Identification and functional analysis of fructosyl amino acid-binding protein from Gram-positive bacterium Arthrobacter sp.

AIM: Fructosyl amino acid-binding protein (FABP) is a substrate-binding protein (SBP), which recognizes fructosyl amino acids (FAs) as its ligands. Although FABP has been shown as a molecular recognition tool of biosensing for glycated proteins, the availability of FABP is still limited and no FABP was reported from Gram-positive bacteria. In this study, a novel FABP from Gram-positive bacteria, Arthrobacter spp., was reported. METHOD AND RESULTS: BLAST analysis revealed that FABP homologues exist in some of Arthrobacter species genomes. An FABP homologue cloned from Arthrobacter sp. FV1-1, FvcA, contained a putative lipoprotein signal sequence, suggesting that it is a lipoprotein anchored to the bacterial cytoplasmic membrane, which is a typical characteristic for SBPs from Gram-positive bacteria. In contrast, FvcA also exhibits high amino acid sequence similarity to a known Gram-negative bacterial FABP, which exists as a free periplasmic protein. FvcA, without the N-terminal anchoring region, was then recombinantly produced as soluble protein and was found to exhibit Nα-FA-specific binding activity by intrinsic fluorescent measurement. CONCLUSION: This study identified a novel FABP from a Gram-positive bacterium, Arthrobacter sp., which exhibited Nα-FA-specific binding ability. This is the first report concerning an FABP from a Gram-positive bacterium, suggesting that FABP-dependent FA catabolism system is also present in Gram-positive bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The novel FABP exhibits the ability to specifically bind to Nα-FA with a high affinity. This selectivity is beneficial for applying FABP in HbA1c sensing. The successful preparation of water-soluble, functionally expressed Gram-negative bacterial FABP may make way for future applications for a variety of SBPs from Gram-positive bacteria employing the same expression strategy. The results obtained here enhance our understanding of bacterial FA catabolism and contribute to the improved development of FABP as Nα-FA-sensing molecules.

Isolation and identification of N-carbamoyl-beta-D-glucopyranosylamine from human serum.

N-carbamoyl-beta-D-glucopyranosylamine (NCG) is a compound, which can chemically be synthesized by the condensation of glucose and urea by heating under acidic condition. In this study, we isolated and identified NCG and its anomer from human serum. This is the first study showing the occurrence and isolation of NCG from a natural source. The NCG level in human serum was estimated to be 71+/-33 microM using a glucose-3-dehydrogenase-based assay. Because NCG is not commercially used in foods or drugs, we conclude that the NCG isolated from human serum is synthesized from blood glucose and urea in vivo.

Increasing the hydrophobic interaction between terminal W-motifs enhances the stability of Salmonella typhimurium sialidase. A general strategy for the stabilization of beta-propeller protein fold.

Protein engineering of the beta-propeller protein aimed at enhancing the structural stability of the protein was carried out using a monomeric single domain beta-propeller protein, Salmonella typhimurium sialidase, as a model. Ala53 and Ala69 each located at strands B and C of the W1 motif were mutated to Leu and Val, respectively, to increase the hydrophobic interaction between W1 and W6 motifs. The mutants showed enhanced stability towards guanidine hydrochloride and thermal unfolding. Ala53Leu showed higher stability, probably owing to the capability of the mutated Leu to interact extensively with more residues involved in the hydrophobic interactions between the terminal W-motifs. The mutations, which are located far from the active site, have no significant effect on the enzymatic properties. The strategy to enhance the stability proposed here might be applied to the other beta-propeller proteins.

Cloning and expression of glucose 3-dehydrogenase from Halomonas sp. alpha-15 in Escherichia coli.

The gene encoding glucose 3-dehydrogenase (G3DH) from Halomonas sp. alpha-15 was cloned and expressed in Escherichia coli. An open reading frame of 1686 nucleotides was shown to encode G3DH. The flavine adenine dinucleotide binding motif was found in the N-terminal region of G3DH. The deduced primary structure of G3DH showed about 30% identity to sorbitol dehydrogenase from Gluconobacter oxydans and 2-keto-d-gluconate dehydrogenases from Erwinia herbicola and Pantoea citrea. The folding prediction of G3DH suggested that the 3D structure of G3DH was similar with cholesterol oxidase from Brevibacterium sterolicum or glucose oxidase from Aspergillus niger.

Enzymatic synthesis of a novel trehalose derivative, 3,3'-diketotrehalose, and its potential application as the trehalase enzyme inhibitor.

We reported the preparation of a novel trehalose derivative based on enzymatic oxidation of trehalose by water-soluble glucose-3-dehydrogenase (G3DH) from marine bacterium Halomonas sp. alpha-15 cells. The product of G3DH enzymatic conversion was 3,3'-diketotrehalose (3,3'dkT), a novel trehalose derivative of which both third hydroxy groups of glucopyranosides were oxidized. 3,3'dkT was revealed to show an inhibitory effect toward pig-kidney and Bombyx mori trehalases. The IC(50) values of 3,3'dkT were 0.8 and 2.5 mM and K(i) values were 0.2 and 0.6 mM for pig-kidney and for B. mori trehalases, respectively. In addition, 3,3'dkT did not show any inhibitory effect on both maltase and mannosidase activities. Therefore, 3,3'dkT was a specific inhibitor of trehalases.

Biodegradation of formaldehyde by a formaldehyde-resistant bacterium isolated from seawater.

A formaldehyde-tolerant bacterium designated as a DM-2 strain was used to biodegrade formaldehyde. The cells, precultivated in the presence of 400 ppm of formaldehyde, were able to degrade formaldehyde in a minimal medium supplemented with up to 400 ppm of formaldehyde in the presence of 3% NaCl. The rate of formaldehyde degradation achieved in this study was 45 ppm/h when the DM-2 culture's optical density at 660 nm was 1.2.

Screening and characterization of fructosyl-valine-utilizing marine microorganisms.

We describe the isolation of microorganisms utilizing fructosyl-amine (Amadori compound) from the marine environment and of fructosyl-amine oxidase from a marine yeast. Using fructosyl-valine (Fru-Val), a model Amadori compound for glycated hemoglobin, we isolated 12 microbial strains that grow aerobically in a minimal medium supplemented with Fru-Val as the sole nitrogen source. Among these strains, a yeast strain identified as Pichia sp. N1-1, produced a Fru-Val-oxidizing enzyme. The enzyme was purified in its active form, a single-polypeptide water-soluble protein of 54 kDa by gel electrophoresis, producing H(2)O(2) with the oxidation of Fru-Val. By its substrate specificity, the enzyme was categorized as a novel fructosyl-amine oxidase. This is the first study on the isolation of microorganisms utilizing fructosyl-amine in the marine environment and of fructosyl-amine oxidase from budding yeast.

Extended-range glucose sensor employing engineered glucose dehydrogenases.

An enzyme glucose sensor with an expanded dynamic range was constructed using a novel strategy. This strategy was based on a new concept of utilizing protein-engineered enzymes with a different Michaelis constant, which allows for the expanded dynamic range. We used the engineered Escherichia coli pyrroloquinoline quinone glucose dehydrogenase (PQQGDH) of which His775 was substituted for Asp which showed an increased Km value (25-fold). We first constructed the composite colorimetric analytical system employing the wild-type PQQGDH and His775Asp and evaluated its dynamic range. The composite colorimetric analytical system was constructed and showed a wide dynamic range of 0.5-30 mM with less than +/-5% error. The composite colorimetric analytical system, an extended-range colorimetric analytical system, enabled the determination of the concentration of glucose over a 30-fold range that could not have been achieved using the single colorimetric analytical system. Furthermore, we have demonstrated the composite amperometric glucose sensor employing the combination of His775Asn and His775Asp. The extended-range glucose sensor acquired not only the expanded dynamic range (3-70 mM) that covered both dynamic ranges of the single enzyme sensors but also the narrower substrate specificity of glucose due to the inherited property of engineered enzymes.

A Salmonella detection system using an engineered DNA binding protein that specifically captured a DNA sequence.

We have developed a novel method for the detection with high selectivity of a double-stranded DNA fragment using an engineered DNA-binding protein, DnaA IV, a fusion protein of the DNA-binding domain of DnaA and glutathione S-transferase. The DNA fragment detection system is based on DNA-protein interaction and consists of sequence-specific binding of DnaA IV with a DNA fragment containing the DnaA box. DnaA IV, while not capturing other DNA fragments, specifically captured that containing the DnaA box. Because the oriC fragment containing the DnaA box could be specifically amplified by PCR from the genus Salmonella, the DNA fragment detection system was adapted for the detection of Salmonella. The Salmonella detection system using PCR amplification and the engineered DNA-binding protein could distinguish 104 cfu/mL Salmonella from 106 cfu/ mL contaminating bacteria.

Improvement of enantioselectivity of chiral organophosphate insecticide hydrolysis by bacterial phosphotriesterase.

The bacterial phosphotriesterase (PTE) isolated from Flavobacterium sp. can catalyze the cleavage of the P-O bond in a variety of organophosphate triesters and has been shown to be an effective catalyst for the degradation of toxic organophosphate esters. Ethyl 4-nitrophenyl phenylphosphonothioate (EPN) is a chiral organophosphate. Optical isomers of EPN show differences in their toxicity. R-EPN is known to be more toxic to hens and houseflies than S-EPN. We determined the Ki value of each enantiomer toward electric eel acetylcholinesterase. R-EPN (Ki = 6 microM) inhibited acetylcholinesterase much more effectively than S-EPN (Ki = 52 microM) did in vitro. Since PTE has been found to hydrolyze only the S-isomer of EPN, we attempted to alter the enantioselectivity of PTE in order to degrade toxic EPN enantiomer effectively. When PTE hydrolyzed EPN in the presence of dimethyl sulfoxide (DMSO), enzymatic activity toward S-EPN decreased linearly, but enzymatic activity toward R-EPN increased as a function of DMSO concentration. At 20% DMSO, the maximum activity was observed. The kinetic parameters of PTE to EPN isomers clearly indicated that in the presence of 20% DMSO, the enantioselectivity of PTE changed. The Km value for R-EPN decreased from 0.24 to 0.03 mM, and the Vmax value increased from 0.25 to 0.60 U/mg of protein. Vmax/Km values indicated that PTE preferred R-EPN over S-EPN in the presence of DMSO by a factor of 2.

Enzyme electrochemical preparation of a 3-keto derivative of 1,5-anhydro-D-glucitol using glucose-3-dehydrogenase.

A novel enzymatic organic synthesis was reported, utilizing glucose-3-dehydrogenase (G3DH) and its regeneration via electrochemical methods. We combined the water-soluble G3DH prepared from a marine bacterium, Halomonas sp. alpha-15, and electron mediator with the electrode system in order to regenerate the enzyme. Using this system, the conversion of 1,5-anhydro-D-glucitol (1,5AG), a diabetes marker in human blood, was investigated. The final yield of the product, 3-keto anhydroglucitol (3-ketoAG), which was identified by 13C nuclear magnetic resonance, was 82% based on the initial amount of 1,5AG. The electrochemical yield of the reaction proceeded almost stoichiometrically. The electrochemical conversion rate of 1,5AG was 1.24 mmol/(L.h), and the electrochemical yield of 1,5AG consumption was 80%, whereas that for 3-ketoAG was 60%.

Increasing the thermal stability of the water-soluble pyrroloquinoline quinone glucose dehydrogenase by single amino acid replacement.

Based on the characterization of a PCR mutation of water-soluble glucose dehydrogenase possessing pyrroloquinoline quinone (PQQ), PQQGDH-B, Ser231Cys, we have constructed a series of Ser231 variants. The replacement of Ser231 to Cys, Met, Leu, Asp, Asn, His, or Lys resulted in an increase in thermal stability. Among these variants, Ser231Lys showed the highest level of thermal stability and also showed high catalytic activity. Considering that Ser231Lys showed more than an 8-fold increase in its half-life during the thermal inactivation at 55 degrees C compared with the wild-type enzyme, and also retained catalytic activity similar to a wild-type enzyme, the application of this mutant enzyme as a glucose sensor constituent may develop into a stable glucose sensor construction.

Fluorescence resonance energy transfer from pyrene to perylene labels for nucleic acid hybridization assays under homogeneous solution conditions.

We characterized the fluorescence resonance energy transfer (FRET) from pyrene (donor) to perylene (acceptor) for nucleic acid assays under homogeneous solution conditions. We used the hybridization between a target 32 mer and its complementary two sequential 16 mer deoxyribonucleotides whose neighboring terminals were each respectively labeled with a pyrene and a perylene residue. A transfer efficiency of approximately 100% was attained upon the hybridization when observing perylene fluorescence at 459 nm with 347-nm excitation of a pyrene absorption peak. The Förster distance between two dye residues was 22.3 A (the orientation factor of 2/3). We could change the distance between the residues by inserting various numbers of nucleotides into the center of the target, thus creating a gap between the dye residues on a hybrid. Assuming that the number of inserted nucleo-tides is proportional to the distance between the dye residues, the energy transfer efficiency versus number of inserted nucleotides strictly obeyed the Förster theory. The mean inter-nucleotide distance of the single-stranded portion was estimated to be 2.1 A. Comparison between the fluorescent properties of a pyrene-perylene pair with those of a widely used fluorescein-rhodamine pair showed that the pyrene-perylene FRET is suitable for hybridization assays.

Construction and characterization of mutant water-soluble PQQ glucose dehydrogenases with altered K(m) values--site-directed mutagenesis studies on the putative active site.

Based on a PCR mutant enzyme of water-soluble glucose dehydrogenase-harboring pyrroloquinoline quinone as the prosthetic group, PQQGDH-B, a site-directed mutagenesis study was carried out. The substitution of Glu277 residue with Gly resulted in a decrease in the K(m) value for glucose and altered the substrate specificity profile, compared with the wild-type enzyme. Mutational analyses on the neighboring amino acid residues of Glu277 were also carried out and constructed Asp275Glu, Asp276Glu, Ile278Phe, and Asn279His. Considering that Asp275Glu, Asp276Glu and also Glu277Gly showed drastic decreases in EDTA tolerance, this region may construct a PQQGDH-B putative active site, such as a binding site for Ca(2+), which is responsible for the binding PQQ. A series of Glu277 variants, Glu277 substituted by Ala, Val, Asp, Asn, His, Gln, or to Lys, was constructed and they all showed decreased K(m) values and altered substrate specificity profiles. Among them, Glu277Lys showed similar thermal stability with the wild-type enzyme, but its catalytic efficiency increased significantly, compared with the wild-type enzyme. The potential applications of Glu277Lys in analytical use are also discussed.

Site-directed mutagenesis study on the thermal stability of a chimeric PQQ glucose dehydrogenase and its structural interpretation.

We have previously reported that a chimeric pyrroloquinoline quinone (PQQ) glucose dehydrogenase (GDH), E97A3, which was made up of 97% of Escherichia coli PQQGDH sequence and 3% of Acinetobacter calcoaceticus PQQGDH, showed increased thermal stability compared with both parental enzymes. Site-directed mutagenesis studies were carried out in order to investigate the role of amino-acid substitution at the C-terminal region, Ser771, of a chimeric PQQGDHs on their thermal stability. A series of Ser771 substitutions of a chimeric PQQGDH, E99A1, confirmed that hydrophobic interaction governs the thermal stability of the chimeric enzymes. Comparison of the thermal denaturation of E. coli PQQGDH and E97A3 followed by far-ultraviolet (UV) circular dichroism (CD) spectroscopy revealed that E97A3 acquired stability at the first step of denaturation, which is reversible, and where no significant secondary structure change was observed. These results suggested that the interaction between C-terminal and N-terminal regions may play a crucial role in maintaining the overall structure of beta-propeller proteins.

Engineering a chimeric pyrroloquinoline quinone glucose dehydrogenase: improvement of EDTA tolerance, thermal stability and substrate specificity.

An engineered Escherichia coli PQQ glucose dehydrogenase (PQQGDH) with improved enzymatic characteristics was constructed by substituting and combining the gene-encoding protein regions responsible for EDTA tolerance, thermal stability and substrate specificity. The protein region responsible for complete EDTA tolerance in Acinetobacter calcoaceticus, which is recognized as the indicator of high stability in co-factor binding, was elucidated. The region is located between 32 and 59% from the N-terminus of A. calcoaceticus PQQGDH(A27 region) and also corresponds to the same position from 32 to 59% from the N-terminus in E. coli PQQGDH, though E. coli PQQGDH is EDTA sensitive. We previously reported that the C-terminal 3% region of A. calcoaceticus (A3 region) played an important role in the increase of thermal stability, and that His775Asn substitution in E. coli PQQGDH resulted in an increase in the substrate specificity of E. coli PQQGDH towards glucose. Based on these findings, chimeric and/or mutated PQQGDHs, E97A3 H775N, E32A27E41 H782N, E32A27E38A3 and E32A27E38A3 H782N were constructed to investigate the compatibility of two protein regions and one amino acid substitution. His775 substitution to Asn corresponded to His782 substitution to Asn (H782N) in chimeric enzymes harbouring the A27 region. Since all the chimeric PQQGDHs harbouring the A27 region were EDTA tolerant, the A27 region was found to be compatible with the other region and substituted amino acid responsible for the improvement of enzymatic properties. The contribution of the A3 region to thermal stability complemented the decrease in the thermal stability due to the His775 or His782 substitution to Asn. E32A27E38A3 H782N, which harbours all the above mentioned three regions, showed improved EDTA tolerance, thermal stability and substrate specificity. These results suggested a strategy for the construction of a semi-artificial enzyme by substituting and combining the gene-encoding protein regions responsible for the improvement of enzyme characteristics. The characteristics of constructed chimeric PQQGDH are discussed based on the predicted model, beta-propeller structure.

Effect of growth substrates on production of new soluble glucose 3-dehydrogenase in Halomonas (Deleya) sp. alpha-15.

Halomonas (Deleya) sp. alpha-15 produces new co-factor binding soluble glucose 3-dehydrogenase (G3DH), which oxidizes the third hydroxy group of pyranose. This study investigated the condition of efficient production of G3DH using Halomonas (Deleya) sp. alpha-15. This enzyme was inducible, and alpha-methyl-D-glucoside, isopropyl-thiogalactopyranoside (IPTG) and lactose were revealed to be suitable carbon sources for G3DH induction. Maximum G3DH production was achieved by using minimal medium containing 0.8% (w/v) lactose with a productivity of 470U/l.

Construction of a marine cyanobacterial strain with increased heavy metal ion tolerance by introducing exogenic metallothionein gene.

A marine cyanobacterial strain with enhanced heavy metal ion tolerance was constructed by introducing an exogenous cyanobacterial metallothionein gene, smtA, from a freshwater unicellular cyanobacterium, Synechococcus sp. PCC 7942. An expression vector harboring the c-phycocyanin (cpc) promoter and cpc N-terminal region was constructed and smtA was inserted into its multiple cloning site. The marine cyanobacterium, Synechococcus sp. NKBG 15041c, was highly sensitive to the heavy metal ions present in the medium. However, the recombinant marine cyanobacteria harboring the expression vector with smtA could grow even in the presence of 4 µM of CdCl2, at which concentration the wild-type strain did not grow.

Increased production of recombinant pyrroloquinoline quinone (PQQ) glucose dehydrogenase by metabolically engineered Escherichia coli strain capable of PQQ biosynthesis.

We have previously shown that the production of recombinant Escherichia coli PQQGDH was greatly improved by using a medium supplemented with the cofactor PQQ, which is not synthesized in E. coli. We show here that the increase in the accumulated PQQGDH is due to the increased stability of the holo-enzyme over apo-enzyme, using recombinant Acinetobacter calcoaceticus PQQGDH. In order to achieve cost-effective PQQGDH production, we incorporated the genes for PQQ biosynthetic pathway from Klebsiella pneumoniae into E. coli, which as a result allowed E. coli to produce PQQ. Using this metabolically engineered E. coli strain as a host, a 10-fold increase in the production of recombinant A. calcoaceticus PQQGDH was achieved, compared to the condition without PQQ and MgCl2.