RESUMEN

Hydroxynitrile lyases (HNLs) catalyze the cleavage of cyanohydrin into cyanide and the corresponding aldehyde or ketone. Moreover, they catalyze the synthesis of cyanohydrin in the reverse reaction, utilized in industry for preparation of enantiomeric pure pharmaceutical ingredients and fine chemicals. We discovered a new HNL from the cyanogenic millipede, Chamberlinius hualienensis. The enzyme displays several features including a new primary structure, high stability, and the highest specific activity in (R)-mandelonitrile ((R)-MAN) synthesis (7420 U·mg-1 ) among the reported HNLs. In this study, we elucidated the crystal structure and reaction mechanism of natural ChuaHNL in ligand-free form and its complexes with acetate, cyanide ion, and inhibitors (thiocyanate or iodoacetate) at 1.6, 1.5, 2.1, 1.55, and 1.55 Å resolutions, respectively. The structure of ChuaHNL revealed that it belongs to the lipocalin superfamily, despite low amino acid sequence identity. The docking model of (R)-MAN with ChuaHNL suggested that the hydroxyl group forms hydrogen bonds with R38 and K117, and the nitrile group forms hydrogen bonds with R38 and Y103. The mutational analysis showed the importance of these residues in the enzymatic reaction. From these results, we propose that K117 acts as a base to abstract a proton from the hydroxyl group of cyanohydrins and R38 acts as an acid to donate a proton to the cyanide ion during the cleavage reaction of cyanohydrins. The reverse mechanism would occur during the cyanohydrin synthesis. (Photo: Dr. Yuko Ishida) DATABASES: Structural data are available in PDB database under the accession numbers 6JHC, 6KFA, 6KFB, 6KFC, and 6KFD.

Asunto(s)

Acetonitrilos/química , Aldehído-Liasas/química , Proteínas de Artrópodos/química , Artrópodos/química , Lipocalinas/química , Acetonitrilos/metabolismo , Aldehído-Liasas/genética , Aldehído-Liasas/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/metabolismo , Artrópodos/enzimología , Sitios de Unión , Biocatálisis , Clonación Molecular , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Ácido Yodoacético/química , Ácido Yodoacético/metabolismo , Cinética , Lipocalinas/genética , Lipocalinas/metabolismo , Simulación del Acoplamiento Molecular , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Tiocianatos/química , Tiocianatos/metabolismo

RESUMEN

Hydroxynitrile lyase (HNL) catalyzes the reversible synthesis and degradation of cyanohydrins, which are important synthetic intermediates for fine chemical and pharmaceutical industries. Here, we report the discovery of HNL from Parafontaria laminata (PlamHNL) millipedes, purification of the HNL to homogeneity, expression of the gene for the enzyme in heterologous expression hosts, and increase in the reaction rate and enantioselectivity in the synthesis of 2-chloromandelonitrile by protein engineering. The recombinant PlamHNL expressed in Pichia pastoris is glycosylated and has a higher thermostability and pH stability than the nonglycosylated HNL expressed in Escherichia coli. PlamHNL showed a unique wide substrate specificity among other millipede HNLs acting on various cyanohydrins, including 2-chloromandelonitrile, a key intermediate for the antithrombotic agent clopidogrel. We solved the X-ray crystal structure of the PlamHNL and found that the catalytic residues were almost identical to those of HNL from Chamberlinius hualienensis, although the forming binding cavity was different. In order to improve the catalytic activity and stereoselectivity, a computational structure-guided directed evolution approach was performed by an enzyme-substrate docking simulation at all of the residues that were exposed on the surface of the active site. The PlamHNL-N85Y mutant showed higher conversion (91% conversion with 98.2% ee of the product) than the wild type (76% conversion with 90% ee of the product) at pH 3.5 and 25 °C for 30 min of incubation. This study shows the diversity of millipede HNLs and reveals the molecular basis for improvement of the activity and stereoselectivity of the wild-type HNL to increase the reaction rate and enantioselectivity in the synthesis of 2-chloromandelonitrile.

RESUMEN

Because the synthesis of chiral compounds generally requires a broad range of substrate specificity and stable enzymes, screening for better enzymes and/or improvement of enzyme properties through molecular approaches is necessary for sustainable industrial development. Herein, the discovery of unique hydroxynitrile lyases (HNLs) from two species of passion fruits, Passiflora edulis forma flavicarpa (yellow passion fruit, PeHNL-Ny) and Passiflora edulis Sims (purple passion fruit, PeHNL-Np), isolated and purified from passion fruit leaves is reported. These are the smallest HNLs (comprising 121 amino acids). Amino acid sequences of both enzymes are 99 % identical; there is a difference of one amino acid in a consensus sequence. PeHNL-Np has an Ala residue at position 107 and is nonglycosylated at Asn105. Because it was confirmed that natural and glycosylated PeHNL-Ny showed superior thermostability, pH stability, and organic tolerance to that of PeHNL-Np, it has been speculated that protein engineering around the only glycosylation site, Asn105, located at the C-terminal region of PeHNL-Ny, might contribute to the stabilization of PeHNL. Therefore, the focus is on improved stability of the nonglycosylated PeHNL by truncating its C-terminal region. The C-terminal-truncated PeHNLΔ107 was obtained by truncating 15 amino acids from the C terminus followed by expression in Escherichia coli. PeHNLΔ107 expressed in E. coli was not glycosylated, and showed improved thermostability, solvent stability, and reusability similar to that of the wild-type glycosylated form of PeHNL expressed in Pichia pastoris. These data reveal that the lack of the high-flexibility region at the C terminus of PeHNL might be a possible reason for improving the stability of PeHNL.

Asunto(s)

Aldehído-Liasas/metabolismo , Frutas/enzimología , Passiflora/enzimología , Aldehído-Liasas/química , Aldehído-Liasas/aislamiento & purificación , Modelos Moleculares , Hojas de la Planta/enzimología , Estabilidad Proteica , Temperatura

RESUMEN

A hydroxynitrile lyase (HNL) from the millipede Chamberlinius hualienensis has high potential for industrial use in the synthesis of cyanohydrins. However, obtaining sufficient amounts of millipedes is difficult, and the production of the Chamberlinius hualienensis HNL (ChuaHNL) in E. coli has not been very successful. Therefore, we investigated the conditions required for high-yield heterologous production of this enzyme using Pichia pastoris. When we employed P. pastoris to express His-ChuaHNL, the yield was very low (22.6 ± 3.8 U/L culture). Hence, we investigated the effects of ChuaHNL codon optimization and the co-production of two protein disulfide isomerases (PDIs) [from P. pastoris (PpPDI) and C. hualienensis (ChuaPDI1, ChuaPDI2)] on His-ChuaHNL production. The productivity of His-ChuaHNL was increased approximately 140 times per unit culture to 3170 ± 144.7 U/L by the co-expression of codon-optimized ChuaHNL and PpPDI. Moreover, we revealed that the N-glycosylation on ChuaHNL had a large effect on the stability, enzyme secretion, and catalytic properties of ChuaHNL in P. pastoris. This study demonstrates an economical and efficient approach for the production of HNL, and the data show that glycosylation has a large effect on the enzyme properties and the P. pastoris expression system.

Asunto(s)

Aldehído-Liasas/metabolismo , Pichia/enzimología , Codón , Glicosilación , Pichia/genética , Proteína Disulfuro Isomerasas/genética

RESUMEN

Mixtures of saturated and unsaturated 1-methoxyalkanes (alkyl methyl ethers, representing more than 45.4% of the millipede hexane extracts) were newly identified from the Thai polydesmid millipede, Orthomorpha communis, in addition to well-known polydesmid defense allomones (benzaldehyde, benzoyl cyanide, benzoic acid, mandelonitrile, and mandelonitrile benzoate) and phenolics (phenol, o- and p-cresol, 2-methoxyphenol, 2-methoxy-5-methylphenol and 3-methoxy-4-methylphenol). The major compound was 1-methoxy-n-hexadecane (32.9%), and the mixture might function as "raincoat compounds" for the species to keep off water penetration and also to prevent desiccation.

Asunto(s)

Artrópodos/metabolismo , Animales , Cresoles/análisis , Fenoles/análisis , Feromonas/análisis , Tailandia

RESUMEN

Hydroxynitrile lyases (HNLs), which are key enzymes in cyanogenesis, catalyze the cleavage of cyanohydrins into carbonyl compounds and hydrogen cyanide. Since HNLs also catalyze the reverse reaction, they are used industrially for the asymmetric synthesis of cyanohydrins, which are valuable building blocks of pharmaceuticals and fine chemicals. HNLs have been isolated from cyanogenic plants and bacteria. Recently, an HNL from the cyanogenic millipede Chamberlinius hualienensis was shown to have the highest specific activity for (R)-mandelonitrile synthesis, along with high stability and enantioselectivity. However, no HNLs have been isolated from other cyanogenic millipedes. We identified and characterized HNLs from 10 cyanogenic millipedes in the Paradoxosomatidae and Xystodesmidae. Sequence analyses showed that HNLs are conserved among cyanogenic millipedes and likely evolved from one ancestral gene. The HNL from Parafontaria tonominea was expressed in Escherichia coli SHuffle T7 and showed high specific activity for (R)-mandelonitrile synthesis and stability at a range of pHs and temperatures. The stability of millipede HNLs is likely due to disulfide bond(s). The E. coli cells expressing HNL produced (R)-mandelonitrile with 97.6% enantiomeric excess without organic solvents. These results demonstrate that cyanogenic millipedes are a valuable source of HNLs with high specific activity and stability.

Asunto(s)

Acetonitrilos/síntesis química , Aldehído-Liasas/química , Artrópodos/enzimología , Aldehído-Liasas/biosíntesis , Aldehído-Liasas/metabolismo , Animales , Artrópodos/genética , Biocatálisis , Catálisis , Clonación Molecular/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Nitrilos/metabolismo

RESUMEN

Hydroxynitrile lyases (HNLs) are enzymes used in the synthesis of chiral cyanohydrins. The HNL from Passiflora edulis (PeHNL) is R-selective and is the smallest HNL known to date. The crystal structures of PeHNL and its C-terminal peptide depleted derivative were determined by molecular replacement method using the template structure of a heat stable protein, SP1, from Populus tremula at 2.8 and 1.8 Å resolution, respectively. PeHNL belongs to dimeric α+ß barrel superfamily consisting of a central ß-barrel in the middle of a dimer. The structure of PeHNL complexed with (R)-mandelonitrile ((R)-MAN) was also determined. The hydroxyl group of (R)-MAN forms hydrogen bonds with His8 and Tyr30 in the active site, whereas the nitrile group is oriented toward the carboxyl group of Glu54, unlike other HNLs, where it interacts with basic residues typically. The results of mutational analysis indicate that the catalytic dyad of His8-Asn101 is critical for the enzymatic reaction. The length of the hydrogen bond between His-Nδ1 and Asn101-Oδ1 is short in the PeHNL-(R)-MAN complex (~ 2.6 Å), which would increase the basicity of His8 to abstract a proton from the hydroxyl group of (R)-MAN. The cyanide ion released from the nitrile group abstracts a proton from the protonated His8 to generate a hydrogen cyanide. Thus, the His8 in the active site of PeHNL acts both as a general acid and a general base in the reaction. ENZYMES: EC 4.1.2.10 DATABASE: Structural data are available in PDB database under the accession numbers 5XZQ, 5XZT, and 5Y02.

Asunto(s)

Aldehído-Liasas/química , Aldehído-Liasas/metabolismo , Passiflora/enzimología , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Aldehído-Liasas/genética , Secuencia de Aminoácidos , Catálisis , Cristalografía por Rayos X , Enlace de Hidrógeno , Modelos Moleculares , Mutación , Proteínas de Plantas/genética , Conformación Proteica , Homología de Secuencia de Aminoácido

RESUMEN

A hydroxynitrile lyase from the passion fruit Passiflora edulis (PeHNL) was isolated from the leaves and showed high stability in biphasic co-organic solvent systems for cyanohydrin synthesis. Cyanohydrins are important building blocks for the production of fine chemicals and pharmaceuticals. Thus, to enhance production yields of PeHNL for industrial applications, we cloned and expressed recombinant PeHNL in Escherichia coli BL21(DE3) and Pichia pastoris GS115 cells without a signal peptide sequence. The aim of this study is to determine the effect of N-glycosylation on enzyme stability and catalytic properties in microbial expression systems. PeHNL from leaves (PeHNL-N) and that expressed in P. pastoris (PeHNL-P) were glycosylated, whereas that expressed in E. coli (PeHNL-E) was not. The enzymes PeHNL-N and PeHNL-P showed much better thermostability, pH stability, and organic solvent tolerance than the deglycosylated enzyme PeHNL-E and the deglycosylated mutant N105Q from P. pastoris (PeHNL-P-N105Q). The glycosylated PeHNL-P also efficiently performed transcyanation of (R)-mandelonitrile with a 98 % enantiomeric excess in a biphasic system with diisopropyl ether. These data demonstrate the efficacy of these methods for improving enzyme expression and stability for industrial application through N-glycosylation.

Asunto(s)

Aldehído-Liasas/metabolismo , Passiflora/enzimología , Aldehído-Liasas/química , Aldehído-Liasas/genética , Biocatálisis , Estabilidad de Enzimas , Escherichia coli/metabolismo , Glicosilación , Concentración de Iones de Hidrógeno , Cinética , Pichia/metabolismo , Hojas de la Planta/enzimología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Solventes/química , Estereoisomerismo , Temperatura

RESUMEN

Histamine formation and bacteriological changes caused by temperature abuse commonly occurring in the manufacturing process of standard canned tuna was assessed in microbiologically challenged tonggol (Thunnus tonggol). The in situ challenge was performed by water-soaking at 26-28 °C for 7 h to ensure the multiplication and active phase of fish microflora. Right after pre-cooking to back-bone temperature (BBT) of 50-52 °C, histamine dropped to 5.17 ± 2.71 ppm, and slowly reached 6.84 ± 1.69 ppm at 16 h abuse. On the contrary, histamine was reduced to 2.87 ± 1.23 ppm and eventually reached 5.01 ± 1.32 ppm at 24 h abuse in the pre-cooked fish previously frozen. The numbers of total aerobic bacteria, Enterobactericeae, psychrotroph, histamine forming bacteria (HFB) and diversity of fish microflora were revealed by cultural and nested PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) techniques. Interestingly, frozen storage effectively halted histamine formation in raw fish throughout 16 h abuse despite the presence of HFB. These included the prolific strains of Morganella morganii, Proteus penneri, Proteus mirabilin, Citrobacter spp. The nested PCR-DGGE profile confirmed the presence of M. morganii and Citrobacter spp. in raw fish. These prolific strains were hardly observed in the precooked fish previously frozen. Frozen storage did not only promote even histamine distribution throughout fish muscle but also enhanced histamine loss during thawing and pre-cooking. Therefore, pre-cooking and frozen storage were proven to be the effective combined hurdles not only to reduce but also prolong histamine formation of the challenged toggol throughout 24 h of temperature abuse during canning process.