α-Hydroxynitrile lyase protein from Xylella fastidiosa: Cloning, expression, and characterization

Caruso, Célia Sulzbacher; Travensolo, Regiane de Fátima; Bicudo, Rogério de Campus; Lemos, Eliana Gertrudes de Macedo; Araújo, Ana Paula Ulian de; Carrilho, Emanuel

doi:10.1016/j.micpath.2009.06.007

Cited by 16 publications

(11 citation statements)

References 22 publications

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“…Its main studied properties are included in Table 5. 26 There are also examples of very highly soluble expression of the putative HNL genes from Oryza sativa in E. coli, which show no HNL activity, either in synthesis or in decomposition of several cyanohydrins. 157…”

Section: Screening For Discovery Of New and Potent Hnlsmentioning

confidence: 99%

Hydroxynitrile Lyases: Insights into Biochemistry, Discovery, and Engineering

2011

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Hydroxynitrile lyases are valuable enzymes for asymmetric synthesis of cyanohydrins. These hydroxyl and nitrileÀcontaining compounds are being used in production of very useful pharmaceuticals, agrochemicals, and other biologically active compounds using chemical or chemoenzymatic follow-up reactions in industry. Although a huge amount of information exists on the reaction parameters of these enzymes, including stability to pH and organic solvents, yield, reaction time, and valuable data on the enantiopurity of their products, cyanohydrins, there is a lack of update on the biochemistry, discovery, and engineering of the HNLs. Therefore, in the Introduction, we will have a look into these enzymes, cyanohydrins, and aldoxime-nitrile pathways. A brief view of functional groups and several examples of cyanohydrin-based chemicals and pharmaceuticals will also be described. Then we will present characteristics of many S-and R-selective HNLs with comparative tables for several enzymatic properties under biochemistry section. The methods of screening and discovery of these enzymes both from nature and a library of mutants will be described as well as their potential in the synthesis of chemicals. Cloning and expression of new HNLs will also be described under the discovery section. A pool of successful applications of protein engineering methods and the subsequent improvement in the properties of mutant HNLs will be reviewed in detail afterward.

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Section: Screening For Discovery Of New and Potent Hnlsmentioning

confidence: 99%

Hydroxynitrile Lyases: Insights into Biochemistry, Discovery, and Engineering

2011

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“…Hydroxynitrile lyases are widespread in plants playing a major role in disease suppression, and only recently a bacterial protein with HNL activity in the cyanohydrin cleavage reaction was reported (16). Usually plants are colonized by a range of different bacteria (14).…”

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confidence: 99%

Characterization of Two Bacterial Hydroxynitrile Lyases with High Similarity to Cupin Superfamily Proteins

Hussain

Wiedner

Steiner

et al. 2012

Appl Environ Microbiol

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Hydroxynitrile lyases (HNLs) catalyze the cleavage of cyanohydrins. In the reverse reaction, they catalyze the formation of carbon-carbon bonds by enantioselective condensation of hydrocyanic acid with carbonyls. In this study, we describe two proteins from endophytic bacteria that display activity in the cleavage and the synthesis reaction of (R)-mandelonitrile with up to 74% conversion of benzaldehyde (enantiopreference ee 89%). Both showed high similarity to proteins of the cupin superfamily which so far were not known to exhibit HNL activity. Hydroxynitrile lyases (HNLs) catalyze the reversible cleavage of cyanohydrins, yielding the respective carbonyl compound and HCN. They are of great relevance in organic synthesis, in which the ability of the enzymes to catalyze the reverse reaction forming C-C bonds in a stereoselective manner is of substantial industrial relevance (9,11,19). Hydroxynitrile lyases are quite heterogeneous exhibiting remarkable diversity with respect to their substrate specificity, mass, glycosylation, and amino acid sequence, and their similarity to oxidoreductases, ␣/␤-hydrolases, carboxypeptidases, or Zn 2ϩ -containing alcohol dehydrogenases has been reported (3,4,8,17,18,19).Hydroxynitrile lyases are widespread in plants playing a major role in disease suppression, and only recently a bacterial protein with HNL activity in the cyanohydrin cleavage reaction was reported (16). Usually plants are colonized by a range of different bacteria (14). Endophytes in particular live in close association with their host and promote plant growth and health by various mechanisms, including the production of substances with phytohormonal activity or antimicrobial substances such as antibiotics (13,15,21). The production of HCN in pseudomonads has been reported (5), for example, but it has also been reported in other bacteria. In contrast to plants, cyanogenesis in bacteria usually follows a completely different biosynthetic pathway involving a HCN synthase (2,20). In this work, we present the discovery of a new bacterial enzyme class with hydroxynitrile lyase activity initially identified by function-based screening of a gene library of an isolated bacterial endophyte related to Pseudomonas mephitica and further investigated with a second highly similar protein from Burkholderia phytofirmans strain PsJN.Gene libraries of different endophytic bacteria isolated from potato were constructed in the pZero-2 vector by standard procedures (see supplemental material) and screened for HNL activity toward (R/S)-mandelonitrile using a colony-based colorimetric filter assay [67 mM (R/S)-mandelonitrile in 30 mM citratephosphate buffer (pH 3.5) at room temperature (RT)] (10). In the gene library of a strain related to Pseudomonas mephitica, HNL activity was detected. After subcloning and rescreening, the transformant exhibiting the strongest activity toward mandelonitrile was selected for further characterization. Sequencing revealed that the plasmid contained a 1,659-bp insert, which carried two open reading fram...

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“…26,27 From Sorghum bicolor SbHNL was isolated and shown to be (S)-selective; 28,29 structurally it is related to serine carboxypeptidase with an a/b-hydrolase fold. Similarly several other HNLs, namely Hevea brasiliensis HbHNL, 27 Manihot esculenta MeHNL, 30 Arabidopsis thaliana AtHNL, 31,32 Xylella fastidiosa XfHNL 33 and Baliospermum montanum BmHNL, 34 have an a/b-hydrolase fold; they are related to the esterase/ lipase superfamily. Interestingly, of these 5 structurally closely related HNL's some are (R)-and others (S)-selective.…”

Section: Hydroxynitrile Lyases (Hnls)mentioning

confidence: 99%

Immobilisation of hydroxynitrile lyases

Hanefeld

2013

Chem. Soc. Rev.

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Hydroxynitrile lyases are a versatile group of enzymes that are applied both in the laboratory and on an industrial scale. What makes them particularly interesting is that to date five structurally unrelated categories of hydroxynitrile lyases have been discovered. Given their great importance they have often been immobilised utilising many different methodologies. Therefore the hydroxynitrile lyases are ideally suited to compare different immobilisation methods and their dependence on the structural features of the enzyme in question, since the activity is the same in all cases. This review examines all the different immobilisation methods applied to hydroxynitrile lyases and draws conclusions on the effect of the approach.

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α-Hydroxynitrile lyase protein from Xylella fastidiosa: Cloning, expression, and characterization

Cited by 16 publications

References 22 publications

Hydroxynitrile Lyases: Insights into Biochemistry, Discovery, and Engineering

Hydroxynitrile Lyases: Insights into Biochemistry, Discovery, and Engineering

Characterization of Two Bacterial Hydroxynitrile Lyases with High Similarity to Cupin Superfamily Proteins

Immobilisation of hydroxynitrile lyases

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