Moonlighting protein in Starkeyomyces koorchalomoides: Characterization of dihydrolipoamide dehydrogenase as a protein acetyltransferase utilizing acetoxycoumarin as the acetyl group donor

In this report we have identified for the first time a transacetylase (TAase) in a mesophilic fungi Starkeyomyces koorchalomoides catalyzing the transfer of acetyl group from polyphenolic acetate (PA) to a receptor protein glutathione S-transferase (GST). An elegant assay procedure was established for TAase based on its ability to mediate inhibition of GST by 7,8-diacetoxy-4-methylcoumarin (DAMC), a model PA. Utilizing this assay procedure, S. koorchalomoides TAase was purified to homogeneity. TAase was found to have MW of 50 kDa. The purified enzyme exhibited maximum activity at 45 °C at pH 6.8. The N-terminal sequence of purified fungal TAase (ANDASTVED) showed identity with corresponding N-terminal sequence of dihydrolipoamide dehydrogenase (LADH), a mitochondrial matrix enzyme and an E3 component of pyruvate dehydrogenase complex (PDHC). TAase was found to have all the properties of LADH and avidly interacted with the anti-LADH antibody. TAase catalyzed acetylation of GST by DAMC was identified by LC–MS/MS and a single lysine residue (Lys-113) was found to be acetylated. Further, recombinant LADH from Streptococcus pneumoniae lacking lipoyl domain was found to exhibit little TAase activity, suggesting the role of lipoyl domain in the TAase activity of LADH. These observations bear evidence for the protein acetyltransferase activity of LADH. Such an activity of LADH can be attributed as a moonlighting function of the enzyme.     

Lipases fromRhizomucor miehei andHumicola lanuginosa: Modification of the lid covering the active site alters enantioselectivity

The homologous lipases fromRhizomucor miehei andHumicola lanuginosa showed approximately the same enantioselectivity when 2-methyldecanoic acid esters were used as substrates. Both lipases preferentially hydrolyzed theS-enantiomer of 1-heptyl 2-methyldecanoate (R. miehei:E _S =8.5;H. lanuginosa:E _S =10.5), but theR-enantiomer of phenyl 2-methyldecanoate (E _R =2.9). Chemical arginine specific modification of theR. miehei lipase with 1,2-cyclohexanedione resulted in a decreased enantioselectivity (E _R =2.0), only when the phenyl ester was used as a substrate. In contrast, treatment with phenylglyoxal showed a decreased enantioselectivity (E _S =2.5) only when the heptyl ester was used as a substrate. The presence of guanidine, an arginine side chain analog, decreased the enantioselectivity with the heptyl ester (E _S =1.9) and increased the enantioselectivity with the aromatic ester (E _R =4.4) as substrates. The mutation, Glu 87 Ala, in the lid of theH. lanuginosa lipase, which might decrease the electrostatic stabilization of the open-lid conformation of the lipase, resulted in 47% activity compared to the native lipase, in a tributyrin assay. The Glu 87 Ala mutant showed an increased enantioselectivity with the heptyl ester (E _S =17.4) and a decreased enantioselectivity with the phenyl ester (E _R =2.5) as substrates, compared to native lipase. The enantioselectivities of both lipases in the esterification of 2-methyldecanoic acid with 1-heptanol were unaffected by the lid modifications.     

CSF1R-dependent macrophages control postnatal somatic growth and organ maturation

Homozygous mutation of the Csf1r locus (Csf1rko) in mice, rats and humans leads to multiple postnatal developmental abnormalities. To enable analysis of the mechanisms underlying the phenotypic impacts of Csf1r mutation, we bred a rat Csf1rko allele to the inbred dark agouti (DA) genetic background and to a Csf1r-mApple reporter transgene. The Csf1rko led to almost complete loss of embryonic macrophages and ablation of most adult tissue macrophage populations. We extended previous analysis of the Csf1rko phenotype to early postnatal development to reveal impacts on musculoskeletal development and proliferation and morphogenesis in multiple organs. Expression profiling of 3-week old wild-type (WT) and Csf1rko livers identified 2760 differentially expressed genes associated with the loss of macrophages, severe hypoplasia, delayed hepatocyte maturation, disrupted lipid metabolism and the IGF1/IGF binding protein system. Older Csf1rko rats developed severe hepatic steatosis. Consistent with the developmental delay in the liver Csf1rko rats had greatly-reduced circulating IGF1. Transfer of WT bone marrow (BM) cells at weaning without conditioning repopulated resident macrophages in all organs, including microglia in the brain, and reversed the mutant phenotypes enabling long term survival and fertility. WT BM transfer restored osteoclasts, eliminated osteopetrosis, restored bone marrow cellularity and architecture and reversed granulocytosis and B cell deficiency. Csf1rko rats had an elevated circulating CSF1 concentration which was rapidly reduced to WT levels following BM transfer. However, CD43^hi non-classical monocytes, absent in the Csf1rko, were not rescued and bone marrow progenitors remained unresponsive to CSF1. The results demonstrate that the Csf1rko phenotype is autonomous to BM-derived cells and indicate that BM contains a progenitor of tissue macrophages distinct from hematopoietic stem cells. The model provides a unique system in which to define the pathways of development of resident tissue macrophages and their local and systemic roles in growth and organ maturation.     

Optimization of cyclodextrin glycosyltransferase production from Klebsiella pneumoniae AS-22 in batch, fed-batch, and continuous cultures

Production of a novel cyclodextrin glycosyltransferase (CGTase) from Klebsiella pneumoniae AS-22 strain, which converts starch predominantly to alpha-CD at high conversion yields, in batch, fed-batch, and continuous cultures, is presented. In batch fermentations, optimization of different operating parameters such as temperature, pH, agitation speed, and carbon-source concentration resulted in more than 6-fold increase in CGTase activity. The enzyme production was further improved by two fed-batch approaches. First, using glucose-based feed to increase cell density, followed by starch-based feed to induce enzyme production, resulted in high cell density of 76 g dry cell weight/L, although the CGTase production was low. Using the second approach of a single dextrin-based feed, 20-fold higher CGTase was produced compared to that in batch fermentations with media containing tapioca starch. In continuous operation, more than 8-fold increase in volumetric CGTase productivity was obtained using dextrin-based media compared to that in batch culture using starch-based media.     

Effect of spice essential oils on growth and aflatoxin B1 production by Aspergillus flavus

Aflatoxin B¹ production by Aspergillus flavus was studied in yeast extract sucrose broth in the presence of cinnamon, clove, almond and cardamom oils. Growth and aflatoxin B₁ production was inhibited by 0.5 μl cinnamon oil ml^-1 medium and by 1 μl clove oil ml_-1. Almond and cardamom oils only affected growth when their concentration exceeded 1.25 μl ml^-1 medium. Aflatoxin B₁ production was stimulated by 0.75 and 1 μl almond oil ml^-1 medium or by 0.25 and 0.5 μl cardamom oil ml^-1.     

Synthesis of a carboxyl linker containing Pk trisaccharide

An efficient synthesis of a Pk trisaccharide with a functionalized side arm at the reducing end for conjugation to other molecules is presented. Construction of the Pk trisaccharide with a high alpha-selectivity was achieved in high yield by coupling a reactive galactosyl phosphite donor with a lactosyl acceptor.     

Structural origins of the interfacial activation in Thermomyces (Humicola) lanuginosa lipase

The already known X-ray structures of lipases provide little evidence about initial, discrete structural steps occurring in the first phases of their activation in the presence of lipids (process referred to as interfacial activation). To address this problem, five new Thermomyces (formerly Humicola) lanuginosa lipase (TlL) crystal structures have been solved and compared with four previously reported structures of this enzyme. The bias coming from different crystallization media has been minimized by the growth of all crystals under the same crystallization conditions, in the presence of detergent/lipid analogues, with low or high ionic strength as the only main variable. Resulting structures and their characteristic features allowed the identification of three structurally distinct species of this enzyme: low activity form (LA), activated form (A), and fully Active (FA) form. The isomerization of the Cys268-Cys22 disulfide, synchronized with the formation of a new, short alpha(0) helix and flipping of the Arg84 (Arginine switch) located in the lid's proximal hinge, have been postulated as the key, structural factors of the initial transitions between LA and A forms. The experimental results were supplemented by theoretical calculations. The magnitude of the activation barrier between LA (ground state) and A (end state) forms of TlL (10.6 kcal/mol) is comparable to the enthalpic barriers typical for ring flips and disulfide isomerizations at ambient temperatures. This suggests that the sequence of the structural changes, as exemplified in various TlL crystal structures, mirror those that may occur during interfacial activation.     

Interfacial control of lid opening in Thermomyces lanuginosa lipase

Small unilamelar vesicles of anionic phospholipids (SUV), such as 1-palmitoyl-2-oleoylglycero-sn-3-phosphoglycerol (POPG), provide an interface where Thermomyces lanuginosa triglyceride lipase (TlL) binds and adopts a catalytically active conformation for the hydrolysis of substrate partitioned in the interface, such as tributyrin or p-nitrophenylbutyrate, with an increase in catalytic rate of more than 100-fold for the same concentration of substrate [Berg et al. (1998) Biochemistry 37, 6615-6627.]. This interfacial activation is not seen with large unilamelar vesicles (LUV) of the same composition, or with vesicles of zwitterionic phospholipids such as 1-palmitoyl-2-oleoylglycero-sn-3-phosphocholine (POPC), independently of the vesicle size. Tryptophan fluorescence experiments show that lipase binds to all those types of vesicles with similar affinity, but it adopts different forms that can be correlated with the enzyme catalytic activity. The spectral change on binding to anionic SUV corresponds to the catalytically active, or "open" form of the enzyme, and it is not modified in the presence of substrate partitioned in the vesicles, as demonstrated with inactive mutants. This indicates that the displacement of the lid characteristic of lipase interfacial activation is induced by the anionic phospholipid interface without blocking the accessibility of the active site to the substrate. Experiments with a mutant containing only Trp89 in the lid show that most of the spectral changes on binding to POPG-SUVs take place in the lid region that covers the active site; an increase in Trp anisotropy indicates that the lid becomes less flexible in the active form, and quenching experiments show that it is significantly buried from the aqueous phase. On the other hand, results with a mutant where Trp89 is changed to Leu show that the environment of the structural tryptophans in positions 117, 221, and 260 is somehow altered on binding, although their mobility and solvent accessibility remains the same as in the inactive form in solution. The form of TlL bound to POPC-SUV or -LUV vesicles as well as to LUV vesicles of POPG has the same spectral signatures and corresponds to an inactive or "closed" form of the enzyme. In these interfaces, the lid is highly flexible, and Trp89 remains accessible to solvent. Resonance energy transfer experiments show that the orientation of TlL in the interface is different in the active and inactive forms. A model of interaction consistent with these data and the available X-ray structures is proposed. This is a unique system where the composition and physical properties of the lipid interface control the enzyme activity.     

Characterization of protein transacetylase from human placenta as a signaling molecule calreticulin using polyphenolic peracetates as the acetyl group donors

We have earlier shown that a unique membrane-bound enzyme mediates the transfer of acetyl group(s) from polyphenolic peracetates (PA) to functional proteins, which was termed acetoxy drug: protein transacetylase (TAase) because it acted upon several classes of PA. Here, we report the purification of TAase from human placental microsomes to homogeneity with molecular mass of 60 kDa, exhibiting varying degrees of specificity to several classes of PA confirming the structure-activity relationship for the microsome-bound TAase. The TAase catalyzed protein acetylation by a model acetoxy drug, 7,8-diacetoxy-4-methyl coumarin (DAMC) was established by the demonstration of immunoreactivity of the acetylated target protein with anti-acetyl lysine antibody. TAase activity was severely inhibited in calcium-aggregated microsomes as well as when Ca2+ was added to purified TAase, suggesting that TAase could be a calcium binding protein. Furthermore, the N-terminal sequence analysis of purified TAase (EPAVYFKEQFLD) using Swiss Prot Database perfectly matched with calreticulin (CRT), a major microsomal calcium binding protein of the endoplasmic reticulum (ER). The identity of TAase with CRT was substantiated by the observation that the purified TAase avidly reacted with commercially available antibody raised against the C-terminus of human CRT (13 residues peptide, DEEDATGQAKDEL). Purified TAase also showed Ca2+ binding and acted as a substrate for phosphorylation catalyzed by protein kinase C (PKC), which are hallmark characteristics of CRT. Further, purified placental CRT as well as the commercially procured pure CRT yielded significant TAase catalytic activity and were also found effective in mediating the acetylation of the target protein NADPH cytochrome P-450 reductase by DAMC as detected by Western blot using anti-acetyl lysine antibody. These observations for the first time convincingly attribute the transacetylase function to CRT. Hence, this transacetylase function of CRT is designated calreticulin transacetylase (CRTAase). We envisage that CRTAase plays an important role in protein modification by way of acetylation independent of Acetyl CoA.