Structure-Based Phylogeny as a Diagnostic for Functional Characterization of Proteins with a Cupin Fold

Agarwal, Garima; Rajavel, Malligarjunan; Gopal, B.; Srinivasan, Narayanaswamy

doi:10.1371/journal.pone.0005736

Cited by 59 publications

(80 citation statements)

References 41 publications

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“…RmlC-like cupins superfamily comprises several families with members performing diverse functions ranging from enzymatic activities like dioxygenases, decarboxylases, hydrolases, isomerases and epimerases to non-enzymatic functions such as binding to auxin, nuclear transcription factors and seed storage [46]. While, germin-like superfamily members are ubiquitously expressed in various plant species and play important roles in plant development and defence.…”

Section: Discussionmentioning

confidence: 99%

The effects of putrescine are partly overlapping with osmotic stress processes in wheat

et al. 2018

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Section: Discussionmentioning

confidence: 99%

The effects of putrescine are partly overlapping with osmotic stress processes in wheat

et al. 2018

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“…Although cupins are structurally conserved and usually contain two conserved motifs, G-(X)5-H-X-H-(X) 3,4-E-(X)6-G (motif 1) and G-(X)5-P-X-G-(X)2-H-(X) 3-N (motif 2), the overall sequence identity is low among members of this superfamily [13]. The two motifs also include the residues for metal binding.…”

Section: Introductionmentioning

confidence: 99%

Biochemical and structural characterization of a novel bacterial manganese‐dependent hydroxynitrile lyase

et al. 2013

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Hydroxynitrile lyases (HNLs), which catalyse the decomposition of cyanohydrins, are found mainly in plants. In vitro, they are able to catalyse the synthesis of enantiopure cyanohydrins, which are versatile building blocks in the chemical industry. Recently, HNLs have also been discovered in bacteria. Here, we report on the detailed biochemical and structural characterization of a hydroxynitrile lyase from Granulicella tundricola (GtHNL), which was successfully heterologously expressed in Escherichia coli. The crystal structure was solved at a crystallographic resolution of 2.5 Å and exhibits a cupin fold. As GtHNL does not show any sequence or structural similarity to any other HNL and does not contain conserved motifs typical of HNLs, cupins represent a new class of HNLs. GtHNL is metal‐dependent, as confirmed by inductively coupled plasma/optical emission spectroscopy, and in the crystal structure, manganese is bound to three histidine and one glutamine residue. GtHNL displayed a specific activity of 1.74 U·mg−1 at pH 6 with (R)‐mandelonitrile, and synthesized (R)‐mandelonitrile with 90% enantiomeric excess at 80% conversion using 0.5 m benzaldehyde in a biphasic reaction system with methyl tertiary butyl ether.

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“…These proteins are predicted to be members of the cupin superfamily, which contains 50 families, including various enzymes like dioxygenases, decarboxylases, hydrolases, isomerases, and epimerases, but also some nonenzymatic proteins (1,6). Cupins are characterized by a conserved domain, comprising a six-stranded beta-barrel structure, including the motifs [G(X) 5 HXH(X) 3,4 E(X) 6 G] and [G(X) 5 PXG(X) 2 H(X) 3 N] together with a 11-to 16-amino-acid (aa) intermotif region (6, 7).…”

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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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Structure-Based Phylogeny as a Diagnostic for Functional Characterization of Proteins with a Cupin Fold

Cited by 59 publications

References 41 publications

The effects of putrescine are partly overlapping with osmotic stress processes in wheat

The effects of putrescine are partly overlapping with osmotic stress processes in wheat

Biochemical and structural characterization of a novel bacterial manganese‐dependent hydroxynitrile lyase

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

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