Hydrolysis of phosphoether bonds of heme-independent chloride peroxidase from Serratia marcescens

Heme- and metal-independent chloroperoxidase from Serratia marcescens W 250 is shown to be capable of catalyzing the p-nitrophenyl phosphate hydrolysis. The parameters of the phosphatase reaction are determined and inhibitors and activators of the process are found. A hypothetical mechanism of the hydrolysis of phosphoesters by heme- and metal-independent haloperoxidases is suggested. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.     

Mycelial forms of <Emphasis Type="Italic">Pseudallescheria boydii</Emphasis> present ectophosphatase activities

Phosphatase activities were characterized in intact mycelial forms of Pseudallescheria boydii, which are able to hydrolyze the artificial substrate p-nitrophenylphosphate (p-NPP) to p-nitrophenol (p-NP) at a rate of 41.41 ± 2.33 nmol p-NP per h per mg dry weight, linearly with increasing time and with increasing cell density. MgCl₂, MnCl₂ and ZnCl₂ were able to increase the (p-NPP) hydrolysis while CdCl₂ and CuCl₂ inhibited it. The (p-NPP) hydrolysis was enhanced by increasing pH values (2.5-8.5) over an approximately 5-fold range. High sensitivity to specific inhibitors of alkaline and acid phosphatases suggests the presence of both acid and alkaline phosphatase activities on P. boydii mycelia surface. Cytochemical localization of the acid and alkaline phosphatase showed electron-dense cerium phosphate deposits on the cell wall, as visualized by electron microscopy. The product of p-NPP hydrolysis, inorganic phosphate (Pi), and different inhibitors for phosphatase activities inhibited p-NPP hydrolysis in a dose-dependent manner, but only the inhibition promoted by sodium orthovanadate and ammonium molybdate is irreversible. Intact mycelial forms of P. boydii are also able to hydrolyze phosphoaminoacids with different specificity.     

Agars from Chilean Gelidiaceae

Cystocarpic, tetrasporic and vegetative thalli of Gelidium chilense were extracted with water at 95 °C. The contents of 3,6-anhydro-galactose and sulfate group of the hydrocolloids correspond to those of an agar-type polysaccharide. The percentages of 6-O-methyl-galactose and of pyruvic acid ranged between 5.7–6.2% and 0.42–0.54%, respectively. The gel melting and gelation temperatures of Gelidium chilense, G. rex and G. lingulatum agars were determined. A correlation between 6-O-methylgalactose content and gelation temperatures was not observed. It was found by anion-exchange chromatography that 19.8% of tetrasporic and 4.9% of vegetative G. chilense agars are unsulfated polymers. Structural studies on the neutral fraction from tetrasporic G. chilense agar by partial hydrolysis and ¹H NMR spectroscopy have shown that it is mainly composed of agarose. Methylation analysis, oxidative hydrolysis and partial hydrolysis, followed by ¹H NMR spectroscopy, have shown that the neutral fraction of the agar from tetrasporic Gelidium rex is agarose. The results obtained in this work are compared with previously reported data on studies of agars from Chilean Gelidium species.     

Structure and Activity of the Metal-independent Fructose-1,6-bisphosphatase YK23 from Saccharomyces cerevisiae

Fructose-1,6-bisphosphatase (FBPase), a key enzyme of gluconeogenesis and photosynthetic CO₂ fixation, catalyzes the hydrolysis of fructose 1,6-bisphosphate (FBP) to produce fructose 6-phosphate, an important precursor in various biosynthetic pathways. All known FBPases are metal-dependent enzymes, which are classified into five different classes based on their amino acid sequences. Eukaryotes are known to contain only the type-I FBPases, whereas all five types exist in various combinations in prokaryotes. Here we demonstrate that the uncharacterized protein YK23 from Saccharomyces cerevisiae efficiently hydrolyzes FBP in a metal-independent reaction. YK23 is a member of the histidine phosphatase (phosphoglyceromutase) superfamily with homologues found in all organisms. The crystal structure of the YK23 apo-form was solved at 1.75-Å resolution and revealed the core domain with the α/β/α-fold covered by two small cap domains. Two liganded structures of this protein show the presence of two phosphate molecules (an inhibitor) or FBP (a substrate) bound to the active site. FBP is bound in its linear, open conformation with the cleavable C1-phosphate positioned deep in the active site. Alanine replacement mutagenesis of YK23 identified six conserved residues absolutely required for activity and suggested that His¹³ and Glu⁹⁹ are the primary catalytic residues. Thus, YK23 represents the first family of metal-independent FBPases and a second FBPase family in eukaryotes.     

Coronatine analogues produced by Xanthomonas campestris pv Phormiicola

Liquid cultures of Xanthomonas campestris pv phormiicola were found to contain two analogues of coronatine lacking the cyclopropane ring structure, and no trace of either coronatine or norcoronatine. The two compounds were isolated and fully characterised by NMR, MS, hydrolysis and GC of hydrolysis products, as N-coronafacoyl- -valine and N-coronafacoyl- -isoleucine. A survey of 12 strains from 10 other X. campestris pathovars did not locate another source of production of these compounds, whereas all three strains of X. campestris pv phormiicola examined produced comparable levels of both compounds. This is the first report of phytotoxins biosynthetically derived from coronafacic acid outside of the genus Pseudomonas. The implications of these findings to the biosynthesis of the cyclopropane ring structure of coronatine are discussed.     

Sequence-specific cleavage of RNA in the absence of divalent metal ions by a DNAzyme incorporating imidazolyl and amino functionalities

Two modified 2′-deoxynucleoside 5′-triphosphates have been used for the in vitro selection of a modified deoxyribozyme (DNAzyme) capable of the sequence-specific cleavage of a 12 nt RNA target in the absence of divalent metal ions. The modified nucleotides, a C5-imidazolyl-modified dUTP and 3-(aminopropynyl)-7-deaza-dATP were used in place of TTP and dATP during the selection and incorporate two extra protein-like functionalities, namely, imidazolyl (histidine analogue) and primary amino (lysine analogue) into the DNAzyme. The functional groups are analogous to the catalytic Lys and His residues employed during the metal-independent cleavage of RNA by the protein enzyme RNaseA. The DNAzyme requires no divalent metal ions or other cofactors for catalysis, remains active at physiological pH and ionic strength and can recognize and cleave a 12 nt RNA substrate with sequence specificity. This is the first example of a functionalized, metal-independent DNAzyme that recognizes and cleaves an all-RNA target in a sequence-specific manner. The selected DNAzyme is two orders of magnitude more efficient in its cleavage of RNA than an unmodified DNAzyme in the absence of metal ions and represents a rate enhancement of 10⁵ compared with the uncatalysed hydrolysis of RNA.     

Enzyme-catalysed deprotection of N-acetyl and N-formyl amino acids

To investigate the full potential of hydrolases for the removal of two amine-protecting groups, 15 different, commercially available lipases, acylases, proteases and esterases were studied for the hydrolyses of N-acetyl and N-formyl protecting groups. In addition to the well-known acylases from porcine kidney and Aspergillus melleus, this screening revealed that porcine liver esterase and the lipases from Rhizomucor miehei and Pseudomonas stutzeri are also catalysts for the hydrolysis of N-acetylalanine. The activity of lipases in this reaction was unexpected, since lipases are commonly believed not to hydrolyse amides. In addition, from these 15 enzymes, three were found to be active in the hydrolysis of N-formylalanine, i.e. porcine liver esterase and the two acylases. This is the first example where esterase is employed to deprotect N-formyl amides.     

Effects of freezing on plasma membrane H<Superscript>+</Superscript>-ATPase of the callus from <Emphasis Type="Italic">Chorispora bungeana</Emphasis>

The influence of freezing treatment on plasma membrane (PM) H⁺-ATPase was investigated using plasma membrane vesicles isolated from calluses from Chorispora bungeana Fisch. & C.A. Mey. by the discontinuous sucrose gradient centrifugation. Freezing treatment (−4 °C) for 5 d resulted in significant increases in the ATPase activity and the activity of p-nitrophenyl phosphate (PNPP) hydrolysis, decreases in the K_m for ATP hydrolysis and PNPP hydrolysis, and the shift of optimal pH from 6.5 to 7.0. Also, the activity PNPP hydrolysis was less sensitive to vanadate after freezing treatment compared to control, while the inhibition of ATP hydrolysis by hydroxylamine was more sensitive. In addition, freezing treatment also decreased the activation effects of trypsin on PNPP hydrolysis, but increased the activation effects of lysophosphatidylcholine on ATP hydrolysis. Taken together, these results suggested that PM H⁺-ATPase might play an important role during adaptation to freezing and enhancing the frost hardness in C. bungeana.     

Structural basis of cyclic oligoadenylate degradation by ancillary Type III CRISPR-Cas ring nucleases

Type III CRISPR-Cas effector systems detect foreign RNA triggering DNA and RNA cleavage and synthesizing cyclic oligoadenylate molecules (cA) in their Cas10 subunit. cAs act as a second messenger activating auxiliary nucleases, leading to an indiscriminate RNA degradation that can end in cell dormancy or death. Standalone ring nucleases are CRISPR ancillary proteins which downregulate the strong immune response of Type III systems by degrading cA. These enzymes contain a CRISPR-associated Rossman-fold (CARF) domain, which binds and cleaves the cA molecule. Here, we present the structures of the standalone ring nuclease from Sulfolobus islandicus (Sis) 0811 in its apo and post-catalytic states. This enzyme is composed by a N-terminal CARF and a C-terminal wHTH domain. Sis0811 presents a phosphodiester hydrolysis metal-independent mechanism, which cleaves cA₄ rings to generate linear adenylate species, thus reducing the levels of the second messenger and switching off the cell antiviral state. The structural and biochemical analysis revealed the coupling of a cork-screw conformational change with the positioning of key catalytic residues to proceed with cA₄ phosphodiester hydrolysis in a non-concerted manner.     

Kinetics of hydrolysis of type I, II, and III collagens by the class I and II Clostridium histolyticum collagenases.

The kinetics of hydrolysis of rat tendon type I, bovine nasal septum type II, and human placental type III collagens by class I and class IIClostridium histolyticum collagenases (CHC) have been investigated. To facilitate this study, radioassays developed previously for the hydrolysis of these [³H]acetylated collagens by tissue collagenases have been adapted for use with the CHC. While the CHC are known to make multiple scissions in these collagens, the assays are shown to monitor the initial proteolytic events. The individual kinetic parametersk _cat andK _M have been determined for the hydrolysis of all three collagens by both class I and class II CHC. The specific activities of these CHC toward fibrillar type I and III collagens have also been measured. In contrast to human tissue collagenases, neither class of CHC exhibits a marked specificity toward any collagen type either in solution or in fibrillar form. The values of the kinetic parametersk _cat andK _M for the CHC are similar in magnitude to those of the human enzymes acting on their preferred substrates. Thus, the widely held view that the CHC are more potent collagenases is not strictly correct. As with the tissue collagenases, the local collagen structure at the cleavage sites is believed to play an important role in determining the rates of the reactions studied.     

Microplate assay of elevated esterase activity in individual pyrethroid-resistant mosquitoes

Involvement of esterase-mediated hydrolysis as a mechanism of pyrethroidresistance in three species of mosquitoes,viz., Aedes aegypti, Culex quinquefasciatus andAnopheles Stephensi was investigated by microplate assay of Β-esterases in individual larva and adult female and male mosquitoes. Assuming an absorbance value of 0.4 and above at 555 nm as the threshold level of elevated esterase activity which confers resistance, frequency distributions of such individual test mosquitoes were constructed in resistant and susceptible populations. The results indicate the involvement of ester hydrolysis of Pyrethroids as a predominant mechanism of pyrethroid-resistance in the larvae ofCulex quinquefasciatus but not inAedes aegypti. However, a marginal role of esterases is indicated in the larvae ofAnopheles stephensi.     

Hydrolysis of organic phosphates by corn and soybean roots

Because of the importance of organic phosphates as sources of P for plants, this work was performed to study the hydrolysis of nine organic phosphates by sterile, intact corn (Zea mays L.) and soybean (Glycine max L.) roots. Results showed that the rates of hydrolysis ofp-nitrophenyl phosphate (PNP) in buffered solutions by roots of three varieties of corn and three varieties of soybean ranged from 13 to 22 μmol PO₄−P g⁻¹ root h⁻¹ and from 2.1 to 2.2 μmol PO₄−P 0.1 g⁻¹ root h⁻¹, respectively. The average rate of hydrolysis of PNP in nonbuffered solutions was 2- to 3-fold lower for corn roots and 6- to 10-fold lower for soybean roots as compared with those obtained with buffered solutions. The orthophosphate released from hydrolysis of organic P compounds in buffered solutions during a 48-h incubation of corn roots showed that the maximum rate of hydrolysis of PNP was 4 to 6 times greater than the commonly used substrates: α- and β-glycerophosphates, phenolphthalein diphosphate, and glucose-6-phosphate. The rates of hydrolysis of glucose-6-phosphate and glucose-1-phosphate were similar and about 6- to 12-fold lower than that of PNP. Phosphoethanolamine and phosphocholine were hydrolyzed slightly, ando-carboxyphenyl phosphate was not hydrolyzed. The rates of hydrolysis of organic P compounds in nonbuffered solutions by corn and soybean roots were 1 to 3 and 1 to 10 times lower than those in buffered solutions, respectively. The trends in rates of hydrolysis by soybean roots of buffered organic P substrates were similar to those observed with corn roots, with the exception of glucose-1-phosphate and phosphoethanolamine.     

Novel products in hyaluronan digested by bovine testicular hyaluronidase

When the products of hyaluronan (HA) digested by bovine testicular hyaluronidase (BTH) were analyzed by high-performance liquid chromatography (HPLC), minor peaks were detected just before the main even-numbered oligosaccharide peaks. The amount of each minor peak was dependent on the reaction conditions for transglycosylation, rather than hydrolysis, by the BTH. Mainly based on HPLC and MS analysis, each minor peak was found to correspond to its oligosaccharide with one N-acetyl group removed from the reducing terminal N-acetylglucosamine. Enzymatic studies showed that the N-deacetylation activity was closely related to reaction temperature, pH, and the concentration of NaCl contained in the buffer, and glycosaminoglycan types and chain lengths of substrates. These findings strongly suggest that the N-deacetylation reaction in minor peaks was due to a novel enzyme contaminant in the BTH, N-deacetylase, that carries out N-deacetylation at the reducing terminal N-acetylglucosamine of oligosaccharides and is dependent on HA hydrolysis by BTH.     

Detection of a single,metal-independent aminopeptidase activity in polymyxin-extracts from Escherichia coli B

Summary The supernatant of Escherichia coli B autolysed in the presence of polymyxin B contains a single, metal-independent aminopeptidase activity (E.C.-group 3.4.1). The enzyme cleaves the 4-nitroanilides of L-alanine, L-lysine, L-leucine, glycine, and weakly L-phenylalanine. The corresponding N-acetyl-L-alanine-, L-glutamic acid- and L-cysteine-derivatives are not attacked.Dedicated to Dr. Hans Poschenrieder on the occasion of his 75th birthday.     

The detection of <Emphasis Type="Italic">hip</Emphasis>O gene by real-time PCR in thermophilic <Emphasis Type="Italic">Campylobacter</Emphasis> spp. with very weak and negative reaction of hippurate hydrolysis

A total of 190 Campylobacter spp. isolates, of which 34 gave the result of very weak activity, and 156 gave the negative activity in the test for hippurate hydrolysis were characterized. The genomic DNA was isolated from a fresh culture of each isolate and the real-time PCR, targeting the hipO gene, was used to confirm the species distribution of Campylobacter isolates. The hipO gene was detected in 17 isolates (11%) within the total of 156 negative isolates for hippurate hydrolysis. Out of 34 isolates with very weak activity, 19 isolates (56%) were also found to be positive for hipO gene and characterized as C. jejuni. The real-time PCR assay used in this study could be employed for more accurate diagnosis of Campylobacter infections at species level after the biochemical characterization based on hippuricase activity of the isolates. This could also provide important data for the epidemiology of infections associated with these zoonotic pathogens.     

Biocatalytic preparation of chiral epichlorohydrins using recombinant<Emphasis Type="Italic">Pichia pastoris</Emphasis> expressing epoxide hydrolase of<Emphasis Type="Italic">Rhodotorula glutinis</Emphasis>

The use of enantioselective hydrolysis for preparing chiral epichlorohydrins was investigated using recombinantPichia pastoris with the enantioselective epoxide hydrolase ofRhodotorula glutinis. The rate of the recombinant epoxide hydrolase-catalyzed enantioselective hydrolysis of racemic epichlorohydrins was enhanced by the addition of 5% (v/v) Tween 20. Enantiopure (R)-epichlorohydrins with an enantiopurity of 100%ee and a yield of 26% were obtained within 5 min from 50 mM racemates.     

Hydrolysis of Chlorella biomass for fermentable sugars in the presence of HCl and MgCl2

When Chlorella biomass was hydrolyzed in the presence of 2% HCl and 2.5% MgCl₂, a sugar concentration of nearly 12%, and a sugar recovery of about 83% was obtained. Fermentation experiments demonstrated that glucose in the Chlorella biomass hydrolysates was converted into ethanol by Saccharomyces cerevisiae with a yield of 0.47 g g⁻¹, which is 91% of the theoretical yield. This chemical hydrolysis approach is thus a novel route for the hydrolysis of biomass to generate fermentable sugars.     

Structure of acinetoferrin,a new citrate-based dihydroxamate siderophore fromAcinetobacter haemolyticus

From low-iron cultures of Acinetobacter haemolyticus ATCC 17906, a new hydroxamate siderophore was purified by XAD-7 adsorption followed by preparative thin layer chromatography. The siderophore, named acinetoferrin, released citric acid, 1,3-diaminopropane and (E)-2-octenoic acid upon hydrolysis with HCl, reductive hydrolysis with HI and oxidation with periodate, respectively. Structure elucidation by a combination of NMR spectroscopy and positive fast atom bombardment mass spectrometry revealed that acinetoferrin is a derivative of citric acid, both of its terminal carboxyl groups being symmetrically amide-linked with the 1-amino-3-(N-hydroxy-N-2-octenylamino)propane residues. The (E)-2-octenoic acid is novel as a component of the siderophores.