Enzyme discovery beyond homology: a unique hydroxynitrile lyase in the Bet v1 superfamily

Abstract

Homology and similarity based approaches are most widely used for the identification of new enzymes for biocatalysis. However, they are not suitable to find truly novel scaffolds with a desired function and this averts options and diversity. Hydroxynitrile lyases (HNLs) are an example of non-homologous isofunctional enzymes for the synthesis of chiral cyanohydrins. Due to their convergent evolution, finding new representatives is challenging. Here we show the discovery of unique HNL enzymes from the fern Davallia tyermannii by coalescence of transcriptomics, proteomics and enzymatic screening. It is the first protein with a Bet v1-like protein fold exhibiting HNL activity, and has a new catalytic center, as shown by protein crystallography. Biochemical properties of D. tyermannii HNLs open perspectives for the development of a complementary class of biocatalysts for the stereoselective synthesis of cyanohydrins. This work shows that systematic integration of -omics data facilitates discovery of enzymes with unpredictable sequences and helps to extend our knowledge about enzyme diversity.

Figure 1. Identification of the DtHNL sequence.

A: Blue Native PAGE followed by HNL activity assay. (a) I: Concentrated fractions of anion exchange purification were applied separately on BN PAGE. NativeMark^™ Unstained Protein Standard (Thermo Fisher Scientific); total protein extract from D. tyermannii leaves (1); flow through (2); elution fractions (3–8); active bands at 20 kDa are highlighted in the box. (a) II: HNL activity is depicted by the blurred blue spot corresponding to purification fractions 2–7. The total protein extract shows a weak signal (1). Assay conditions: 100 mM citrate buffer pH 4.0; substrate: racemic mandelonitrile. Incubation time 8 min. (b) Screening of putative HNL sequences. Cell free lysate of E. coli TOP 10 F’ strains expressing six putative HNL proteins. Each sample was tested in triplicate: 30 μL (1), 20 μL (2), 10 μL (3) of cell free lysate, respectively. E. coli TOP 10F’ transformed with pMS vector was used as negative control. Assay conditions: 100 mM citrate buffer pH 4.5; substrate: racemic mandelonitrile 13 mM. The intensive blue spots developed after few seconds of incubation. Proteins with unknown function were named as the relative isotig or contig number found in the transcriptome. (c) Nucleotide and amino acid sequence of isotig 02643 (DtHNL1). Fragments detected by mass spectrometry are labeled. Indicated results were obtained from the protein bands excised from lanes 4 (red) and 5 (red and blue). The peptides identified by mass spectrometry cover 72% of the open reading frame.

Figure 2. Biochemical properties of DtHNL isoenzymes.

DtHNL1 ; DtHNL2 ; DtHNL3 ; DtHNL4 . Grey dashed lines indicate the spontaneous degradation of racemic mandelonitrile in a negative control reaction without enzyme addition (background reaction). Standard enzymatic assay was performed by monitoring benzaldehyde formation at 280 nm. Values were obtained from the average of a minimal of two and a maximum of three independent samples, each of which is the average of two or three technical replicates. Standard deviations are within the 20% threshold (or 25% for temperature profile). For clarity, error bars have been omitted. (a) pH profile. Relative activity of DtHNL isoenzymes at different pH values from 2.0 to 7.0. The assay was performed in HCl-potassium chloride buffer (filled symbols), or sodium citrate-phosphate buffer (empty symbols). Activity of DtHNL1 and 2 at pH 7.0 is not depicted due to high standard deviations. (b) Temperature profile. Relative activity of DtHNL isoenzymes at different temperatures from 10 to 50 °C. The assay was performed at pH 5.0. Omitted points are due to high standard deviations. Enzyme stability at pH 2.5 (c) and at pH 4.0 (d). Activity after incubation of DtHNL isoenzymes at pH 2.5 or 4.0, respectively, and 8 °C. Relative activity is based on the activity before incubation.

Figure 3. Crystal structure DtHNL1.

(a) Overall structure of the DtHNL1 dimer. Individual protomers are shown in a grey or blue ribbon representation. Bound ligands are shown in a stick representation (mandelonitrile in yellow and benzaldehyde in cyan). (b) Close-up view of the active site. Residues potentially important for the enzymatic activity are shown in a sticks representation and water molecules are depicted as red spheres. The bridging water is labeled W1. Dashed lines indicate plausible hydrogen bonding interactions. Bound benzaldehyde (cyan, right) and (R)-mandelonitrile (yellow, left) as observed in a crystal soaked with (R)-mandelonitrile are drawn in a sticks representation. (c) Access to the active site. Water channel extending from the active site to the surface of the protein as calculated using the program CAVER.

Figure 4. Proposed catalytic mechanism for the DtHNL1.

Cyanohydrin cleavage of (R)-mandelonitrile based on complex-crystal structures and mutagenesis experiments.

Figure 5. Multiple sequence alignment.

Isotig02775 and isotig02778 are proteins with similarity to DtHNL1 from the transcriptome of P. aquilinum leaves. Contig4149 was found in the transcriptome obtained from a P. aquilinum gametophyte. Isotig04300 is the sequence most similar to isotig02775 and isotig02778, which was found in D. tyermannii. Isotig translation frame is indicated in brackets. Conservation % is indicated by a color code. Residues involved in substrate binding and catalysis are highlighted. Alignment was built with CLC Main Workbench 7.6.2 (QIAGEN). Proteins with unknown function were named as the relative isotig or contig number found in the transcriptome.