(m)(app) of 12 × 10(6) M(-1) s(-1), k(cat)(app) of 1300 s(-1), and K(m)(app) of 110 µm. Anerobic reduction of the enzyme with P3N yields a flavosemiquinone, which is formed within 7.5 ms, consistent with this species being a catalytic intermediate. Absorption spectroscopy, mass spectrometry, and x-ray crystallography demonstrate a tightly, non-covalently bound FMN in the active site of the enzyme. Thus, PA4202 is the first NMO identified and characterized in bacteria. The x-ray crystal structure of the enzyme was solved at 1.44 Å, showing a TIM barrel-fold. Four motifs in common with the biochemically characterized NMO from Cyberlindnera saturnus are identified in the structure of bacterial NMO, defining Class I NMO, which includes bacterial, fungal, and two animal NMOs. Notably, the only other NMO from Neurospora crassa for which biochemical evidence is available lacks the four motifs, defining Class II NMO.

Structure of choline oxidase in complex with the reaction product glycine betaine.

Choline oxidase from Arthrobacter globiformis, which is involved in the biosynthesis of glycine betaine from choline, has been extensively characterized in its mechanistic and structural properties. Despite the knowledge gained on the enzyme, the details of substrate access to the active site are not fully understood. The `loop-and-lid' mechanism described for the glucose-methanol-choline enzyme superfamily has not been confirmed for choline oxidase. Instead, a hydrophobic cluster on the solvent-accessible surface of the enzyme has been proposed by molecular dynamics to control substrate access to the active site. Here, the crystal structure of the enzyme was solved in complex with glycine betaine at pH 6.0 at 1.95 Šresolution, allowing a structural description of the ligand-enzyme interactions in the active site. This structure is the first of choline oxidase in complex with a physiologically relevant ligand. The protein structures with and without ligand are virtually identical, with the exception of a loop at the dimer interface, which assumes two distinct conformations. The different conformations of loop 250-255 define different accessibilities of the proposed active-site entrance delimited by the hydrophobic cluster on the other subunit of the dimer, suggesting a role in regulating substrate access to the active site.

Human choline dehydrogenase: medical promises and biochemical challenges.

Human choline dehydrogenase (CHD) is located in the inner membrane of mitochondria primarily in liver and kidney and catalyzes the oxidation of choline to glycine betaine. Its physiological role is to regulate the concentrations of choline and glycine betaine in the blood and cells. Choline is important for regulation of gene expression, the biosynthesis of lipoproteins and membrane phospholipids and for the biosynthesis of the neurotransmitter acetylcholine; glycine betaine plays important roles as a primary intracellular osmoprotectant and as methyl donor for the biosynthesis of methionine from homocysteine, a required step for the synthesis of the ubiquitous methyl donor S-adenosyl methionine. Recently, CHD has generated considerable medical attention due to its association with various human pathologies, including male infertility, homocysteinuria, breast cancer and metabolic syndrome. Despite the renewed interest, the biochemical characterization of the enzyme has lagged behind due to difficulties in the obtainment of purified, active and stable enzyme. This review article summarizes the medical relevance and the physiological roles of human CHD, highlights the biochemical knowledge on the enzyme, and provides an analysis based on the comparison of the protein sequence with that of bacterial choline oxidase, for which structural and biochemical information is available.