Molecular cloning and expression of novel fructosyl peptide oxidases and their application for the measurement of glycated protein

Fructosyl peptide oxidases, enzymes that are active against a model compound of glycated hemoglobin, N(alpha)-fructosyl valyl-histidine, were characterized. To identify the primary structure of fructosyl peptide oxidases, we have prepared cDNA libraries from Eupenicillium terrenum ATCC18547 and Coniochaeta sp. NISL9330. The coding regions, both fungal fructosyl peptide oxidases consisting of 1314-bp, were obtained with degenerated primers based on the amino acid sequences and specific primers by 3(') and 5(') RACE (rapid amplification of cDNA ends). By their sequence similarities and substrate specificities, fructosyl peptide oxidases and their homologs could be categorized into two groups: (A) enzymes that preferably oxidize alpha-glycated molecules and (B) enzymes that preferably oxidize epsilon-glycated molecules. We showed that recombinant fructosyl peptide oxidases could be used to detect protease-treated fructosyl-hexapeptide, a glycated peptide that is released from HbA(1C) by endoproteinase Glu-C, suggesting these enzymes could be useful for the enzymatic measurement of HbA(1C).     

Enhancement of thermostability of fungal deglycating enzymes by directed evolution

Fructosyl peptide oxidases are valuable for the determination of glycoproteins such as hemoglobin A1c. For practical use in clinical diagnosis, we applied directed evolution to improve the thermostability of these enzymes. After two rounds of random mutagenesis and high-throughput screening, six thermostabilizing amino acid substitutions were identified. Therefore, site-directed and cassette mutageneses were applied to combine these six stabilizing mutations. The simultaneous mutants showed that the stabilizing effect of the amino acid replacement was cumulative. The sextuple mutant enzyme, R94K/G184D/F265L/N272D/H302R/H388Y, had a half-life of thermal inactivation at 50°C that was 79.8-fold longer than that of the parental fructosyl peptide oxidase. The thermostable variants also showed increased tolerance to digestion by a protease. The sextuple mutant enzyme did not lose its activity on incubation with neutral protease, while the wild-type enzyme almost completely lost its activity. Furthermore, three amino acid substitutions were introduced into another fructosyl peptide oxidase with a different substrate specificity. The half-life of inactivation at 50°C was 3.61-fold longer than that of the parent enzyme. These engineered fructosyl peptide oxidases will be useful for industrial application to clinical diagnosis.     

Motif‐based search for a novel fructosyl peptide oxidase from genome databases

The measurement of glycated hemoglobin A1c (HbA1c) has important implications for diagnosis of diabetes and assessment of treatment effectiveness. We proposed specific sequence motifs to identify enzymes that oxidize glycated compounds from genome database searches. The gene encoding a putative fructosyl amino acid oxidase was found in the Phaeosphaeria nodorum SN15 genome and successfully expressed in Escherichia coli. The recombinant protein (XP_001798711) was confirmed to be a novel fructosyl peptide oxidase (FPOX) with high specificity for α‐glycated compounds, such as HbA1c model compounds fructosyl‐^αN‐valine (f‐^αVal) and fructosyl‐^αN‐valyl‐histidine (f‐^αVal‐His). Unlike previously reported FPOXs, the P. nodorum FPOX has a K_m value for f‐^αVal‐His (0.185 mM) that is considerably lower than that for f‐^αVal (0.458 mM). Based on amino acid sequence alignment, three dimensional structural modeling, and site‐directed mutagenesis, Gly60 was found to be a determining residue for the activity towards f‐^αVal‐His. A flexible surface loop region was also found to likely play an important role in accepting f‐^αVal‐His. Biotechnol. Bioeng. 2010; 106: 358–366. © 2010 Wiley Periodicals, Inc.     

SocA is a novel periplasmic binding protein for fructosyl amino acid

Bacterial periplasmic proteins (bPBPs) undergo drastic conformational changes upon binding substrate, making them appealing as novel molecular recognition tools for biosensing. A putative bPBP-encoding gene, socA, belongs to the soc operon responsible for santhopine (fructosyl glutamine, FQ) catabolism of Agrobacterium tumefaciens. The socA gene was isolated and expressed in Escherichia coli as a soluble 28.8kDa periplasmic protein to investigate its properties as a potential bPBP for fructosyl amino acid (FA). The autofluorescence of SocA was used to monitor the protein's conformational change resulting from substrate binding. The fluorescence intensity changed upon binding FQ in a concentration dependent manner with a calculated K(d) of 2.1muM, but was unaffected by the presence of sugars or amino acid. Our results demonstrate that SocA is a novel FA bPBP that can be utilized as a novel molecular recognition element for the monitoring of FA.     

An enzymatic method for the determination of hemoglobinA<Subscript>1C</Subscript>

Fructosyl peptide oxidase is a flavoenzyme that catalyzes the oxidative deglycation of N-(1-deoxyfructosyl)-Val-His, a model compound of hemoglobin (Hb)A_1C. To develop an enzymatic method for the measurement of HbA_1C, we screened for a proper protease using N-(1-deoxyfructosyl)-hexapeptide as a substrate. Several proteases, including Neutral protease from Bacillus polymyxa, were found to release N-(1-deoxyfructosyl)-Val-His efficiently, however no protease was found to release N-(1-deoxyfructosyl)-Val. Neutral protease also digested HbA_1C to release N-(1-deoxyfructosyl)-Val-His, and then the fructosyl peptide was detected using fructosyl peptide oxidase. The linear relationship was observed between the concentration of HbA_1C and the absorbancy of fructosyl peptide oxidase reaction, hence this new method is a practical means for measuring HbA_1C.     

Enzymes used for the determination of HbA1C

To develop an enzymatic measurement of HbA(1C), two key enzymes, i.e., fructosyl peptide oxidase and Aspergillus protease were characterized. Fructosyl peptide oxidase from Eupenicillium terrenum was a flavoenzyme that could catalyze the oxidation of N-(1-deoxyfructosyl)-Val-His. The enzyme showed high specificity toward alpha-glycated molecules, therefore it seemed suitable for the HbA(1C) assay. Since high levels of FPOX expression seemed toxic to host cells, we applied a gene expression system using a bacteriophage vector and achieved high levels of expression in Escherichia coli. Next, we found that Aspergillus protease was able to digest N-(1-deoxyfructosyl)-hexapeptide, a glycated peptide that was released from the beta-chain of HbA(1C) by Glu-C endoproteinase. We showed that the N-(1-deoxyfructosyl)-Val-His released from N-(1-deoxyfructosyl)-hexapeptide by Aspergillus protease could be assayed enzymatically using fructosyl peptide oxidase, therefore these enzymes could be applied to the enzymatic measurement of HbA(1C).     

Structures and mechanisms of enzymes in the leukotriene cascade

Leukotrienes are a family of proinflammatory lipid mediators of the innate immune response and are important signaling molecules in inflammatory and allergic conditions. The leukotrienes are formed from arachidonic acid, which is released from membranes by cPLA₂, and further converted by 5-lipoxygenase to form the labile epoxide leukotriene (LT) A₄. This intermediate is converted by either of the two enzymes, LTA₄ hydrolase or LTC₄ synthase, to form LTB₄ or LTC₄, respectively. In order for 5-lipoxygenase to work efficiently in cells, five-lipoxygenase-activating protein needs to be present. LTB₄ is one of the most powerful chemotactic agents whereas LTC₄ induces smooth muscle contractions, for example in the airways causing bronchoconstriction in asthmatic patients. The leukotrienes and the five enzymes/proteins involved in their formation have been subject to intense studies including drug design programs. Compounds blocking the formation or action of leukotrienes are potentially beneficial in treatment of several acute and chronic inflammatory diseases of the cardiovascular and respiratory systems. In order to succeed with drug development studies, knowledge of the molecular characteristics of the targets is indispensable. This chapter reviews the biochemistry, catalytic, and structural properties of the enzymes in the leukotriene cascade.     

Heterogeneity of hemoglobin A1d: assessment and partial characterization of two new minor hemoglobins, A1d3a and A1d3b, increased in uremic and diabetic patients, respectively

We have separated and quantified two new minor hemoglobins named HbA_1d3a and HbA_1d3b. The level of HbA_1d3a was significantly higher in uremic than in non-uremic patients (3.00 ± 0.50% vs. 1.28 ± 0.26% of total hemoglobin). It correlated well with carbamylated hemoglobin (r=0.80, n=81, p<0.002) and with plasma urea concentration (r=0.78, n=81, p<0.002). These data and the electrospray ionization mass spectrometric analysis provide strong evidence that HbA_1d3a is an α-chain modified by carbamylation. The HbA_1d3b level in the diabetic patients was found to be 1.6-fold that in non-diabetic subjects (3.00 ± 0.49 vs. 1.90 ± 0.33). This was attributed to HbA_1d3 modified by glycation. Indeed HbA_1d3b correlated significantly with HbA_1c (r=0.71, p<0.002) and with serum glucose level (r=0.62, p<0.002). These two new minor hemoglobins may serve as complements for the objective assessment of averagd long-term uremia and glycemia in uremic and diabetic patients.     

A novel phospholipase A2/esterase from hyperthermophilic archaeon Aeropyrum pernix K1

An open reading frame of the hyperthermophilic archaeon Aeropyrum pernix K1 APE2325, which composed of 474 bases, was cloned and expressed in Escherichia coli BL21 (DE3) Codon Plus-RIL. The recombinant protein was purified by Ni-chelation affinity chromatography. It showed a single band with a molecular mass of 18kDa in SDS-PAGE. The purified enzyme exhibited both phospholipase A(2) and esterase activities with the optimal catalytic temperature at 90 degrees C. The enzyme activity was Ca(2+)-independent. Kinetic analysis revealed its Km, k cat, and Vm for the p-nitrophenyl propionate substrate were 103microM, 39s(-1), and 249micromol/min/mg, respectively. The recombinant protein was thermostable and its half-life at 100 degrees C was about 1h.     

Immunocytochemical localization of enzymes involved in the C3 and C4 pathways in the photosynthetic cells of an amphibious sedge, Eleocharis vivipara

Eleocharis vivipara link, an amphibious leafless sedge, develops traits of C₄ photosynthesis and Kranz anatomy in the terrestrial form but develops C₃-like traits with non-Kranz anatomy when submerged. The cellular localization of C₃ and C₄ enzymes in the photosynthetic cells of the two forms was investigated by immunogold labeling and electron microscopy. The terrestrial form has mesophyll cells and three kinds of bundle sheath cell, namely, parenchyma sheath cells, non-chlorophyllous mestome sheath cells, and Kranz cells. Phosphoenol-pyruvate carboxylase (PEPCase) was present in the cytosol of both the mesophyll cells and the parenchyma sheath cells, with higher-density labeling in the latter, but not in the Kranz cells. Pyruvate, Pi dikinase (PPDK) was found at high levels in the chloroplasts of both the mesophyll cells and the parenchyma sheath cells with some-what stronger labeling in the latter. This enzyme was also absent from the Kranz cells. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was found in the chloroplasts of all types of photosynthetic cell, but labeling was significantly less intense in the parenchyma sheath cells than in other types of cell. The submerged form also has three types of photosynthetic cell, as well as non-chlorophyllous mestome sheath cells, but it lacks the traits of Kranz anatomy as a consequence of modification of the cells. Rubisco was densely distributed in the chloroplasts of all the photosynthetic cells. However, PEPCase and PPDK were found in both the mesophyll cells and the parenchyma sheath cells but at lower levels than in the terrestrial form. These data reveal that the terrestrial form has a unique pattern of cellular localization of C₃ and C₄ enzymes, and they suggest that this pattern and the changes in the extent of accumulation of the various enzymes are the main factors responsible for the difference in photosynthetic traits between the two forms.Abbreviations CAM crassulacean acid metabolism - MC meso phyll cell - PSC parenchyma sheath cell - KC Kranz cell - PEP-Case phosphoenolpyruvate carboxylase - PPDK pyruvate, Pi dikinase - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - LS large subunit - RuBP ribulose-1,5-bisphosphate This study was supported by Grants-in-Aid from the Ministry of Agriculture, Forestry and Fisheries of Japan (Integrated Research Program for the Use of Biotechnological Procedures for Plant Breeding) and from the Science and Technology Agency of Japan (Enhancement of Center-of-Excellence, the Special Coordination Funds for Promoting Science and Technology). The author is grateful to Drs M. Matsuoka and S. Muto for providing the antisera and Dr. M. Samejima for his advice at the early stages of this study.     

Identification of novel phospholipase A2 group IX members in metazoans

Sequence homologues of the bacterium Streptomyces violaceoruber and sea anemone Nematostella vectensis PLA₂ pfam09056 members were identified in several bacteria, fungi and metazoans illustrating the evolution of this PLA₂ sub-family. Comparison of their molecular structures revealed that bacteria and fungi members are part of the GXIV of PLA₂s while metazoan representatives are similar with GIX PLA₂ of the marine snail Conus magus. Members of GXIV and GIX PLA₂s show modest overall sequence similarity (21–35%) but considerable motif conservation within the putative Ca²⁺-binding, catalytic sites and cysteine residue positions which are essential for enzyme function. GXIV PLA₂s of bacteria and fungi typically contain four cysteine residues composing two intramolecular disulphide bonds. GIX PLA₂ homologues were identified in cnidarians and molluscs and in a single tunicate but appear to be absent from other metazoan genomes. The mature GIX PLA₂ deduced peptides contain up to ten cysteine residues capable of forming five putative disulphide bonds. Three disulphide bonds were identified in GIX PLA₂s, two of which correspond to those localized in GXIV PLA₂s. Phylogenetic analysis demonstrates that metazoan GIX PLA₂s cluster separate from the bacterial and fungal GXIV PLA₂s and both pfam09056 members form a group separate from the prokaryote and eukaryote GXIIA PLA₂ pfam06951. Duplicate PLA₂ pfam09056 genes were identified in the genomes of sea anemone N. vectensis and oyster Crassostrea gigas suggest that members of this family evolved via species-specific duplication events. These observations indicate that the newly identified metazoan pfam09056 members may be classified as GIX PLA₂s and support the idea of the common evolutionary origin of GXIV and GIX PLA₂ pfam09056 members, which emerged early in bacteria and were maintained in the genomes of fungi and selected extant metazoan taxa.     

Susceptibility of Helicoverpa zea and Heliothis virescens (Lepidoptera: Noctuidae) to Vip3A insecticidal protein expressed in VipCot™ cotton

Susceptibility of laboratory and field colonies of Helicoverpa zea (Boddie) and Heliothis virescens F. to Vip3A insecticidal protein was studied in diet incorporation and diet overlay assays from 2004 to 2008. Responses of field populations were compared to paired responses of University of Arkansas laboratory susceptible H. zea (LabZA) and H. virescens (LabVR) colonies. After 7 d of exposure, observations were made on number of dead larvae (M) and the number of larvae alive but remaining as first instars (L1). Regression estimates using M (LC₅₀) and M plus L1 (MIC₅₀) data were developed for laboratory and field populations. Susceptibility of laboratory and field populations exposed to Vip3A varied among different batches of protein used over the study period. Within the same batch of Vip3A protein, susceptibilities of laboratory colonies of both species (LabZA and LabVR) were similar. Field colonies were significantly more susceptible to Vip3A than the respective reference colonies of both species. Within field populations, susceptibility to Vip3A varied up to 75-fold in H. zea and 132-fold in H. virescens in LC₅₀ estimates. Variabilities in MIC₅₀s were up to 59- and 11-fold for H. zea and H. virescens, respectively.     

Photosynthetic light and CO2 utilization and C4 traits of two novel super-rice hybrids

Characteristics of photosynthetic light and CO2 use efficiency from seedling to heading stage, and C4 pathway enzyme activities in both flag leaves and lemma were compared between two newly developed super-rice hybrids (Oryza sativa L.), Liangyoupeijiu and Hua-an 3, and a traditional rice hybrid, Shanyou 63. At seedling and tillering stages, Liangyoupeijiu and Hua-an 3 had higher net photosynthetic rates (Pn) and light saturated assimilation rates (Asat) than did Shanyou 63, at both normal (360 micromol mol(-1)) and doubled (720 micromol mol(-1)) CO2 concentrations. At the heading stage, the flag leaves of all three rice hybrids had similar Pn and Asat. However, the two super-rice hybrids had higher apparent quantum yield (AQY) and carboxylation efficiency (CE) during all three typical developmental stages, and higher quantum yield of CO2 fixation (PhiCO2) at the tillering and heading stages. In addition, Liangyoupeijiu showed significantly higher activities of the C(4) pathway enzymes in both flag leaves and lemmas than did Shanyou 63. As a result, flag leaves of the two super-rice hybrids had higher Pn at morning, noontime and late afternoon during the daily cycle. Since most of the grain yield of rice comes from the photosynthesis of flag leaves, the similar Asat and much higher AQY, CE and PhiCO2 at heading stage of the two super-rice hybrids indicates that higher photosynthetic efficiency rather than higher photosynthetic capacity may be the primary factor contributing to their higher grain yields.     

Screening and development of enzymes for determination and transformation of amino acids

ABSTRACT

The high stereo- and substrate specificities of enzymes have been utilized for micro-determination of amino acids. Here, I review the discovery of l-Phe dehydrogenase and its practical use in the diagnosis of phenylketonuria in more than 5,400,000 neonates over two decades in Japan. Screening and uses of other selective enzymes for micro-determination of amino acids have also been discussed. In addition, novel enzymatic assays with the systematic use of known enzymes, including assays based on a pyrophosphate detection system using pyrophosphate dikinase for a variety of l-amino acids with amino-acyl-tRNA synthetase have been reviewed. Finally, I review the substrate specificities of a few amino acid-metabolizing enzymes that have been altered, using protein engineering techniques, mainly for production of useful chemicals, thus enabling the wider use of natural enzymes.     

Purification and properties of intracellular fructosyl transferase fromAureobasidium pullulans

Intracellular frustosyl transferase was purified fromAureobasidium pullulans C-23 by ethanol fractionation, CM-Sephadex chromatography and preparative disc gel electrophoresis. It was shown to be homogeneous on disc polyacrylamide gel electrophoresis, with a molecular size of 190kDa. The pI value of the enzyme was about 3.7. The enzyme has aK _m value of 0.43 mM for sucrose and was optimally active at pH 5.0 and 60°C. The enzyme was stable from pH 2.5 to 12. It was almost completely inhibited by 5mM Hg²⁺ but was not significantly affected by other cations. The transferase was inactivated by treatment with the tryptophan-specific reagentN-bromosuccinimide and the tyrosine-specific reagent, I₂, suggesting that tryptophan and tyrosine residues are probably located at or near the active site of the enzyme.     

Emerging roles of secreted phospholipase A2 enzymes: An update

Phospholipase A₂ (PLA₂) enzymes catalyze the hydrolysis of the sn-2 position of glycerophospholipids to produce free fatty acids and lysophospholipids. More than one third of the mammalian PLA₂ enzymes belong to the secreted PLA₂ (sPLA₂) family, which consists of low molecular mass, Ca²⁺-requiring enzymes with a His–Asp catalytic dyad. Individual sPLA₂ enzymes exhibit unique tissue and cellular localizations and specific enzymatic properties, suggesting their distinct biological roles. The past decade has met a new era of the sPLA₂ research field toward deciphering their in vivo functions by developing several specific tools and methods. These include i) the production of transgenic and knockout mouse lines for several sPLA₂s, ii) the development of specific analytical tools including the production of large amounts of recombinant sPLA₂ proteins, and iii) applying mass spectrometry lipidomics to unveil their specific enzymatic properties occurring in vivo. It is now obvious that individual sPLA₂s are involved in diverse biological events through lipid mediator-dependent and -independent processes, act redundantly or non-redundantly in the context of physiology and pathophysiology, and may represent potential drug targets or novel bioactive molecules in certain situations. In this review, we will highlight the newest understanding of the biological roles of sPLA₂s in the past few years.     

In situ hybridisation in filamentous fungi using peptide nucleic acid probes

Fluorescent DNA and peptide nucleic acid (PNA) probes were used for in situ hybridisations in colonies of Schizophyllum commune and Aspergillus niger. DNA probes for 18S rRNA did not diffuse through the cell wall after mild chemical fixation. After permeabilising the cell wall with lysing enzymes or slow freezing and embedding, hybridisation was still poor and not reproducible. In contrast, PNA probes did diffuse through the cell wall after mild chemical fixation and reproducible fluorescent signals were obtained. The rRNA signal was most intense in the apical compartment of hyphae of S. commune. Within this compartment, the signal was lower at the extreme apex. Apparently, ribosomes are unevenly distributed in hyphae. In S. commune, the mRNA of the SC3 gene was also detected with a PNA probe. The ratio between 18S rRNA and SC3 mRNA signals were variable between hyphae and their compartments. This is the first report of using PNA probes for in situ hybridisation of mRNA in fungi. The method provides a powerful tool to study gene expression.     

Supercomplex organization of the oxidative phosphorylation enzymes in yeast mitochondria

Accumulating evidence indicates that the enzymes involved in mitochondrial oxidative phosphorylation (OXPHOS) are co-assembled into higher-ordered supercomplexes within the mitochondrial inner membrane. This review will focus largely on the OXPHOS supercomplexes of the yeast Saccharomyces cerevisiae. The recent evidence to indicate that diversity in the populations of the cytochrome bc (1)-COX supercomplexes exist shall be outlined. In addition, the existence of dimeric/oligomeric F(1)F(o)-ATP synthase complexes and their proposed role in establishment of the cristae architecture of the inner mitochondrial membrane shall also be discussed.     

Distribution of photorespiratory enzymes between bundle-sheath and mesophyll cells in leaves of the C3−C4 intermediate species Moricandia arvensis (L.) DC

In order to study the location of enzymes of photorespiration in leaves of the C₃–C₄ intermediate species Moricandia arvensis (L.). DC, protoplast fractions enriched in mesophyll or bundlesheath cells have been prepared by a combination of mechanical and enzymic techniques. The activities of the mitochondrial enzymes fumarase (EC 4.2.1.2) and glycine decarboxylase (EC 2.1.2.10) were enriched by 3.0- and 7.5-fold, respectively, in the bundle-sheath relative to the mesophyll fraction. Enrichment of fumarase is consistent with the larger number of mitochondria in bundle-sheath cells relative to mesophyll cells. The greater enrichment of glycine decarboxylase indicates that the activity is considerably higher on a mitochondrial basis in bundle-sheath than in mesophyll cells. Serine hydroxymethyltransferase (EC 2.1.2.1) activity was enriched by 5.3-fold and glutamate-dependent glyoxylate-aminotransferase (EC 2.6.1.4) activity by 2.6-fold in the bundle-sheath relative to the mesophyll fraction. Activities of serine- and alanine-dependent glyoxylate aminotransferase (EC 2.6.1.45 and EC 2.6.1.4), glycollate oxidase (EC 1.1.3.1), hydroxypyruvate reductase (EC 1.1.1.81), glutamine synthetase (EC 6.3.1.2) and phosphoribulokinase (EC 2.7.1.19) were not significantly different in the two fractions. These data provide further independent evidence to complement earlier immunocytochemical studies of the distribution of photorespiratory enzymes in the leaves of this species, and indicate that while mesophyll cells of M. arvensis have the capacity to synthesize glycine during photorespiration, they have only a low capacity to metabolize it. We suggest that glycine produced by photorespiratory metabolism in the mesophyll is decarboxylated predominantly by the mitochondria in the bundle sheath.Abbreviation RuBP ribulose 1,5-bisphosphate     

抗菌肽AMP-17抗白念珠菌作用靶点鉴定

白念珠菌(Candida albicans)是侵袭性真菌感染的主要致病性病原体。抗菌肽AMP-17有较强的抗念珠菌活性,经AMP-17作用白念珠菌后的蛋白组学结果显示,细胞壁(XOG1)和氧化应激(SRR1)蛋白基因表达差异显著,提示AMP-17可能通过影响XOG1和SRR1基因发挥其抗白念珠菌作用。为进一步探究XOG1和SRR1基因是否为AMP-17的作用靶点,本研究利用规律成簇间隔短回文重复序列相关蛋白9(clustered regulatory interspaced short palindromic repeats-associated protein 9,CRISPR/Cas9)系统构建了白念珠菌xog1Δ/Δ和srr1Δ/Δ缺失菌;表型观察结果发现除XOG1基因缺失可影响白念珠菌的体外应激和菌丝形成,2个基因缺失对白念珠菌生长繁殖和生物膜形成无明显影响;药敏实验分析显示AMP-17对xog1Δ/Δ和srr1Δ/Δ缺失菌的MIC₈₀值从野生菌的8μg/mL增至16μg/mL,而对srr1Δ/Δ::srr1回补菌的MIC₈₀值降至野...