Lactose metabolism by Staphylococcus aureus: characterization of lacABCD, the structural genes of the tagatose 6-phosphate pathway.

The nucleotide and deduced amino acid sequences of the lacA and lacB genes of the Staphylococcus aureus lactose operon (lacABCDFEG) are presented. The primary translation products are polypeptides of 142 (Mr = 15,425) and 171 (Mr = 18,953) amino acids, respectively. The lacABCD loci were shown to encode enzymes of the tagatose 6-phosphate pathway through both in vitro studies and complementation analysis in Escherichia coli. A serum aldolase assay, modified to allow detection of the tagatose 6-phosphate pathway enzymes utilizing galactose 6-phosphate or fructose phosphate analogs as substrate, is described. Expression of both lacA and lacB was required for galactose 6-phosphate isomerase activity. LacC (34 kDa) demonstrated tagatose 6-phosphate kinase activity and was found to share significant homology with LacC from Lactococcus lactis and with both the minor 6-phosphofructokinase (PfkB) and 1-phosphofructokinase (FruK) from E. coli. Detection of tagatose 1,6-bisphosphate aldolase activity was dependent on expression of the 36-kDa protein specified by lacD. The LacD protein is highly homologous with LacD of L. lactis. Thus, the lacABCD genes comprise the tagatose 6-phosphate pathway and are cotranscribed with genes lacFEG, which specify proteins for transport and cleavage of lactose in S. aureus.     

Crystal structures of LacD from Staphylococcus aureus and LacD.1 from Streptococcus pyogenes: insights into substrate specificity and virulence gene regulation

Staphylococcus aureus LacD, a Class I tagatose-1,6-bisphosphate (TBP) aldolase, shows broadened substrate specificity by catalyzing the cleavage of 1,6-bisphosphate derivatives of d-tagatose, d-fructose, d-sorbose, and d-psicose. LacD.1 and LacD.2 are two closely-related Class I TBP aldolases in Streptococcus pyogenes. Here we have determined the crystal structures of S. aureus LacD and S. pyogenes LacD.1. Monomers of both enzymes are folded into a (β/α)₈ barrel and two monomers associate tightly to form a dimer in the crystals. The structures suggest that the residues E189 and S300 of rabbit muscle Class I fructose-1,6-bisphosphate (FBP) aldolase are important for substrate specificity. When we mutated the corresponding residues of S. aureus LacD, the mutants (L165E, L275S, and L165E/L275S) showed enhanced substrate specificity toward FBP.

Structured summary

lacDbinds to lacD by X-ray crystallography(View interaction)lacD1binds to lacD1 by X-ray crystallography(View interaction)     

High-yield production of pure tagatose from fructose by a three-step enzymatic cascade reaction

Objective

To produce tagatose from fructose with a high conversion rate and to establish a high-yield purification method of tagatose from the reaction mixture.

Results

Fructose at 1 M (180 g l^?1) was converted to 0.8 M (144 g l^?1) tagatose by a three-step enzymatic cascade reaction, involving hexokinase, plus ATP, fructose-1,6-biphosphate aldolase, phytase, over 16 h with a productivity of 9 g l^?1 h^?1. No byproducts were detected. Tagatose was recrystallized from ethanol to a purity of 99.9% and a yield of 96.3%. Overall, tagatose at 99.9% purity was obtained from fructose with a yield of 77%.

Conclusion

This is the first biotechnological production of tagatose from fructose and the first application of solvent recrystallization for the purification of rare sugars.

     

Structure of a Class I Tagatose-1,6-bisphosphate Aldolase: INVESTIGATION INTO AN APPARENT LOSS OF STEREOSPECIFICITY*

Tagatose-1,6-bisphosphate aldolase from Streptococcus pyogenes is a class I aldolase that exhibits a remarkable lack of chiral discrimination with respect to the configuration of hydroxyl groups at both C3 and C4 positions. The enzyme catalyzes the reversible cleavage of four diastereoisomers (fructose 1,6-bisphosphate (FBP), psicose 1,6-bisphosphate, sorbose 1,6-bisphosphate, and tagatose 1,6-bisphosphate) to dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde 3-phosphate with high catalytic efficiency. To investigate its enzymatic mechanism, high resolution crystal structures were determined of both native enzyme and native enzyme in complex with dihydroxyacetone-P. The electron density map revealed a (α/β)₈ fold in each dimeric subunit. Flash-cooled crystals of native enzyme soaked with dihydroxyacetone phosphate trapped a covalent intermediate with carbanionic character at Lys²⁰⁵, different from the enamine mesomer bound in stereospecific class I FBP aldolase. Structural analysis indicates extensive active site conservation with respect to class I FBP aldolases, including conserved conformational responses to DHAP binding and conserved stereospecific proton transfer at the DHAP C3 carbon mediated by a proximal water molecule. Exchange reactions with tritiated water and tritium-labeled DHAP at C3 hydrogen were carried out in both solution and crystalline state to assess stereochemical control at C3. The kinetic studies show labeling at both pro-R and pro-S C3 positions of DHAP yet detritiation only at the C3 pro-S-labeled position. Detritiation of the C3 pro-R label was not detected and is consistent with preferential cis-trans isomerism about the C2–C3 bond in the carbanion as the mechanism responsible for C3 epimerization in tagatose-1,6-bisphosphate aldolase.     

Purification and characterization of l-allo-threonine aldolase from Aeromonas jandaei DK-39

l-allo-Threonine aldolase (l-allo-threonine acetaldehyde-lyase), which exhibited specificity for l-allo-threonine but not for l-threonine, was purified from a cell-free extract of Aeromonas jandaei DK-39. The purified enzyme catalyzed the aldol cleavage reaction of l-allo-threonine (K_m=1.45 mM, V_max=45.2 μmol min⁻¹ mg⁻¹). The activity of the enzyme was inhibited by carbonyl reagents, which suggests that pyridoxal-5′-phosphate participates in the enzymatic reaction. The enzyme does not act on either l-serine or l-threonine, and thus it can be distinguished from serine hydroxy-methyltransferase (l-serine:tetrahydrofolate 5,10-hydroxy-methyltransferase, EC 2.1.2.1) or l-threonine aldolase (EC 4.1.2.5).     

Lactose and D0galactose metabolism in Staphylococcus aureus: pathway of D-galactose 6-phosphate degradation

The pathway by which D-galactose 6-phosphate is degraded in Staphylococcus aureus has been elucidated. Galactose 6-phosphate is isomerized to tagatose 6-phosphate, which is phosphorylated with adenosine 5′-triphosphate, and the resulting tagatose 1,6-diphosphate is cleaved to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. The isomerase, kinase, and aldolase that catalyze these reactions are inducible and are distinct from the corresponding enzymes of glucose 6-phosphate metabolism.     

Investigation of the role of lysine in the subunit contact regions of rabbit muscle aldolase.

Rabbit muscle aldolase (E.C. 4. 1. 2. 13) was guanidinated by reaction with O-methylisourea. Up to 60% of the lysine residues can be guanidinated without any dissociation of the tetramer but with a complete loss of enzymatic activity. Native and guanidinated aldolase can be dissociated into monomers in 2.4 m MgCl₂ with only slight change in conformation of the subunit. Nitrotroponylation of guanidinated aldolase in dilute buffer gives no reaction whereas in 2.4 m MgCl₂ nitrotroponlylation modifies another 8–12% of the lysine residues. Removal of MgCl₂ by dialysis affords 100% recovery of activity and tetrameric structure for native aldolase and 100% recovery of tetrameric structure for guanidinated aldolase. In contrast nitrotroponylated and guanidinated aldolase remains monomeric before precipitating as the MgCl₂ concentration is lowered. It is concluded that lysine may be involved in the protein-protein interaction of the subunit contact domains of muscle aldolase.     

Streptococcus mutans serotype c tagatose 6-phosphate pathway gene cluster.

DNA cloned into Escherichia coli K-12 from a serotype c strain of Streptococcus mutans encodes three enzyme activities for galactose utilization via the tagatose 6-phosphate pathway: galactose 6-phosphate isomerase, tagatose 6-phosphate kinase, and tagatose-1,6-bisphosphate aldolase. The genes coding for the tagatose 6-phosphate pathway were located on a 3.28-kb HindIII DNA fragment. Analysis of the tagatose proteins expressed by recombinant plasmids in minicells was used to determine the sizes of the various gene products. Mutagenesis of these plasmids with transposon Tn5 was used to determine the order of the tagatose genes. Tagatose 6-phosphate isomerase appears to be composed of 14- and 19-kDa subunits. The sizes of the kinase and aldolase were found to be 34 and 36 kDa, respectively. These values correspond to those reported previously for the tagatose pathway enzymes in Staphylococcus aureus and Lactococcus lactis.     

Identification of Fructose-1,6-bisphosphate aldolase cytosolic class I as an NMH7 MADS domain associated protein

We are interested in identifying proteins that interact with the MADS domain protein NMH7 of Medicago sativa. We use an affinity column with a synthetic peptide derived from the MADS domain of NMH7 which has been reported to mediate protein-protein interaction with non-MADS domain interacting proteins. We identified ∼40 and ∼80 kDa specifically bound proteins as the monomeric and dimeric forms of Fructose-1,6-bisphosphate aldolase cytosolic class I. NiNTA pull down assays revealed that K- and C-terminus regions of NMH7 are not required for the interaction with aldolase. Aldolase enzymatic activity is not required for the interaction with NMH7. NMH7 and aldolase were coimmunoprecipitated from non-inoculated seed and seedlings extracts. Colocalization studies using confocal microscopy showed that aldolase and NMH7 are localized in the cytoplasm and the nucleus of the cortical cells. These data together show that M. sativa aldolase is a novel MADS domain binding protein, and suggest a broader functional repertory for this enzyme, as has been proposed for other glycolytic enzymes.     

Horizontal Gene Transfer and Gene Duplication of β-Fructofuranosidase Confer Lepidopteran Insects Metabolic Benefits

Horizontal gene transfer (HGT) is a potentially critical source of material for ecological adaptation and the evolution of novel genetic traits. However, reports on posttransfer duplication in organism genomes are lacking, and the evolutionary advantages conferred on the recipient are generally poorly understood. Sucrase plays an important role in insect physiological growth and development. Here, we performed a comprehensive analysis of the evolution of insect β-fructofuranosidase transferred from bacteria via HGT. We found that posttransfer duplications of β-fructofuranosidase were widespread in Lepidoptera and sporadic occurrences of β-fructofuranosidase were found in Coleoptera and Hymenoptera. β-fructofuranosidase genes often undergo modifications, such as gene duplication, differential gene loss, and changes in mutation rates. Lepidopteran β-fructofuranosidase gene (SUC) clusters showed marked divergence in gene expression patterns and enzymatic properties in Bombyx mori (moth) and Papilio xuthus (butterfly). We generated SUC1 mutations in B. mori using CRISPR/Cas9 to thoroughly examine the physiological function of SUC. BmSUC1 mutant larvae were viable but displayed delayed growth and reduced sucrase activities that included susceptibility to the sugar mimic alkaloid found in high concentrations in mulberry. BmSUC1 served as a critical sucrase and supported metabolic homeostasis in the larval midgut and silk gland, suggesting that gene transfer of β-fructofuranosidase enhanced the digestive and metabolic adaptation of lepidopteran insects. These findings highlight not only the universal function of β-fructofuranosidase with a link to the maintenance of carbohydrate metabolism but also an underexplored function in the silk gland. This study expands our knowledge of posttransfer duplication and subsequent functional diversification in the adaptive evolution and lineage-specific adaptation of organisms.     

Gene duplication and deletion,not horizontal transfer,drove intra-species mosaicism of Bartonella henselae

Bartonella henselae is a facultative intracellular pathogen that occurs worldwide and is responsible primarily for cat-scratch disease in young people and bacillary angiomatosis in immunocompromised patients. The principal source of genome-level diversity that contributes to B. henselae's host-adaptive features is thought to be horizontal gene transfer events. However, our analyses did not reveal the acquisition of horizontally-transferred islands in B. henselae after its divergence from other Bartonella. Rather, diversity in gene content and genome size was apparently acquired through two alternative mechanisms, including deletion and, more predominantly, duplication of genes. Interestingly, a majority of these events occurred in regions that were horizontally transferred long before B. henselae's divergence from other Bartonella species. Our study indicates the possibility that gene duplication, in response to positive selection pressures in specific clones of B. henselae, might be linked to the pathogen's adaptation to arthropod vectors, the cat reservoir, or humans as incidental host-species.     

Identification of Stringent Response-Related and Potential Serological Proteins Released from Bacillus anthracis Overexpressing the RelA/SpoT Homolog,Rsh Bant

RelA and SpoT synthesize ppGpp, a key effector molecule that facilitates the adaptation of bacteria to nutrient starvation and other stresses, known as the stringent response. To investigate the role of Rsh _Bant , a putative RelA/SpoT homolog (encoded by BAS4302) in Bacillus anthracis, we examined the alteration of the secretome profiles after the overexpression of a functional His-Rsh _Bant protein in the B. anthracis strain Sterne at the stationary growth phase. In the ppGpp-deficient E. coli mutant strain CF1693, overexpression of Rsh _Bant restored a ppGpp-dependent growth defect on minimal glucose media. The secretome profiles obtained using a two-dimensional electrophoresis (2-DE) analysis were altered by overexpression of Rsh _Bant in B. anthracis. Among the 66 protein spots differentially expressed >1.5-fold, the 29 proteins were abundant for further identification using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Functional categorization of those proteins implicated their involvement in various biological activities. Taken together, our results imply that overexpression of a functional His-Rsh _Bant can lead to the increased levels of intracellular ppGpp in B. anthracis, resulting in the significant changes in its secretome profiling. The stringent response-controlled proteins identified are likely useful as potential targets for serodiagnostic applications.     

Chaperone-like activity of the acute-phase component human serum α1-acid glycoprotein: Inhibition of thermal- and chemical-induced aggregation of various proteins

In vitro chaperone-like activity of the acute-phase component and plasma drug transporter human α₁-acid glycoprotein (AAG) has been shown for the first time. AAG suppressed thermal aggregation of a variety of unrelated enzymatic (e.g., aldolase, catalase, enolase, carbonic anhydrase) and non-enzymatic proteins (β-lactoglobulin, ovotransferrin) and it also prevented dithiothreitol induced aggregation of insulin. The anti-aggregation ability of AAG was abolished/reduced upon drug binding suggesting that protein–protein interactions established between the lipocalin β-barrel fold of AAG and hydrophobic surfaces of the stressed proteins are involved in the chaperone-like activity. The results shed some light on the possible biological function of this enigmatic protein and suggest that besides haptoglobin, clusterin, fibrinogen and α₂-macroglobulin AAG can be considered as a novel member of the extracellular molecular chaperones found in human body fluids.     

Sensitivity changes of photoreceptor cells of Hirudo medicinalis caused by changes in extracellular calcium concentration

Extracellular recordings from the vacoule of photoreceptor cells of Hirudo medicinalis L. were performed using microelectrodes. The cells were adapted by white light flashes given at constant intervals (20 s). Response height versus relative intensity curves obtained from the same cell in physiological saline (PS) and in bathing solutions of either a) lowered calcium contents (2 ΜM/1 or less) or b) raised calcium contents (15 mM/1) were compared. The cells' adaptation state in PS was operationally defined by the ratio Q=h _A /h _S where h _A is the response height evoked by the adapting flashes, and h _S is the corresponding saturation response height. Sensitivity changes were measured by the half saturation intensity shift. Lowering extracellular calcium resulted in:

1. The response height increased and the shape of the response became more rounded and prolonged.
2. The total resistance between the vacuole and outside decreased from 8.2±1.4 MΩ (n=6) in PS to 4.6±0.4 MΩ (n=5). The resistance was independent of the cells' adaptation state.
3. A change of the cells' sensitivity occured either in direction to light adaptation or in direction to dark adaptation. It depended functionally on the ratio Q:

a) if Q was less or equal to about 0.6 the cells' sensitivity increased. b) if Q was greater than 0.6 the cells' sensitivity diminished. Raising extracellular calcium decreased the sensitivity of all cells tested independent of their adaptation states in PS. The results can be interpreted under the assumptions that 1. the sensitivity of leech photoreceptor cells is inversely proportional to the intracellular free calcium concentration and Z. intracellular calcium can interact with extracellular calcium in relatively dark adapted cells whereas in relatively light adapted cells the raise of intracellular free calcium is mainly effected by a release from intracellular stores. It is assumed that a Q value of about 0.6 separates relatively light adapted cells from relatively dark adapted cells.     

Creatine kinase and aldolase isoenzyme transitions in cultures of chick skeletal muscle cells

Changes in the isoenzyme patterns and activities of the two enzymes creatine kinase (CPK) and fructose diphosphate aldolase have been followed during the course of differentiation of chick skeletal muscle cells in vitro. The characteristic isoenzyme transitions of both of these enzymes known to occur in developing muscle in situ can be demonstrated in extracts of cultured myogenic cells by cellulose polyacetate electrophoresis followed by specific enzymatic staining: MM-CPK replaces the embryonic BB-CPK, while aldolase isoenzymes containing A subunits replace the C-containing forms which predominate at earlier stages. The specific activities of both enzymes increase during in vitro differentiation. Although the major part of these concomitant changes occurs after myoblast fusion has reached a maximum level, analysis of their timing relative to the process of fusion indicates that the increases in the activities of both enzymes, as well as the accumulation of nuclei within myotubes, proceed exponentially from the beginning of the second day in culture. Fusion and enzyme accumulation are unaffected by addition of dibutyryl cyclic AMP (1 × 10^?4M) to the medium. In calcium-deficient medium, or in media containing 5-bromodeoxyuridine (BrdUrd) at concentrations from 0.2 to 7 × 10^?5M, fusion is almost completely blocked, while cell viability is maintained. The CPK and aldolase isoenzyme transitions fail to occur normally in both fusion-preventing media. This blockage of the normal differentiative changes is, however, less complete in the calcium-deficient cultures, which, in contrast to the BrdUrd containing cultures, contained a number of long bipolar cells thought to be able to differentiate without fusion. These results are interpreted as indicating that for most, but possibly not for all, myogenic cells in typical primary muscle cell cultures, fusion is a prerequisite for the parallel differentiative changes in CPK and aldolase isoenzymes. The possibility is discussed that a “cluster” of proteins, including CPK and aldolase, may be coordinately regulated during myogenesis.     

Inhibition of heat- and chemical-induced aggregation of various proteins reveals chaperone-like activity of the acute-phase component and serine protease inhibitor human α1-antitrypsin

In vitro chaperone-like activity of the serpin family member and plasma acute-phase component human α₁-antitrypsin (AAT) has been shown for the first time. Results of light-scattering experiments demonstrated that AAT efficiently inhibits both heat- and chemical-induced aggregation of various test proteins including alcohol dehydrogenase, aldolase, carbonic anhydrase, catalase, citrate synthase, enolase, glutathione S-transferase, l-lactate dehydrogenase, and β_L-crystallin. The results suggest that the unique metastable serpin architecture enables dual function, protease inhibiton as well as chaperone activity and highlight the serpin superfamily as a possible source of additional intra- and extracellular chaperones (e.g. α₁-antichymotrypsin). The present finding is surprising in the light of the well-known role of mutated forms of AAT and other serpins in the pathogenesis of diseases called serpinopathies that featured with aberrant conformational transitions and consequent self-aggregation of serpin proteins.