Natural nitriles and their metabolism

Legras, Jean Luc; Chuzel, Gérard; Arnaud, A.; Galzy, P.

doi:10.1007/bf01200927

Cited by 74 publications

(51 citation statements)

References 117 publications

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“…These compounds are intermediates in the metabolism of cyanide by plants, animals, and microbes. Cyanogenic glycosides and cyanolipids are found in plants, aminonitriles and cyanohydrins are found in fungi, mandelonitriles are found in arthropods, and a variety of nitrile compounds are found in microorganisms (15). It has been speculated that microbial nitrile metabolism is, at least in part, a consequence of eukaryotic-prokary-otic ecological relationships and that members of the nitrilase superfamily of genes have been horizontally transferred from plants to bacteria and archaea (18).…”

mentioning

confidence: 99%

Exploring Nitrilase Sequence Space for Enantioselective Catalysis

Robertson

Chaplin

DeSantis

et al. 2004

Appl Environ Microbiol

207

116

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Nitrilases are important in the biosphere as participants in synthesis and degradation pathways for naturally occurring, as well as xenobiotically derived, nitriles. Because of their inherent enantioselectivity, nitrilases are also attractive as mild, selective catalysts for setting chiral centers in fine chemical synthesis. Unfortunately, <20 nitrilases have been reported in the scientific and patent literature, and because of stability or specificity shortcomings, their utility has been largely unrealized. In this study, 137 unique nitrilases, discovered from screening of >600 biotope-specific environmental DNA (eDNA) libraries, were characterized. Using culture-independent means, phylogenetically diverse genomes were captured from entire biotopes, and their genes were expressed heterologously in a common cloning host. Nitrilase genes were targeted in a selection-based expression assay of clonal populations numbering 10 6 to 10 10 members per eDNA library. A phylogenetic analysis of the novel sequences discovered revealed the presence of at least five major sequence clades within the nitrilase subfamily. Using three nitrile substrates targeted for their potential in chiral pharmaceutical synthesis, the enzymes were characterized for substrate specificity and stereospecificity. A number of important correlations were found between sequence clades and the selective properties of these nitrilases. These enzymes, discovered using a high-throughput, culture-independent method, provide a catalytic toolbox for enantiospecific synthesis of a variety of carboxylic acid derivatives, as well as an intriguing library for evolutionary and structural analyses.An inherent macromolecular asymmetry, determined by the primordial choice of L-amino acids as protein structural determinants, results in a bias in the populations of chiral molecular moieties produced by stereoselective enzymatic catalysis and in the specificities of those enantiomers in their subsequent interactions with proteins in complex natural systems (16). The principle of chirality and the natural predisposition for shape and handedness in molecular binding by receptors, pumps, and enzymes have been recognized as essential principles for effective drug design (6). In turn, asymmetric enzymatic catalysis, unlike scalar chemical methods, is the optimal tool for synthesis of these enantiopure, pharmaceutically optimal molecules (24). However, the synthetic potential of biocatalysts has yet to be fully realized due to the paucity of available enzymes.The protein sequence space parsed by modern science represents a small fraction of the genetic information available in the biosphere. This has become apparent from recent microbiological efforts targeting a spectrum of physical and chemical environments. These studies have shown that biotopes at extremes of temperature, pressure, pH, salinity, etc., are rich in microbial biodiversity. It is also clear that the physical properties and chemical specificities of the associated gene products reflect the extrinsic and int...

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mentioning

confidence: 99%

Exploring Nitrilase Sequence Space for Enantioselective Catalysis

Robertson

Chaplin

DeSantis

et al. 2004

Appl Environ Microbiol

207

116

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“…aliphatic components, are less evident. In all sample analyses, cyanogenic glycosides were detected, which might represent the important part of organic nitrilated compounds, whose origin can be related to amino acids (Legras et al 1990). The presence of carbohydrates in all colloid fractions was also confirmed by higher UV absorption spectral analyses (at λ = 250 nm; Binkley & Binkley 1999, Giani et al 2005b.…”

Section: Structure and Chemical Composition Of Macroaggregatesmentioning

confidence: 97%

Composition and function of mucilage macroaggregates in the northern Adriatic

Turk¹,

Hagström²,

Kovač³

et al. 2010

Aquat. Microb. Ecol.

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The episodic hyperproduction of mucilage macroaggregates in the northern Adriatic Sea creates an important site for the accumulation, transformation, and degradation of organic matter. In this review, the structure and function of macroaggregate components in relation to their macrogel and colloidal fractions are discussed. High resolution electron microscopy showed a very complex structure, a honeycomb-like structure of the mucus macroagregates that might grow to macroscopic sizes. The process of the formation and microbial interaction with the physicochemical diversity of the organic matter pool is poorly understood. Whether the in situ bacteria react to the carbohydrate-rich mucus as an imbalance in its C:N:P ratio or whether the mucus is in fact largely a bacterial construct in relation to high dissolved organic carbon levels is unknown. The majority of carbohydrate and protein macroaggregate pools are potentially degradable, while the great majority of lipids can be preserved in the water column and exported away or finally deposited on the seabed. Our present knowledge indicates that different macroaggregate fractions and components are subjected to compositional selective reactivity, with important implications for macroaggregate persistence. Future work should reconcile the discrepancies between bacterial ectoenzyme potential activities and biogeochemical degradation sequences based on actual measurements. The determination of biofilm architecture, particularly the spatial arrangement of microcolonies, has profound implications for the function of these complex communities. We need to improve our understanding of the dynamic relationship among bacteria, other microorganisms, and a variety of organic matter forms.

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“…ally, PAN is an intermediate of different value-added and bioactive compounds; therefore, this engineered pathway has the potential to be applied to the microbial production of PAN derivatives such as amides, carboxylic acids, and cyanohydrins from L-Phe by the introduction of other plant and bacterial enzymes (9,12). The production of other amino acid-derived nitriles (such as L-Trp, L-Tyr, and aliphatic nitriles) may also be possible by replacing CYP79A2 and Oxd with the corresponding plant and bacterial enzymes (9,10,12,22).…”

Section: Discussionmentioning

confidence: 99%

“…The biosynthetic pathway of R,S-mandelonitrile (MAN; 2-hydroxy-2-phenylacetonitrile), a defensive substance of some cyanogenic plants, has long been studied; L-Phe is first converted to E,Z-phenylacetaldoxime (PAOx), and then E,Z-PAOx is converted to MAN, probably via formation of PAN as an intermediate (Fig. 1, top) (9)(10)(11)(12). Recently, similar pathways from L-Phe to PAN via E,ZPAOx were also identified in Populus nigra and Fallopia sachalinensis, which activate the pathway in response to stress induced by herbivores to generate volatile PAN as a defensive substance (13,14).…”

mentioning

confidence: 99%

Biosynthetic Pathway for the Cyanide-Free Production of Phenylacetonitrile in Escherichia coli by Utilizing Plant Cytochrome P450 79A2 and Bacterial Aldoxime Dehydratase

Miki

Asano

2014

Appl Environ Microbiol

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The biosynthetic pathway for the production of phenylacetonitrile (PAN), which has a wide variety of uses in chemical and pharmaceutical industries, was constructed in Escherichia coli utilizing enzymes from the plant glucosinolate-biosynthetic and bacterial aldoxime-nitrile pathways. First, the single-step reaction to produce E,Z-phenylacetaldoxime (PAOx) from L-Phe was constructed in E. coli by introducing the genes encoding cytochrome P450 (CYP) 79A2 and CYP reductase from Arabidopsis thaliana, yielding the E,Z-PAOx-producing transformant. Second, this step was expanded to the production of PAN by further introducing the aldoxime dehydratase (Oxd) gene from Bacillus sp. strain OxB-1, yielding the PAN-producing transformant. The E,Z-PAOx-producing transformant also produced phenethyl alcohol and PAN as by-products, which were suggested to be the metabolites of E,Z-PAOx produced by E. coli enzymes, while the PAN-producing transformant accumulated only PAN in the culture broth, which suggested that the CYP79A2 reaction (the conversion of L-Phe to E,Z-PAOx) was a potential bottleneck in the PAN production pathway. Expression of active CYP79A2 and concentration of biomass were improved by the combination of the autoinduction method, coexpression of groE, encoding the heat shock protein GroEL/GroES, N-terminal truncation of CYP79A2, and optimization of the culture conditions, yielding a >60-fold concentration of E,Z-PAOx (up to 2.9 mM). The concentration of PAN was 4.9 mM under the optimized conditions. These achievements show the potential of this bioprocess to produce nitriles and nitrile derivatives in the absence of toxic chemicals.

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Natural nitriles and their metabolism

Cited by 74 publications

References 117 publications

Exploring Nitrilase Sequence Space for Enantioselective Catalysis

Exploring Nitrilase Sequence Space for Enantioselective Catalysis

Composition and function of mucilage macroaggregates in the northern Adriatic

Biosynthetic Pathway for the Cyanide-Free Production of Phenylacetonitrile in Escherichia coli by Utilizing Plant Cytochrome P450 79A2 and Bacterial Aldoxime Dehydratase

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