High Substrate Specificity Research Articles

Activation of a GH43 β-xylosidase by divalent metal cations: Slow binding of divalent metal and high substrate specificity

RS223-BX of glycoside hydrolase family 43 is a β-d-xylosidase that is strongly activated (kcat/Km as much as 116-fold) by the addition of divalent metal cations, Ca2+, Co2+, Fe2+, Mg2+, Mn2+ and Ni2+. Slow activation by Mg2+ was demonstrated (kon 0.013s−1mM−1, koff 0.008s−1) at pH 7.0 and 25°C. koff and kon values are independent of Mg2+ concentration, but koff and kon are slower in the presence of increasing levels of substrate 4-nitrophenyl-β-d-xylopyranoside. The kinetics strongly suggest that M2+ binds to the enzyme rapidly, forming E M2+, followed by slow isomerization to the activated enzyme, E∗ M2+. Moderately high values of kcat (7–30s−1) were found for M2+-activated RS223-BX acting on xylobiose (natural substrate) at pH 7.0 and 25°C. Certain M2+-activated RS223-BX exhibit the highest reported values of kcat/Km of any β-xylosidase acting on natural substrates: for example, at pH 7.0 and 25°C, xylobiose (Mn2+, 190s−1mM−1), xylotriose (Ca2+, 150s−1mM−1) and xylotetraose (Ca2+, 260s−1mM−1). There is potential for the enzyme to add value to industrial saccharification operations at low substrate and high d-glucose and high d-xylose concentrations.

The Arabidopsis cer26 mutant, like the cer2 mutant, is specifically affected in the very long chain fatty acid elongation process

Plant aerial organs are covered by cuticular waxes, which form a hydrophobic crystal layer that mainly serves as a waterproof barrier. Cuticular wax is a complex mixture of very long chain lipids deriving from fatty acids, predominantly of chain lengths from 26 to 34 carbons, which result from acyl-CoA elongase activity. The biochemical mechanism of elongation is well characterized; however, little is known about the specific proteins involved in the elongation of compounds with more than 26 carbons available as precursors of wax synthesis. In this context, we characterized the three Arabidopsis genes of the CER2-like family: CER2, CER26 and CER26-like . Expression pattern analysis showed that the three genes are differentially expressed in an organ- and tissue-specific manner. Using individual T-DNA insertion mutants, together with a cer2 cer26 double mutant, we characterized the specific impact of the inactivation of the different genes on cuticular waxes. In particular, whereas the cer2 mutation impaired the production of wax components longer than 28 carbons, the cer26 mutant was found to be affected in the production of wax components longer than 30 carbons. The analysis of the acyl-CoA pool in the respective transgenic lines confirmed that inactivation of both genes specifically affects the fatty acid elongation process beyond 26 carbons. Furthermore, ectopic expression of CER26 in transgenic plants demonstrates that CER26 facilitates the elongation of the very long chain fatty acids of 30 carbons or more, with high tissular and substrate specificity.

Immobilization Method to Preserve Enzyme Specificity in Biosensors: Consequences for Brain Glutamate Detection

Microelectrode biosensors are a promising technique to probe the brain interstitial fluid and estimate the extracellular concentration of neurotransmitters like glutamate. Their selectivity is largely based on maintaining high substrate specificity for the enzymes immobilized on microelectrodes. However, the effect of enzyme immobilization on substrate specificity is poorly understood. Furthermore, the accuracy of biosensor measurements for brain biological extracts has not been reliably established in comparison with conventional analytical techniques. In this study, microelectrode biosensors were prepared using different enzyme immobilization methods, including glutaraldehyde, a conventional cross-linker, and poly(ethylene glycol) diglycidyl ether (PEGDE), a milder immobilization reagent. Glutaraldehyde, but not PEGDE, significantly decreased the apparent substrate specificity of glutamate and glucose oxidase. For glutaraldehyde prepared biosensors, detection of secondary substrates by glutamate oxidase increased, resulting in a significant overestimate of glutamate levels. This effect was not observed with PEGDE-based biosensors, and when brain microdialysates were analyzed, the levels of glutamate detected by biosensors were consistent with those detected by capillary electrophoresis. In addition, basal concentrations of glutamate detected in vivo were approximately 10-fold lower than the levels detected with glutaraldehyde-based biosensors (e.g., 1.2 μM vs 16 μM, respectively). Overall, enzyme immobilization can significantly impact substrate specificity, and PEGDE is well-suited for the preparation of stable and selective biosensors. This development questions some of the previous biosensor studies aimed at detecting glutamate in the brain and opens new possibilities for specific neurotransmitter detection.

Characterization of a methyl jasmonate specific esterase in arabidopsis

Methyl jasmonate (MeJA)-specific methyl esterase of Arabidopsis (AtMJE) was identified and characterized. AtMJE has high substrate specificity to MeJA compared to other related substrates, methyl indole-3-acetate (MeIAA) and methyl salicylate (MeSA). Through enzyme kinetics analysis, we found AtMJE has similar level of substrate affinity to JA carboxyl methyltransferase (AtJMT). However, AtMJE has 10 times lower catalytic efficiency than AtJMT at low substrate concentrations. AtMJE gene expression was suppressed for 2 h after MeJA treatment, even though its expression recovered and was induced to maximum level within 8 h after treatment. AtMJE overexpressing plants (AtMJEox) showed enhanced MeJA methyl esterase activity demonstrating esterase activity of AtMJE in vivo. AtMJEox plants responded differentially to JA and MeJA in root growth. MeJA in the media could be a source for more JA production in AtMJEox plants, which resulted in root growth inhibition. In contrast, AtMJEox plants grown on JA containing media showed similar root growth inhibition as wild-type. These results show that AtMJE functions in altering JA/MeJA ratios in Arabidopsis and increased JA, because the conversion of MeJA to JA enhances JA responsive gene expression.

High and stable substrate specificities of microorganisms in enhanced biological phosphorus removal plants

Microbial communities are typically characterized by conditions of nutrient limitation so the availability of the resources is likely a key factor in the niche differentiation across all species and in the regulation of the community structure. In this study we have investigated whether four species exhibit any in situ short-term changes in substrate uptake pattern when exposed to variations in substrate and growth conditions. Microautoradiography was combined with fluorescence in situ hybridization to investigate in situ cell-specific substrate uptake profiles of four probe-defined coexisting species in a wastewater treatment plant with enhanced biological phosphorus removal. These were the filamentous 'Candidatus Microthrix' and Caldilinea (type 0803), the polyphosphate-accumulating organism 'Candidatus Accumulibacter', and the denitrifying Azoarcus. The experimental conditions mimicked the conditions potentially encountered in the respective environment (starvation, high/low substrate concentration, induction with specific substrates, and single/multiple substrates). The results showed that each probe-defined species exhibited very distinct and constant substrate uptake profile in time and space, which hardly changed under any of the conditions tested. Such niche partitioning implies that a significant change in substrate composition will be reflected in a changed community structure rather than the substrate uptake response from the different species.

Arxula adeninivorans Recombinant Urate Oxidase and Its Application in the Production of Food with Low Uric Acid Content

Hyperuricemia and its symptoms are becoming increasingly common worldwide. Elevated serum uric acid levels are caused by increased uric acid synthesis from food constituents and reduced renal excretion. Treatment in most cases involves reducing alcohol intake and consumption of meat and fish or treatment with pharmaceuticals. Another approach could be to reduce uric acid level in food, either during production or consumption. This work reports the production of recombinant urate oxidase by Arxula adeninivorans and its application to reduce uric acid in a food product. The A. adeninivorans urate oxidase amino acid sequence was found to be similar to urate oxidases from other fungi (61-65% identity). In media supplemented with adenine, hypoxanthine or uric acid, induction of the urate oxidase (AUOX) gene and intracellular accumulation of urate oxidase (Auoxp) was observed. The enzyme characteristics were analyzed from isolates of the wild-type strain A. adeninivorans LS3, as well as from those of transgenic strains expressing the AUOX gene under control of the strong constitutive TEF1 promoter or the inducible AYNI1 promoter. The enzyme showed high substrate specificity for uric acid, a broad temperature and pH range, high thermostability and the ability to reduce uric acid content in food.

Enzymatical and microbial degradation of cyclic dipeptides (diketopiperazines)

Diketopiperazines (DKPs) are cyclic dipeptides, representing an abundant class of biologically active natural compounds. Despite their widespread occurrence in nature, little is known about their degradation. In this study, the enzymatical and microbial cleavage of DKPs was investigated. Peptidase catalyzed hydrolysis of certain DKPs was formerly reported, but could not be confirmed in this study. While testing additional peptidases and DKPs no degradation was detected, indicating peptidase stability of the peptide bond in cyclic dipeptides. Besides confirmation of the reported degradation of cyclo(l-Asp-l-Phe) by Paenibacillus chibensis (DSM 329) and Streptomyces flavovirens (DSM 40062), cleavage of cyclo(l-Asp-l-Asp) by DSM 329 was detected. Other DKPs were not hydrolyzed by both strains, demonstrating high substrate specificity. The degradation of cyclo(l-Asp-l-Phe) by DSM 40062 was shown to be inducible. Three strains, which are able to hydrolyze hydantoins and dihydropyrimidines, were identified for the degradation of DKPs: Leifsonia sp. K3 (DSM 27212) and Bacillus sp. A16 (DSM 25052) cleaved cyclo(dl-Ala-dl-Ala) and cyclo(l-Gly-l-Phe), and Rhizobium sp. NA04-01 (DSM 24917) degraded cyclo(l-Asp-l-Phe), cyclo(l-Gly-l-Phe) and cyclo(l-Asp-l-Asp). The first enantioselective cleavage of cyclo(dl-Ala-dl-Ala) was detected with the newly isolated strains Paenibacillus sp. 32A (DSM 27214) and Microbacterium sp. 40A (DSM 27211). Cyclo(l-Ala-d-Ala) and cyclo(l-Ala-l-Ala) were completely degraded, whereas the enantiomer cyclo(d-Ala-d-Ala) was not attacked. Altogether, five bacterial strains were newly identified for the cleavage of DKPs. These bacteria may be of value for industrial purposes, such as degradation of undesirable DKPs in food and drugs and production of (enantiopure) dipeptides and amino acids.

Structures of trans-2-enoyl-CoA reductases from Clostridium acetobutylicum and Treponema denticola: insights into the substrate specificity and the catalytic mechanism

TERs (trans-2-enoyl-CoA reductases; EC 1.3.1.44), which specifically catalyse the reduction of crotonyl-CoA to butyryl-CoA using NADH as cofactor, have recently been applied in the design of robust synthetic pathways to produce butan-1-ol as a biofuel. We report in the present paper the characterization of a CaTER (a TER homologue in Clostridium acetobutylicum), the structures of CaTER in apo form and in complexes with NADH and NAD+, and the structure of TdTER (Treponema denticola TER) in complex with NAD+. Structural and sequence comparisons show that CaTER and TdTER share approximately 45% overall sequence identity and high structural similarities with the FabV class enoyl-acyl carrier protein reductases in the bacterial fatty acid synthesis pathway, suggesting that both typesof enzymes belong to the same family. CaTER and TdTER function as monomers and consist of a cofactor-binding domain and a substrate-binding domain with the catalytic active site located at the interface of the two domains. Structural analyses of CaTER together with mutagenesis and biochemical data indicate that the conserved Glu75 determines the cofactor specificity, and the conserved Tyr225, Tyr235 and Lys244 play critical roles in catalysis. Upon cofactor binding, the substrate-binding loop changes from an open conformation to a closed conformation, narrowing a hydrophobic channel to the catalytic site. A modelling study shows that the hydrophobic channel is optimal in both width and length for the binding of crotonyl-CoA. These results provide molecular bases for the high substrate specificity and the catalytic mechanism of TERs.

Purification and characterization of a β-N-acetylhexosaminidase from wheat bran and its applicability to biocontrol of Fusarium solani

N-acetyl-β-d-hexosaminidase was purified from wheat bran and characterized. The purified enzyme showed two protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with molecular mass of 75 and 78 kDa. The enzyme exhibited optimum pH and temperature at 5.0 and 50°C, respectively. The enzyme was active on the substrates of p-nitrophenyl-N-acetyl-β-d-glucosaminide (pNP-GlcNAc) and p-nitrophenyl-N-acetyl-β-d-galactosaminide (pNP-GalNAc), whereas inactive on pNP-β-d-glucopyranoside, pNP-β-d-galactopyranoside, swollen chitin, and colloidal chitin, suggesting high substrate specificity. The enzyme activity for pNP-GlcNAc was stable at pH 3–6 and under 50°C. The K m , V max and K cat for pNP-GlcNAc were 0.014 mM, 0.05 μmol/min, and 3.01×106 min−1, respectively. The enzyme could be completely inhibited at 1–10 mM HgCl2 and AgNO3, suggesting that the intact thiol group is essential for activity. β-N-Acetylhexosaminidase from wheat bran could inhibit the conidial germination and digest the hyphae of Fusarium solani.

English

Recombinant a-glucosidase III (rHBGase III) from Apis cerana indica Fabricus (rAciHBGase III) was expressed in the yeast Pichia pastoris GS115, enriched and characterized. The full length cDNA of AciHbgase III (~1.8 kb) was amplified by RT-PCR, cloned into the pPICZaA expression vector and used to transform P. pastorisGS115. The maximum secreted expression level of rAciHBGase III [as an N terminal (His)6 tagged chimera] was found 144 h after induction by 1% (v/v) methanol. Enrichment of the enzyme using histrap affinity purification revealed a single active glucosidase band with a molecular mass of ~68 kDa. The optimal pH and temperature for glucosidase activity of the enriched rAciHBGase III were pH 5.0 and 37°C, respectively, whilst the enzyme showed a good pH stability between pH 5.0 to 7.5, but not below pH 5.0, and a poor thermotolerance with < 10% and 0% residual activity at 40 and >50°C, respectively. The rAciHBGase showed a relatively high substrate specificity for maltose (Km of 4.5 mM) and p-nitrophenyl a-D-glucoside (Kmof 4.4 mM) compared to other reported HBGase enzymes.  Key words: a-Glucosidase, Apis cerana indica, expression, kinetics, recombinant enzyme

One Substrate, Five Products: Reactions Catalyzed by the Dihydroneopterin Aldolase from Mycobacterium tuberculosis

Tetrahydrofolate cofactors are required for one carbon transfer reaction involved in the synthesis of purines, amino acids, and thymidine. Inhibition of tetrahydrofolate biosynthesis is a powerful therapeutic strategy in the treatment of several diseases, and the possibility of using antifolates to inhibit enzymes from Mycobacterium tuberculosis has been explored. This work focuses on the study of the first enzyme in tetrahydrofolate biosynthesis that is unique to bacteria, dihydroneopterin aldolase (MtDHNA). This enzyme requires no metals or cofactors and does not form a protein-mediated Schiff base with the substrate, unlike most aldolases. Here, we were able to demonstrate that the reaction catalyzed by MtDHNA generates three different pterin products, one of which is not produced by other wild-type DHNAs. The enzyme-substrate complex partitions 51% in the first turnover to form the aldolase products, 24% to the epimerase product and 25% to the oxygenase products. The aldolase reaction is strongly pH dependent, and apparent pK(a) values were obtained for the first time for this class of enzyme. Furthermore, chemistry is rate limiting for the aldolase reaction, and the analysis of solvent kinetic isotope effects in steady-state and pre-steady-state conditions, combined with proton inventory studies, revealed that two protons and a likely solvent contribution are involved in formation and breakage of a common intermediate. This study provides information about the plasticity required from a catalyst that possesses high substrate specificity while being capable of utilizing two distinct epimers with the same efficiency to generate five distinct products.

Two novel Physcomitrella patens fatty acid elongases (ELOs): identification and functional characterization

The lower plant Physcomitrella patens synthesizes several long-chain polyunsaturated fatty acids (LC-PUFAs) by a series of desaturation and elongation reactions. In the present study, the full-length cDNAs for two novel fatty acid elongases designated PpELO1 and PpELO2 were isolated from P. patens using a PCR-based cloning strategy. These cDNAs encoding proteins of 335 and 280 amino acids with predicted molecular masses of 38.7 and 32.9 kDa, respectively, are predicted to contain seven transmembrane domains with a possible localization in the subcellular endoplasmic reticulum. Sequence comparisons and phylogenetic analysis revealed that they are closely related to other LC-PUFA elongases of the lower eukaryotes such as the Δ(5)- and Δ(6)-elongases of Marchantia polymorpha as well as the Δ(6)-elongase of P. patens. Heterologous expression of the PpELO1 in Saccharomyces cerevisiae led to the elongation of Δ(9)-, Δ(6)-C18, and Δ(5)-C20 LC-PUFAs, whereas only Δ(9)- and Δ(6)-C18 LC-PUFA substrates were used by PpELO2. Chimeric proteins were constructed to identify the amino acid regions most likely to be involved in the determination of the fatty acid substrate specificity. The expression of eight chimeric proteins in yeast revealed that substitution of the C-terminal 50 amino acids from PpELO1 into PpELO2 resulted in a high specificity for C20 fatty acid substrates. As a result, we suggest that the C-terminal region of PpELO1 is sufficient for C20 substrate elongation. Overall, these results provide important insights into the structural basis for substrate specificity of PUFA-generating ELO enzymes.

Halogenating Enzymes for Selective Halogenation Reactions

Haloperoxidases have been the only halogenating enzymes know for more than 35 years. They produce in general free hypohalous acid which acts as the halogenating agent and leads thus to a product formation almost identical to chemical halogenation reactions using electrophilic halogen species. With the detection of FADH2-dependent halogenases a type of enzymes was found that shows high substrate specificity and catalyzes regioselective halogenation reactions. FADH2-dependent halogenases are involved in many biosynthetic pathways and catalyze the halogenation of aromatic and aliphatic substrates activated for electrophilic attack. These enzymes also produce hypohalous acids. However, in contrast to haloperoxidases, the hypohalous acid generated cannot leave the active site but can only react with substrates at the active site resulting in selective halogenation reactions. FADH2-dependent halogenases are a twocomponent system with a flavin reductase as the second component. They show similarity to flavin-dependent monooxygenases in forming an enzyme-bound flavin hydroperoxide intermediate. This flavin hydroperoxide reacts with a halide to form hypohalous acid which may then react in a selective reaction with the organic substrate. For halogenation of unactivated carbon atoms, non-heme iron, α- ketoglutarate- and O2-dependent halogenases use a radical mechanism. A substrate radical intermediate which is formed by abstraction of a H. abstracts a halide radical from the non-heme iron coordination complex resulting in the formation of a halogenated methyl group. While in vitro application of both types of halogenases is problematic, due to issues with cofactor recycling, in vivo use which overcomes this problem seems to have a very high potential. Keywords: Haloperoxidase, FADH2-dependent, Halogenase, Non-heme iron, Radical mechanism, Hypohalous acid, Regioselectivity

Preparation of A2 reverse grouping cells from A2B red blood cells by alpha-galactosidase.

Correct typing of donors’ and recipients’ blood group is of paramount importance in transfusion services. There are two distinct parts in ABO typing, forward and reverse typing, both of which are routinely carried out1. Reverse typing is obligatory, because it can help to reveal mistyping, weak A subgroups with anti-A1 and unexpected IgM antibodies. Any discrepancy between the results of the tests with serum or plasma and red cells should be investigated. Reverse grouping cells, including A, B, O and A2 type red blood cells (RBC), are important to resolve ABO discrepancies. Anti-A and/or anti-B antibodies can be easily detected by red blood cells with A and/or B blood group antigens. The presence of an anti-A1 should be confirmed by testing serum against A1, A2, and O red cells. This method necessitates A2 reverse grouping cells. Very few people in China have the A2 blood group. In the Xi’an area (north-western China) only about 0.0006% of the population have A2 RBC, while about 0.003% of the population have the A2B group are. The ratio of the prevalence of A2B:A2 is 5 (unpublished data). In the Shanghai area (eastern China), this same ratio is about 2.5. Thus, there are more people with A2B group RBC than there are with A2 RBC2,3. As it is known that α-galactosidase, a kind of exoglycosidase, can remove the reducing end α-galactose residues of group B antigen by hydrolysis and based on the characteristics of the group B epitopes and α-galactosidase hydrolysis reaction, α-galactosidase has been used in B to O RBC conversion study since the 1980s4. Our group has been researching in this area for more than 10 years5,6 and we recently obtained a novel α-galactosidase from B. fragilis (which belong to CAZy GH110) with highly specific activity, greatly restricted substrate specificity and a neutral pH optimum7,8. Our research showed that this novel α-galactosidase can convert B RBC to O RBC with high substrate specificity and at low cost7,8. We, therefore, started to study the A2B to A2 conversion using α-galactosidase in order to broaden the source of A2 RBC.

Complete genome, catabolic sub‐proteomes and key‐metabolites of Desulfobacula toluolica Tol2, a marine, aromatic compound‐degrading, sulfate‐reducing bacterium

Among the dominant deltaproteobacterial sulfate-reducing bacteria (SRB), members of the genus Desulfobacula are not only present in (hydrocarbon-rich) marine sediments, but occur also frequently in the anoxic water bodies encountered in marine upwelling areas. Here, we present the 5.2 Mbp genome of Desulfobacula toluolica Tol2, which is the first of an aromatic compound-degrading, marine SRB. The genome has apparently been shaped by viral attacks (e.g. CRISPRs) and its high plasticity is reflected by 163 detected genes related to transposases and integrases, a total of 494 paralogous genes and 24 group II introns. Prediction of the catabolic network of strain Tol2 was refined by differential proteome and metabolite analysis of substrate-adapted cells. Toluene and p-cresol are degraded by separate suites of specific enzymes for initial arylsuccinate formation via addition to fumarate (p-cresol-specific enzyme HbsA represents a new phylogenetic branch) as well as for subsequent modified β-oxidation of arylsuccinates to the central intermediate benzoyl-CoA. Proteogenomic evidence suggests specific electron transfer (EtfAB) and membrane proteins to channel electrons from dehydrogenation of both arylsuccinates directly to the membrane redox pool. In contrast to the known anaerobic degradation pathways in other bacteria, strain Tol2 deaminates phenylalanine non-oxidatively to cinnamate by phenylalanine ammonia-lyase and subsequently forms phenylacetate (both metabolites identified in (13) C-labelling experiments). Benzoate degradation involves CoA activation, reductive dearomatization by a class II benzoyl-CoA reductase and hydrolytic ring cleavage as found in the obligate anaerobe Geobacter metallireducens GS-15. The catabolic sub-proteomes displayed high substrate specificity, reflecting the genomically predicted complex and fine-tuned regulatory network of strain Tol2. Despite the genetic equipment for a TCA cycle, proteomic evidence supports complete oxidation of acetyl-CoA to CO2 via the Wood-Ljungdahl pathway. Strain Tol2 possesses transmembrane redox complexes similar to that of other Desulfobacteraceae members. The multiple heterodisulfide reductase-like proteins (more than described for Desulfobacterium autotrophicum HRM2) may constitute a multifaceted cytoplasmic electron transfer network.

Purification and characterization of recombinant glucose dehydrogenase isolated from a hyperthermophilic Sulfolobus-like bacterium

This study aimed to clone and characterize a thermal resistant glucose dehydrogenase (GDH) and investigate its clinical potential. A Sulfolobus -like thermophilic microbe was first isolated from a hot spring in Taitung, Chihpen County, Taiwan. The gene encoding GDH was cloned from the bacterium and expressed in Escherichia coli . The molecular weight of the enzyme was found to be approximately 39,000 kDa. The enzyme is stable over pH 4.0 to 11.0 and has an optimum pH of 8.0. The thermostability range of the enzyme correlated well with that of the natural environment for Sulfolobus . The GDH showed high substrate specificity for glucose. GDH could be useful in biotechnological applications because of its higher thermostability and substrate specificity when compared with that of other glucose-degrading enzymes. Keywords: Glucose dehydrogenase, sequencing, glucose test strip, blood glucose meter, diabetes mellitus

Identification and Characterization of d-Hydroxyproline Dehydrogenase and Δ1-Pyrroline-4-hydroxy-2-carboxylate Deaminase Involved in Novel l-Hydroxyproline Metabolism of Bacteria: METABOLIC CONVERGENT EVOLUTION

L-hydroxyproline (4-hydroxyproline) mainly exists in collagen, and most bacteria cannot metabolize this hydroxyamino acid. Pseudomonas putida and Pseudomonas aeruginosa convert L-hydroxyproline to α-ketoglutarate via four hypothetical enzymatic steps different from known mammalian pathways, but the molecular background is rather unclear. Here, we identified and characterized for the first time two novel enzymes, D-hydroxyproline dehydrogenase and Δ(1)-pyrroline-4-hydroxy-2-carboxylate (Pyr4H2C) deaminase, involved in this hypothetical pathway. These genes were clustered together with genes encoding other catalytic enzymes on the bacterial genomes. D-hydroxyproline dehydrogenases from P. putida and P. aeruginosa were completely different from known bacterial proline dehydrogenases and showed similar high specificity for substrate (D-hydroxyproline) and some artificial electron acceptor(s). On the other hand, the former is a homomeric enzyme only containing FAD as a prosthetic group, whereas the latter is a novel heterododecameric structure consisting of three different subunits (α(4)β(4)γ(4)), and two FADs, FMN, and [2Fe-2S] iron-sulfur cluster were contained in αβγ of the heterotrimeric unit. These results suggested that the L-hydroxyproline pathway clearly evolved convergently in P. putida and P. aeruginosa. Pyr4H2C deaminase is a unique member of the dihydrodipicolinate synthase/N-acetylneuraminate lyase protein family, and its activity was competitively inhibited by pyruvate, a common substrate for other dihydrodipicolinate synthase/N-acetylneuraminate lyase proteins. Furthermore, disruption of Pyr4H2C deaminase genes led to loss of growth on L-hydroxyproline (as well as D-hydroxyproline) but not L- and D-proline, indicating that this pathway is related only to L-hydroxyproline degradation, which is not linked to proline metabolism.

Substrate Specificity of Fluoroacetate Dehalogenase: An Insight from Crystallographic Analysis, Fluorescence Spectroscopy, and Theoretical Computations

The high substrate specificity of fluoroacetate dehalogenase was explored by using crystallographic analysis, fluorescence spectroscopy, and theoretical computations. A crystal structure for the Asp104Ala mutant of the enzyme from Burkholderia sp. FA1 complexed with fluoroacetate was determined at 1.2 Å resolution. The orientation and conformation of bound fluoroacetate is different from those in the crystal structure of the corresponding Asp110Asn mutant of the enzyme from Rhodopseudomonas palustris CGA009 reported recently (J. Am. Chem. Soc. 2011, 133, 7461). The fluorescence of the tryptophan residues of the wild-type and Trp150Phe mutant enzymes from Burkholderia sp. FA1 incubated with fluoroacetate and chloroacetate was measured to gain information on the environment of the tryptophan residues. The environments of the tryptophan residues were found to be different between the fluoroacetate- and chloroacetate-bound enzymes; this would come from different binding modes of these two substrates in the active site. Docking simulations and QM/MM optimizations were performed to predict favorable conformations and orientations of the substrates. The F atom of the substrate is oriented toward Arg108 in the most stable enzyme-fluoroacetate complex. This is a stable but unreactive conformation, in which the small O-C-F angle is not suitable for the S(N)2 displacement of the F(-) ion. The cleavage of the C-F bond is initiated by the conformational change of the substrate to a near attack conformation (NAC) in the active site. The second lowest energy conformation is appropriate for NAC; the C-O distance and the O-C-F angle are reasonable for the S(N) 2 reaction. The activation energy is greatly reduced in this conformation because of three hydrogen bonds between the leaving F atom and surrounding amino acid residues. Chloroacetate cannot reach the reactive conformation, due to the longer C-Cl bond; this results in an increase of the activation energy despite the weaker C-Cl bond.

Purification and Characterization of a 56 kDa Chitinase Isozyme (PaChiB) from the Stomach of the Silver Croaker,Pennahia argentatus

A 56 kDa chitinase isozyme (PaChiB) was purified from the stomach of the silver croaker Pennahia argentatus. The optimum pH and pH stability of PaChiB were observed in an acidic pH range. When N-acetylchitooligosaccharides ((GlcNAc)n, n=2 -6) were used as substrates, PaChiB degraded (GlcNAc)4 -6 and produced (GlcNAc)2,3. It degraded (GlcNAc)5 to produce (GlcNAc)2 (23.2%) and (GlcNAc)3 (76.8%). The ability to degrade p-nitrophenyl N-acetylchitooligosaccharides (pNp-(GlcNAc)n, n=2 -4) fell in the following order: pNp-(GlcNAc)3≫ pNp-(GlcNAc)2 pNp-(GlcNAc)4. Based on these results, we concluded that PaChiB is an endo-type chitinolytic enzyme, and that it preferentially hydrolyzes the third glycosidic bond from the non-reducing end of (GlcNAc)n. Activity toward crystalline α- and β-chitin was activated at 124%-185% in the presence of 0.5 M NaCl. PaChiB exhibited markedly high substrate specificity toward crab-shell α-chitin.

Identification and characterization of a GDSL lipase‐like protein that catalyzes the ester‐forming reaction for pyrethrin biosynthesis in Tanacetum cinerariifolium– a new target for plant protection

Although natural insecticides pyrethrins produced by Tanacetum cinerariifolium are used worldwide to control insect pest species, little information is known of their biosynthesis. From the buds of T. cinerariifolium, we have purified a protein that is able to transfer the chrysanthemoyl group from the coenzyme A (CoA) thioester to pyrethrolone to produce pyrethrin I and have isolated cDNAs that encode the enzyme. To our surprise, the active principle was not a member of a known acyltransferase family but a member of the GDSL lipase family. The recombinant enzyme (TcGLIP) was expressed in Escherichia coli and displayed the acyltransferase reaction with high substrate specificity, recognized the absolute configurations of three asymmetric carbons and also showed esterase activity. A S40A mutation in the Block I domain reduced both acyltransferase and esterase activities, which suggested an important role of this serine residue in these two activities. The signal peptide directed the localization of TcGLIP::enhanced green fluorescent protein (EGFP) fusion, as well as EGFP, to the extracellular space. High TcGLIP gene expression was observed in the leaves of mature plants and seedlings as well as in buds and flowers, a finding that was consistent with the pyrethrin I content in these parts. Expression was enhanced in response to wounding, which suggested that the enzyme plays a key role in the defense mechanism of T. cinerariifolium.