Functional genomic, biochemical, and genetic characterization of the Salmonella pduO gene, an ATP:cob(I)alamin adenosyltransferase gene

Salmonella enterica degrades 1,2-propanediol by a pathway dependent on coenzyme B12 (adenosylcobalamin [AdoCb1]). Previous studies showed that 1,2-propanediol utilization (pdu) genes include those for the conversion of inactive cobalamins, such as vitamin B12, to AdoCbl. However, the specific genes involved were not identified. Here we show that the pduO gene encodes a protein with ATP:cob(I)alamin adenosyltransferase activity. The main role of this protein is apparently the conversion of inactive cobalamins to AdoCbl for 1,2-propanediol degradation. Genetic tests showed that the function of the pduO gene was partially replaced by the cobA gene (a known ATP:corrinoid adenosyltransferase) but that optimal growth of S. enterica on 1,2-propanediol required a functional pduO gene. Growth studies showed that cobA pduO double mutants were unable to grow on 1,2-propanediol minimal medium supplemented with vitamin B(12) but were capable of growth on similar medium supplemented with AdoCbl. The pduO gene was cloned into a T7 expression vector. The PduO protein was overexpressed, partially purified, and, using an improved assay procedure, shown to have cob(I)alamin adenosyltransferase activity. Analysis of the genomic context of genes encoding PduO and related proteins indicated that particular adenosyltransferases tend to be specialized for particular AdoCbl-dependent enzymes or for the de novo synthesis of AdoCbl. Such analyses also indicated that PduO is a bifunctional enzyme. The possibility that genes of unknown function proximal to adenosyltransferase homologues represent previously unidentified AdoCbl-dependent enzymes is discussed.     

Cohesin-dockerin interactions of cellulosomal subunits of Clostridium cellulovorans

The cellulosome of Clostridium cellulovorans consists of three major subunits: CbpA, EngE, and ExgS. The C. cellulovorans scaffolding protein (CbpA) contains nine hydrophobic repeated domains (cohesins) for the binding of enzymatic subunits. Cohesin domains are quite homologous, but there are some questions regarding their binding specificity because some of the domains have regions of low-level sequence similarity. Two cohesins which exhibit 60% sequence similarity were investigated for their ability to bind cellulosomal enzymes. Cohesin 1 (Coh1) was found to contain amino acid residues corresponding to amino acids 312 to 453 of CbpA, which contains a total of 1,848 amino acid residues. Coh6 was determined to contain amino acid residues corresponding to residues 1113 to 1254 of CbpA. By genetic construction, these two cohesins were each fused to MalE, producing MalE-Coh1 and MalE-Coh6. The abilities of two fusion proteins to bind to EngE, ExgS, and CbpA were compared. Although MalE-Coh6 could bind EngE and ExgS, little or no binding of the enzymatic subunits was observed with MalE-Coh1. Significantly, the abilities of the two fusion proteins to bind CbpA were similar. The binding of dockerin-containing enzymes to cohesin-containing proteins was suggested as a model for assembly of cellulosomes. In our examination of the role of dockerins, it was also shown that the binding of endoglucanase B (EngB) to CbpA was dependent on the presence of EngB's dockerin. These results suggest that different cohesins may function with differing efficiency and specificity, that cohesins may play some role in the formation of polycellulosomes through Coh-CbpA interactions, and that dockerins play an important role during the interaction of cellulosomal enzymes and cohesins present in CbpA.     

Genetic and structural organization of the aminophenol catabolic operon and its implication for evolutionary process

The aminophenol (AP) catabolic operon in Pseudomonas putida HS12 mineralizing nitrobenzene was found to contain all the enzymes responsible for the conversion of AP to pyruvate and acetyl coenzyme A via extradiol meta cleavage of 2-aminophenol. The sequence and functional analyses of the corresponding genes of the operon revealed that the AP catabolic operon consists of one regulatory gene, nbzR, and the following nine structural genes, nbzJCaCbDGFEIH, which encode catabolic enzymes. The NbzR protein, which is divergently transcribed with respect to the structural genes, possesses a leucine zipper motif and a MarR homologous domain. It was also found that NbzR functions as a repressor for the AP catabolic operon through binding to the promoter region of the gene cluster in its dimeric form. A comparative study of the AP catabolic operon with other meta cleavage operons led us to suggest that the regulatory unit (nbzR) was derived from the MarR family and that the structural unit (nbzJCaCbDGFEIH) has evolved from the ancestral meta cleavage gene cluster. It is also proposed that these two functional units assembled through a modular type gene transfer and then have evolved divergently to acquire specialized substrate specificities (NbzCaCb and NbzD) and catalytic function (NbzE), resulting in the creation of the AP catabolic operon. The evolutionary process of the AP operon suggests how bacteria have efficiently acquired genetic diversity and expanded their metabolic capabilities by modular type gene transfer.     

Identification of yacE (coaE) as the structural gene for dephosphocoenzyme A kinase in Escherichia coli K-12

Dephosphocoenzyme A (dephospho-CoA) kinase catalyzes the final step in coenzyme A biosynthesis, the phosphorylation of the 3'-hydroxy group of the ribose sugar moiety. Wild-type dephospho-CoA kinase from Corynebacterium ammoniagenes was purified to homogeneity and subjected to N-terminal sequence analysis. A BLAST search identified a gene from Escherichia coli previously designated yacE encoding a highly homologous protein. Amplification of the gene and overexpression yielded recombinant dephospho-CoA kinase as a 22.6-kDa monomer. Enzyme assay and nuclear magnetic resonance analyses of the product demonstrated that the recombinant enzyme is indeed dephospho-CoA kinase. The activities with adenosine, AMP, and adenosine phosphosulfate were 4 to 8% of the activity with dephospho-CoA. Homologues of the E. coli dephospho-CoA kinase were identified in a diverse range of organisms.     

Phosphatidylinositol 4-phosphate 5-kinase type I is regulated through phosphorylation response by extracellular stimuli

Phosphatidylinositol 4-phosphate 5-kinase (PIPK) catalyzes a final step in the synthesis of phosphatidylinositol 4,5-bisphosphate (PIP(2)), a lipid signaling molecule. Strict regulation of PIPK activity is thought to be essential in intact cells. Here we show that type I enzymes of PIPK (PIPKI) are phosphorylated by cyclic AMP-dependent protein kinase (PKA), and phosphorylation of PIPKI suppresses its activity. Serine 214 was found to be a major phosphorylation site of PIPK type Ialpha (PIPKIalpha) that is catalyzed by PKA. In contrast, lysophosphatidic acid-induced protein kinase C activation increased PIPKIalpha activity. Activation of PIPKIalpha was induced by dephosphorylation, which was catalyzed by an okadaic acid-sensitive phosphatase, protein phosphatase 1 (PP1). In vitro dephosphorylation of PIPKIalpha with PP1 increased PIPK activity, indicating that PP1 plays a role in lysophosphatidic acid-induced dephosphorylation of PIPKIalpha. These results strongly suggest that activity of PIPKIalpha in NIH 3T3 cells is regulated by the reversible balance between PKA-dependent phosphorylation and PP1-dependent dephosphorylation.     

FLASH coordinates NF-kappa B activity via TRAF2

FLASH is a protein recently shown to interact with the death effector domain of caspase-8 and is likely to be a component of the death-inducing signaling complex in receptor-mediated apoptosis. Here we show that antisense oligonucleotide-induced inhibition of FLASH expression abolished TNF-alpha-induced activation of NF-kappaB in HEK293 cells, as determined by luciferase reporter gene expression driven by a NF-kappaB responsive promoter. Conversely, overexpression of FLASH dose-dependently activated NF-kappaB, an effect suppressed by dominant negative mutants of TRAF2, NIK, and IKKalpha, and partially by those of TRAF5 and TRAF6. TRAF2 was co-immunoprecipitated with FLASH from the cell extracts of HEK293 cells or HeLa cells stably expressing exogenous FLASH (HeLa/HA-FLASH). Furthermore, serial deletion mapping demonstrated that a domain spanning the residues 856-1191 of FLASH activated NF-kappaB as efficiently as the full-length and could directly bind to TRAF2 in vitro and in the transfected cells. Taken together, these results suggest that FLASH coordinates downstream NF-kappaB activity via a TRAF2-dependent pathway in the TNF-alpha signaling.     

Prevention of kidney ischemia/reperfusion-induced functional injury and JNK, p38, and MAPK kinase activation by remote ischemic pretreatment

MAPK activities, including JNK, p38, and ERK, are markedly enhanced after ischemia in vivo and chemical anoxia in vitro. The relative extent of JNK, p38, or ERK activation has been proposed to determine cell fate after injury. A mouse model was established in which prior exposure to ischemia protected against a second ischemic insult imposed 8 or 15 days later. In contrast to what was observed after 30 min of bilateral ischemia, when a second period of ischemia of 30- or 35-min duration was imposed 8 days later, there was no subsequent increase in plasma creatinine, decrease in glomerular filtration rate, or increase in fractional excretion of sodium. A shorter period of prior ischemia (15 min) was partially protective against subsequent ischemic injury 8 days later. Unilateral ischemia was also protective against a subsequent ischemic insult to the same kidney, revealing that systemic uremia is not necessary for protection. The ischemia-related activation of JNK and p38 and outer medullary vascular congestion were markedly mitigated by prior exposure to ischemia, whereas preconditioning had no effect on post-ischemic activation of ERK1/2. The phosphorylation of MKK7, MKK4, and MKK3/6, upstream activators of JNK and p38, was markedly reduced by ischemic preconditioning, whereas the post-ischemic phosphorylation of MEK1/2, the upstream activator of ERK1/2, was unaffected by preconditioning. Pre- and post-ischemic HSP-25 levels were much higher in the preconditioned kidney. In summary, post-ischemic JNK and p38 (but not ERK1/2) activation was markedly reduced in a model of kidney ischemic preconditioning that was established in the mouse. The reduction in JNK and p38 activation can be accounted for by reduced activation of upstream MAPK kinases. The post-ischemic activation patterns of MAPKs may explain the remarkable protection against ischemic injury observed in this model.     

Interaction of HRC (histidine-rich Ca(2+)-binding protein) and triadin in the lumen of sarcoplasmic reticulum

HRC (histidine-rich Ca(2+) binding protein) has been identified from skeletal and cardiac muscle and shown to bind Ca(2+) with high capacity and low affinity. While HRC resides in the lumen of the sarcoplasmic reticulum, the physiological function of HRC is largely unknown. In the present study, we have performed co-immunoprecipitation experiments and show that HRC binds directly to triadin, which is an integral membrane protein of the sarcoplasmic reticulum. Using a fusion protein binding assay, we further identified the histidine-rich acidic repeats of HRC as responsible for the binding of HRC to triadin. These motifs may represent a novel protein-protein interaction domain. The HRC binding domain of triadin was also localized by fusion protein binding assay to the lumenal region containing the KEKE motif that was previously shown to be involved in the binding of triadin to calsequestrin. Notably, the interaction of HRC and triadin is Ca(2+)-sensitive. Our data suggest that HRC may play a role in the regulation of Ca(2+) release from the sarcoplasmic reticulum by interaction with triadin.     

Actin directly interacts with phospholipase D, inhibiting its activity

Mammalian phospholipase D (PLD) plays a key role in several signal transduction pathways and is involved in many diverse functions. To elucidate the complex molecular regulation of PLD, we investigated PLD-binding proteins obtained from rat brain extract. Here we report that a 43-kDa protein in the rat brain, beta-actin, acts as a major PLD2 direct-binding protein as revealed by peptide mass fingerprinting in combination with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. We also determined that the region between amino acids 613 and 723 of PLD2 is required for the direct binding of beta-actin, using bacterially expressed glutathione S-transferase fusion proteins of PLD2 fragments. Intriguingly, purified beta-actin potently inhibited both phosphatidylinositol-4,5-bisphosphate- and oleate-dependent PLD2 activities in a concentration-dependent manner (IC50 = 5 nm). In a previous paper, we reported that alpha-actinin inhibited PLD2 activity in an interaction-dependent and an ADP-ribosylation factor 1 (ARF1)-reversible manner (Park, J. B., Kim, J. H., Kim, Y., Ha, S. H., Kim, J. H., Yoo, J.-S., Du, G., Frohman, M. A., Suh, P.-G., and Ryu, S. H. (2000) J. Biol. Chem. 275, 21295-21301). In vitro binding analyses showed that beta-actin could displace alpha-actinin binding to PLD2, demonstrating independent interaction between cytoskeletal proteins and PLD2. Furthermore, ARF1 could steer the PLD2 activity in a positive direction regardless of the inhibitory effect of beta-actin on PLD2. We also observed that beta-actin regulates PLD1 and PLD2 with similar binding and inhibitory potencies. Immunocytochemical and co-immunoprecipitation studies demonstrated the in vivo interaction between the two PLD isozymes and actin in cells. Taken together, these results suggest that the regulation of PLD by cytoskeletal proteins, beta-actin and alpha-actinin, and ARF1 may play an important role in cytoskeleton-related PLD functions.