Oxaloacetate decarboxylase of Vibrio cholerae: purification, characterization, and expression of the genes in Escherichia coli

The oxaloacetate decarboxylase (OAD) Na+ pump consists of subunits α, β, and γ, which are expressed from an oadGAB gene cluster present in various anaerobic bacteria. Vibrio cholerae has two copies of oad genes, which are termed oad-1 and oad-2. The oad-2 genes are part of the citrate fermentation operon, while the oad-1 genes are flanked by genes encoding products not involved in a catabolic pathway. The gene sequences of oad-1 and oad-2 of V. cholerae strain O395-N1 were determined. The apparent frameshift in the published sequence of the oadA-2 gene from V. cholerae El Tor N16961 was not present in strain O395-N1. Upon anaerobic growth of V. cholerae on citrate, exclusively the oad-2 genes are expressed. OAD was isolated from these cells by monomeric avidin-Sepharose affinity chromatography. The enzyme was of higher specific activity than that from Klebsiella pneumoniae and was significantly more stable. Decarboxylase activity was Na+ dependent, and the activation profile showed strong cooperativity with a Hill coefficient nH=1.8. Oxalate and oxomalonate inhibited the enzyme with half-maximal concentrations of 10μM and 200μM, respectively. After reconstitution into proteoliposomes, the enzyme acted as a Na+ pump. With size-exclusion chromatography, the enzyme eluted in a symmetrical peak at a retention volume corresponding to an apparent molecular mass of approximately 570kDa, suggesting a tetrameric structure for OAD-2. The two oad gene clusters were heterologously expressed in Escherichia coli, and the decarboxylases were isolated from the host cell

Structure-Function Relations in Oxaloacetate Decarboxylase Complex. Fluorescence and Infrared Approaches to Monitor Oxomalonate and Na+ Binding Effect

ions across the membrane, which drives endergonic membrane reactions such as ATP synthesis, transport and motility. OAD is a membrane-bound enzyme composed of α, β and γ subunits. The α subunit contains the carboxyltransferase catalytic site. characteristic of a high content of α helix structures. Addition of oxomalonate induced a shift of the amide-I band of OAD toward higher wavenumbers, interpreted as a slight decrease of β sheet structures and a concomitant increase of α helix structures. Oxomalonate binding to αγand α subunits also provoked secondary structure variations, but these effects were negligible compared to OAD complex. alters the tryptophan environment of the β subunit, consistent with the function of these subunits within the enzyme complex. Formation of a complex between OAD and its substrates elicits structural changes in the α-helical as well as β-strand secondary structure elements

Oxaloacetate decarboxylase of Vibrio cholerae: Purification, characterization, and expression of the genes in Escherichia coli

ISSN:0302-8933ISSN:1432-072

Regulation of brain-type creatine kinase by AMP-activated protein kinase: Interaction, phosphorylation and ER localization

AbstractAMP-activated protein kinase (AMPK) and cytosolic brain-type creatine kinase (BCK) cooperate under energy stress to compensate for loss of adenosine triphosphate (ATP) by either stimulating ATP-generating and inhibiting ATP-consuming pathways, or by direct ATP regeneration from phosphocreatine, respectively. Here we report on AMPK-dependent phosphorylation of BCK from different species identified by in vitro screening for AMPK substrates in mouse brain. Mass spectrometry, protein sequencing, and site-directed mutagenesis identified Ser6 as a relevant residue with one site phosphorylated per BCK dimer. Yeast two-hybrid analysis revealed interaction of active AMPK specifically with non-phosphorylated BCK. Pharmacological activation of AMPK mimicking energy stress led to BCK phosphorylation in astrocytes and fibroblasts, as evidenced with a highly specific phospho-Ser6 antibody. BCK phosphorylation at Ser6 did not affect its enzymatic activity, but led to the appearance of the phosphorylated enzyme at the endoplasmic reticulum (ER), close to the ER calcium pump, a location known for muscle-type cytosolic creatine kinase (CK) to support Ca2+-pumping

Identification of apolipoprotein n-acyltransferase (LNT) in mycobacteria

Lipoproteins of Gram-negative and Gram-positive bacteria carry a thioether bound diacylglyceryl but differ by a fatty acid amide-bound to the alpha-amino group of the universally conserved cysteine. In Escherichia coli the N-terminal acylation is catalyzed by the N-acyltransferase Lnt. Using E. coli Lnt as a query in a BLASTp search, we identified putative lnt genes also in Gram-positive mycobacteria. The Mycobacterium tuberculosis lipoprotein LppX, heterologously expressed in Mycobacterium smegmatis, was N-acylated at the N-terminal cysteine, whereas LppX expressed in a M. smegmatis lnt::aph knock-out mutant was accessible for N-terminal sequencing. Western blot analyses of a truncated and tagged form of LppX indicated a smaller size of about 0.3 kDa in the lnt::aph mutant compared to the parental strain. MALDI-TOF/TOF analyses of a trypsin digest of LppX proved the presence of the diacylglyceryl modification in both strains, parental strain and lnt::aph mutant. N-acylation was found exclusively in the M. smegmatis parental strain. Complementation of the lnt::aph mutant with M. tuberculosis ppm1 restored N-acylation. The substrate for N-acylation is a C16 fatty acid while the two fatty acids of the diacylglyceryl residue were identified as C16 and C19:0 fatty acid, the latter most likely tuberculostearic acid. We demonstrate that mycobacterial lipoproteins are triacylated. For the first time to our knowledge, we identify Lnt activity in Gram-positive bacteria and assigned the responsible genes. In M. smegmatis and M. tuberculosis the open reading frames are annotated as MSMEG_3860 and M. tuberculosis ppm1, respectively

PKA phosphorylates and inactivates AMPKalpha to promote efficient lipolysis

The mobilization of metabolic energy from adipocytes depends on a tightly regulated balance between hydrolysis and resynthesis of triacylglycerides (TAGs). Hydrolysis is stimulated by beta-adrenergic signalling to PKA that mediates phosphorylation of lipolytic enzymes, including hormone-sensitive lipase (HSL). TAG resynthesis is associated with high-energy consumption, which when inordinate, leads to increased AMPK activity that acts to restrain hydrolysis of TAGs by inhibiting PKA-mediated activation of HSL. Here, we report that in primary mouse adipocytes, PKA associates with and phosphorylates AMPKalpha1 at Ser-173 to impede threonine (Thr-172) phosphorylation and thus activation of AMPKalpha1 by LKB1 in response to lipolytic signals. Activation of AMPKalpha1 by LKB1 is also blocked by PKA-mediated phosphorylation of AMPKalpha1 in vitro. Functional analysis of an AMPKalpha1 species carrying a non-phosphorylatable mutation at Ser-173 revealed a critical function of this phosphorylation for efficient release of free fatty acids and glycerol in response to PKA-activating signals. These results suggest a new mechanism of negative regulation of AMPK activity by PKA that is important for converting a lipolytic signal into an effective lipolytic response

AMP-activated protein kinase regulates the vacuolar H+-ATPase via direct phosphorylation of the A subunit (ATP6V1A) in the kidney

The vacuolar H(+)-ATPase (V-ATPase) in intercalated cells contributes to luminal acidification in the kidney collecting duct and nonvolatile acid excretion. We previously showed that the A subunit in the cytoplasmic V1 sector of the V-ATPase (ATP6V1A) is phosphorylated by the metabolic sensor AMP-activated protein kinase (AMPK) in vitro and in kidney cells. Here, we demonstrate that treatment of rabbit isolated, perfused collecting ducts with the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) inhibited V-ATPase-dependent H(+) secretion from intercalated cells after an acid load. We have identified by mass spectrometry that Ser-384 is a major AMPK phosphorylation site in the V-ATPase A subunit, a result confirmed by comparing AMPK-dependent phosphate labeling of wild-type A-subunit (WT-A) with that of a Ser-384-to-Ala A subunit mutant (S384A-A) in vitro and in intact HEK-293 cells. Compared with WT-A-expressing HEK-293 cells, S384A-A-expressing cells exhibited greater steady-state acidification of HCO3(-)-containing media. Moreover, AICAR treatment of clone C rabbit intercalated cells expressing the WT-A subunit reduced V-ATPase-dependent extracellular acidification, an effect that was blocked in cells expressing the phosphorylation-deficient S384A-A mutant. Finally, expression of the S384A-A mutant prevented cytoplasmic redistribution of the V-ATPase by AICAR in clone C cells. In summary, direct phosphorylation of the A subunit at Ser-384 by AMPK represents a novel regulatory mechanism of the V-ATPase in kidney intercalated cells. Regulation of the V-ATPase by AMPK may couple V-ATPase activity to cellular metabolic status with potential relevance to ischemic injury in the kidney and other tissues

A two-dimensional screen for AMPK substrates identifies tumor suppressor fumarate hydratase as a preferential AMPKα2 substrate

AMP-activated protein kinase (AMPK) is emerging as a central cellular signaling hub involved in energy homeostasis and proliferation. The kinase is considered as a suitable target for pharmacological intervention in several energy-related pathologies like diabetes type II and cancer, although its signaling network is still incompletely understood. Here we apply an original two-dimensional in vitro screening approach for AMPK substrates that combines biophysical interaction based on surface plasmon resonance with in vitro phosphorylation. By enriching for proteins that interact with a specific AMPK isoform, we aimed to identify substrates that are also preferentially phosphorylated by this specific AMPK isoform. Application of this screen to full-length AMPK α2β2γ1 and soluble rat liver proteins identified the tumor suppressor fumarate hydratase (FH). FH was confirmed to interact with and to be preferentially phosphorylated by the AMPKα2 isoform by using yeast-two-hybrid and in vitro phosphorylation assays. AMPK-mediated phosphorylation of FH significantly increased enzyme activity in vitro and in vivo, suggesting that it is a bona fide AMPK substrate. In vivo, AMPKα2 is supposed to target the cytosolic/nuclear pools of FH, whose tumor suppressor function relies on DNA damage repair and inhibition of HIF-1α-signaling

PKA Regulates Vacuolar H+-ATPase Localization and Activity via Direct Phosphorylation of the A Subunit in Kidney Cells*

The vacuolar H+-ATPase (V-ATPase) is a major contributor to luminal acidification in epithelia of Wolffian duct origin. In both kidney-intercalated cells and epididymal clear cells, cAMP induces V-ATPase apical membrane accumulation, which is linked to proton secretion. We have shown previously that the A subunit in the cytoplasmic V1 sector of the V-ATPase is phosphorylated by protein kinase A (PKA). Here we have identified by mass spectrometry and mutagenesis that Ser-175 is the major PKA phosphorylation site in the A subunit. Overexpression in HEK-293T cells of either a wild-type (WT) or phosphomimic Ser-175 to Asp (S175D) A subunit mutant caused increased acidification of HCO3−-containing culture medium compared with cells expressing vector alone or a PKA phosphorylation-deficient Ser-175 to Ala (S175A) mutant. Moreover, localization of the S175A A subunit mutant expressed in HEK-293T cells was more diffusely cytosolic than that of WT or S175D A subunit. Acute V-ATPase-mediated, bafilomycin-sensitive H+ secretion was up-regulated by a specific PKA activator in HEK-293T cells expressing WT A subunit in HCO3−-free buffer. In cells expressing the S175D mutant, V-ATPase activity at the membrane was constitutively up-regulated and unresponsive to PKA activators, whereas cells expressing the S175A mutant had decreased V-ATPase activity that was unresponsive to PKA activation. Finally, Ser-175 was necessary for PKA-stimulated apical accumulation of the V-ATPase in a polarized rabbit cell line of collecting duct A-type intercalated cell characteristics (Clone C). In summary, these results indicate a novel mechanism for the regulation of V-ATPase localization and activity in kidney cells via direct PKA-dependent phosphorylation of the A subunit at Ser-175