Construction and characterization of an Escherichia coli mutant producing Kdo2-lipid A.

3-deoxy-D-manno-oct-2-ulosonic acid (Kdo)2-lipid A is the conserved structure domain of lipopolysaccharide found in most Gram-negative bacteria, and it is believed to stimulate the innate immune system through the TLR4/MD2 complex. Therefore, Kdo2-lipid A is an important stimulator for studying the mechanism of the innate immune system and for developing bacterial vaccine adjuvants. Kdo2-lipid A has not been chemically synthesized to date and could only be isolated from an Escherichia coli mutant strain, WBB06. WBB06 cells grow slowly and have to grow in the presence of tetracycline. In this study, a novel E. coli mutant strain, WJW00, that could synthesize Kdo2-lipid A was constructed by deleting the rfaD gene from the genome of E. coli W3110. The rfaD gene encodes ADP-L-glycero-D-manno-heptose-6-epimerase RfaD. Based on the analysis by SDS-PAGE, thin layer chromatography (TLC) and electrospray ionization mass spectrometry (ESI/MS), WJW00 could produce similar levels of Kdo2-lipid A to WBB06. WJW00 cells grow much better than WBB06 cells and do not need to add any antibiotics during growth. Compared with the wild-type strain, W3110, WJW00 showed increased hydrophobicity, higher cell permeability, greater autoaggregation and decreased biofilm-forming ability. Therefore, WJW00 could be a more suitable strain than WBB06 for producing Kdo2-lipid A and a good base strain for developing lipid A adjuvants.

Conversion shift of D-fructose to D-psicose for enzyme-catalyzed epimerization by addition of borate.

The conversion yield of d-psicose from d-fructose by a d-psicose 3-epimerase from Agrobacterium tumefaciens increased with increasing molar ratios of borate to fructose, up to a ratio of 0.6. The formation of the psicose-borate complex was the result of the higher binding affinity of borate for psicose than for fructose. The formed psicose-borate complex did not participate in the conversion reaction, acting instead as if the product had been removed. Thus, more fructose was converted to psicose in order to restore the equilibrium. The maximum conversion yield of psicose with borate was about twofold that obtained without borate and occurred at a 0.6 molar ratio of borate to fructose. Above this ratio, the conversion yield decreased with increasing ratios, because the amount of fructose available decreased through the formation of the initial fructose-borate complex. The structures of the two sugar-borate complexes, determined by nuclear magnetic resonance spectroscopy, were alpha-d-psicofuranose cis-C-3,4 diol borate and beta-d-fructopyranose cis-C-4,5 diol borate.

Effects of nucleotides on N-acetyl-d-glucosamine 2-epimerases (renin-binding proteins): comparative biochemical studies.

Renin-binding protein (RnBP) is an endogenous renin inhibitor originally isolated from porcine kidney as a complex of renin, so-called high molecular weight (HMW) renin. Our recent studies demonstrated that human RnBP is the enzyme N-acetyl-D-glucosamine (GlcNAc) 2-epimerase [Takahashi, S. et al. (1999) J. Biochem. 125, 348-353]. We have purified recombinant human, rat, and porcine RnBPs expressed in Escherichia coli JM 109 cells. The purified recombinant RnBPs existed as dimers and inhibited porcine renin activity strongly. On the other hand, porcine renin inhibited recombinant GlcNAc 2-epimerase activities. The human GlcNAc 2-epimerase activity could not be detected in the absence of a nucleotide, whereas ATP, dATP, ddATP, ADP, and GTP enhanced the human GlcNAc 2-epimerase activity. Other nucleotides had no effect on human GlcNAc 2-epimerase activity. Rat and porcine GlcNAc 2-epimerases were activated by several nucleotides. Nucleotides that enhance the activity of GlcNAc 2-epimerases protect these enzymes against degradation by thermolysin. These results indicate that mammalian RnBPs have GlcNAc 2-epimerase activity and that nucleotides are essential for formation of the catalytic domain of the enzyme.

Glucosamine 6-phosphate deaminase in normal human erythrocytes.

In the course of an investigation of hexosamine catabolism in the human malaria parasite, Plasmodium falciparum, it became apparent that a basic understanding of the relevant enzymatic reactions in the host erythrocyte is lacking. To acquire the necessary basic knowledge, we have determined the activities of several enzymes involved in hexosamine metabolism in normal human red blood cells. In the present communication we report the results of studies of glucosamine 6-phosphate deaminase (GlcN6-P) using a newly developed sensitive radiometric assay. The mean specific activity in extracts of fresh erythrocytes assayed within 4h of collection was 14.7 nmol/h/mg protein, whereas preparations from older erythrocytes that had been stored at 4 degrees C for up to 4 weeks had a mean specific activity of 6.2 nmol/h/mg. Characterization of the deaminase by chromatofocusing gave a pI of 8.55. The enzyme was optimally active at pH 9.0 and had a Km of 41 microM. The metal chelators EDTA and EGTA were non-inhibitory; however, inhibition was observed in the presence of metal ions, especially Cu2+, Ni2+ and Zn2+. In addition, the deaminase was also inhibited by several sugar phosphates including the reaction product, fructose 6-phosphate.

Catabolite repression of the xyl operon in Bacillus megaterium.

We characterized catabolite repression of the genes encoding xylose utilization in Bacillus megaterium. A transcriptional fusion of xylA encoding xylose isomerase to the spoVG-lacZ indicator gene on a plasmid with a temperature-sensitive origin of replication was constructed and efficiently used for single-copy replacement cloning in the B. megaterium chromosome starting from a single transformant. In the resulting strain, beta-galactosidase expression is 150-fold inducible by xylose and 14-fold repressed by glucose, showing that both regulatory effects occur at the level of transcription. Insertion of a kanamycin resistance gene into xylR encoding the xylose-dependent repressor leads to the loss of xylose-dependent regulation and to a small drop in the efficiency of glucose repression to eightfold. Deletion of 184 bp from the 5' part of the xylA reading frame reduces glucose repression to only twofold. A potential glucose-responsive element in this region is discussed on the basis of sequence similarities to other glucose-repressed genes in Bacillus subtilis. The sequence including the glucose-responsive element is also necessary for repression exerted by the carbon sources fructose and mannitol. Their efficiencies of repression correlate to the growth rate of B. megaterium, as is typical for catabolite repression. Glycerol, ribose, and arabinose exert only a basal twofold repression of the xyl operon, which is independent of the presence of the cis-active glucose-responsive element within the xylA reading frame.

Identification of two cysteine residues forming a pair of vicinal thiols in glucosamine-6-phosphate deaminase from Escherichia coli and a study of their functional role by site-directed mutagenesis.

The nucleotide sequence of the nagB gene in Escherichia coli, encoding glucosamine-6-phosphate deaminase, located four cysteinyl residues at positions 118, 219, 228, and 239. Chemical modification studies performed with the purified enzyme had shown that the sulfhydryl groups of two of these residues form a vicinal pair in the enzyme and are easily modified by thiol reagents. The allosteric transition to the more active conformer (R), produced by the binding of homotropic (D-glucosamine 6-phosphate or 2-deoxy-2-amino-D-glucitol 6-phosphate) or heterotropic (N-acetyl-D-glucosamine 6-phosphate) ligands, completely protected these thiols against chemical modification. Selective cyanylation of the vicinal thiols with 2-nitro-5-(thiocyanato)benzoate, followed by alkaline hydrolysis to produce chain cleavage at the modified cysteines, gave a pattern of polypeptides which allowed us to identify Cys118 and Cys239 as the residues forming the thiol pair. Subsequently, three mutated forms of the gene were constructed by oligonucleotide-directed mutagenesis, in which one or both of the cysteine codons were changed to serine. The mutant proteins were overexpressed and purified, and their kinetics were studied. The dithiol formed by Cys118 and Cys239 was necessary for maximum catalytic activity. The single replacements and the double mutation affected catalytic efficiency in a similar way, which was also identical to the effect of the chemical block of the thiol pair. However, only one of these cysteinyl residues, Cys239, had a significant role in the allosteric transition, and its substitution for serine reduced the allosteric interaction energy, due to a lower value of KT.