Gene search in silico from yeast genomes for new β-fructosidases

Figure 12 illustrates the dendrogram of the yeast species with β-fructosidase reported sequences in order to have a reference sequence for the in silico search in the genome. In Figure 12, the closer phylogenetic yeast with reported β-fructosidase sequences were Cryptococcus neoformans var. grubii H99 and Cryptococcus gatty WM276. The accession numbers of β-fructosidase sequences for Cryptococcus neoformans var. grubii H99 and Cryptococcus gatty WM276 are AFR94181.1 and ADV25067.1, respectively. Further, the nucleotide and amino acid sequence of the β-fructosidase were downloaded from the NCBI database. Then, the in silico search was performed using CLC Genomics Workbench 9.5, where a BLAST in the genome of Rhodotorula musilaginosa (GenBank:

JWTJ00000000.1) was performed using β-fructosidases sequences from Cryptococcus neoformans var.

grubii H99 and Cryptococcus gatty WM27.

Figure 12. Dendrogram built with the yeasts species with β-fructosidase reported and with Rhodotorula mucilaginosa with not reported β-fructosidase sequence.

The results of the BLAST in the equivalent genome from R. mucilaginosa indicated that only the β- fructosidase gene from Cryptococcus neoformans var. grubii H99 (accession number AFR94181.1) has been aligned with the R. mucilaginosa genome. Thus, it may be suggested that a β-fructosidase gene is present in the genome from R. mucilaginosa. Figure 13 shows an aligned section between 400- 1000 bp in the counting JWTJ0100029443 from the genome of R. mucilaginosa.

Figure 13. β-fructosidase sequence BLAST in genome from Rhodotorula mucilaginosa using a β-fructosidase from Cryptococcus neoformas var. grubii H99 (accession number AFR94181.1).

Afterwards the gene prediction in Sofberry was performed (http://www.softberry.com), using the counting JWTJ0100029443 and the nucleotide sequence from Cryptococcus neoformas var. grubii H99 (accession number AFR94181.1). Thus, table 18 shows the predicted gene of Rhodotorula mucilaginosa Furthermore, with the catalytic motif according to the GH32 family reported by coloured in blue (Lammens et al. 2009).

Table 18. Predicted β-fructosidase from Rhodotorula mucilaginosa a) nucleotide and b) amino acid sequence.

The catalytic domains are highlighted in blue and the signal peptide in red.

a)

ATGTTTCGCTCTCTCGTCCCCATTACCATCGCGGGTCTGGCATATCTCCTTCAGAGTCCAGCTCAATC GACAACGTCAAGTGCTGCCTCTGTCCCCACGGGCGTTCCCATCGAAGGAGACTACTCTGGACCTTAC CGCCCCAGAATTCATTTCTCACCCCCGAAAGGCTTTATGAACGACCCGAACGGTTGCCACCGCGAC CGCAACGGCACGTATCACCTCTACTACCAGTACAACCCGCTCGAGTACGTCGCCGGGAACCAGCAC TGGGGACACGCCACTTCGGACGACCTGTACCACTGGACGAACCAACCCATCGCCATCTTCCCGCCC AACTCGACCTCGCAGGTCTTCTCCGGTTCGGCAGTGCTCGACCCTAACAACACGTCGGGCTTCTTCC CGAACACGACCGACGGCGTCGTCGCCGTCTACACCCTCAACACGCCGACTCTCCAAGTCCAGGAGG TCGCGTACTCGACCGACGGCGGCTACAATTTCACGCCGTACGAGAACAACCCCGTCCTCTCTGTCGG CAGCAACCAGTTCCGTGACCCCAAGGTCTTCTGGTACGAAGACCACTGGGTCATGGCTGTCGCCGC CGCTAACGACTTTACCATCGAAATCTACACTTCGCCGAACCTCACGTCGTGGACTTTCGCCTCCAAC TTTACGCACCACGGTTTGCTCGGACTCGCGTACGAATGTCCGAACTTGGTCCAGGTGCCGTTCCAGG ACGACCCGTCCAAGTCGGCCTGGCTCATGTACATTTCGATCAACCCCGGCGCGCCACTCGGCGGCA GTGTCGGCCAGTACTTCCCGGGCGACTTTAACGGCACCCACTTTGTCGCGTACGACTCGGCGGCGA GGATCGCGGATTTTGCGAAGGACAACTATGCTTCGCAATGGTTCGCCGACACGGAGAACGGCGAGT CGATCTCGATCGCTTGGGCTTCCAACTGGCAGTACACTCAGCAAGTTCCTACATCAGCCCAAGCTTT CAGATCTGCGATGTCGTTGCCGCGCCGGAACTACCTTACGAACATCACCCGGCTCGGCTGGGATCTC GTCTCGCTTCCGTACGACCTCTCGCCGGTCGTCGGCCCGTCGCTCCTGTCGTCGTCCGAGGCCAACT CGACCGCCGACGTCGACTTTACCAACGTGACTTCGAACGCGGTCTGGTTCAGTCTGAACGTGACCCT CCCCGACGCCGCAATCCAGAACGCTTCGCTCATTTCGGCCGACGCATCGATCAACATCACCTTCCTC CCTTCGACCAAGTGCTCCTCCTCTTCGGGATCGGGGTCCGACTCACCAGCGGCGACCCTGACCTACT TCTACGCGGGTCTGACGAACGGAGCCCTCGCGCTCACTCGACCGGCGGCTTCCTCCTCGTGGGGAG CCGAGAACCCCTTCTTCACCGACAAGTTTTCGTACACGCTCGTCGACCCGCTCACGTCCCTCGTCGG CGTGTTTGATCGGTCGATGCTCGAGGTGTTTGTCAACGAGGGAGCACACTCGGCTACCATGTTGGTG TTCCCCGACTCGCCGGTCGGGAGTATGAAGGTCGCGACTGGGGGTTTGCCTGAGGGCACGCAGGTC AACCTGCAGGTCAACGGCCTCGAGTCGACTTGGCAGTCCTCGTGA

b)

MFRSLVPITIAGLAYLLQSPAQSTTSSAASVPTGVPIEGDYSGPYRPRIHFSPPKGFMNDPNGCHRDRNGT YHLYYQYNPLEYVAGNQHWGHATSDDLYHWTNQPIAIFPPNSTSQVFSGSAVLDPNNTSGFFPNTTDG VVAVYTLNTPTLQVQEVAYSTDGGYNFTPYENNPVLSVGSNQFRDPKVFWYEDHWVMAVAAANDFTI EIYTSPNLTSWTFASNFTHHGLLGLAYECPNLVQVPFQDDPSKSAWLMYISINPGAPLGGSVGQYFPGDF NGTHFVAYDSAARIADFAKDNYASQWFADTENGESISIAWASNWQYTQQVPTSAQAFRSAMSLPRRNY LTNITRLGWDLVSLPYDLSPVVGPSLLSSSEANSTADVDFTNVTSNAVWFSLNVTLPDAAIQNASLISAD ASINITFLPSTKCSSSSGSGSDSPAATLTYFYAGLTNGALALTRPAASSSWGAENPFFTDKFSYTLVDPLTS LVGVFDRSMLEVFVNEGAHSATMLVFPDSPVGSMKVATGGLPEGTQVNLQVNGLESTWQSS

Additionally, a protein BLAST was performed and the homology with enzymes belonging to GH32 family was investigated to verify that the enzyme belongs to this family (Figure 14). As can be seen in the protein BLAST predicted that the β-fructosidase belongs to GH 32 family. Moreover the identity percentage for predicted β-fructosidase from Rhodotorula mucilaginosa indicates 94 % 60 % and 58 % of identity to a hypothetical protein from Rhodotorula sp., Cryptococcus gatty and Cryotococos neoformas, respectively. The protein BLAST further suggests that the predicted sequence from Rhodotorula mucilaginosa could be a functional β-fructosidase. First, it is important to remember the main characteristics of the GH32 family. According to the CAZy classification, GH32 and GH68 families belong to GH-J clan (Cantarel et al. 2009). Thus, the GH-J clan shares a β-propeller catalytic domain with three conserved acidic amino acids referred to as the “catalytic triad.” In the GH32 enzymes, these three residues (including the nucleophilic Asp, the acid-base Glu, and an Asp residue acting as a transition state stabilizer) are located within three conserved sequences, referred to as the WMNDPNG (β-fructosidase motif), EC, and RDP motifs, respectively (Lammens et al. 2009; Lafraya et al. 2011).

Table 19 shows some characteristics of the predicted protein such as, size of sequence in amino acids, isoelectric point (pI) and molecular weight. It is worth to say that pI and molecular weight were calculated by Compute pI/Mw tool Expansy. Furthermore, the invertase from S. cerevisiae was used as a control enzyme. As can be seen the size, pI, molecular weight are similar, according to the literature the sequence amino acid is in average between 500 to 570, pI 4.5-4.9 and the molecular weight is between 58.5 to 59.3 kDa (Nadeem et al. 2015). In addition, the predicted sequence from Rhodotorula mucilaginosa has three conserved motifs (WMNDPNG, EC, and RDP), which are characteristics from GH32 family (Lammens et al. 2009).

Table 19. Predicted β-fructosidases theoretical properties and catalytic motifs.

Enzyme Size

(aa) pI Molecular weight (Da)

Fructosidase motif

RDP Motif

EC Motif Predicted β-fructosidase from

Rhodotorula mucilaginosa 547 4.56 59263.53 FMNDPNG RDP EC Invertase from S. cerevisiae

(control) 512 4.58 58544.72 WMNDPNG RDP EC

Figure 14. BLAST protein performed at NCBI of predicted β-fructosidase from Rhodotorula mucilaginosa.

Afterward, a structure homology-model was performed by SWISS-MODEL Workspace in order to propose a model of the structure of the predicted β-fructosidase. Table 20 shows the results obtained for the predicted sequence, it can be noticed that the homology structure-model for Rhodotorula mucilaginosa was similar to the one of S. cerevisiae (PDB 4eqv.1.A). Wherein, it was possible to observe in the model the β-propeller and β-sandwich domains characteristic of GH32 family (Lammens et al. 2009).

Table 20. Predicted β-fructosidase homology structure-model by SWISS-MODEL Workspace.

Table 21 shows a boxshade analysis for the catalytic domains between some reported and the predicted β-fructosidase. As it can be seen for the predicted β-fructosidase from Rhodotorula mucilaginosa in comparison to reported enzymes, the catalytic motifs are located in almost the same position, these results suggest that this sequence could lead to a functional enzyme.

In conclusion, it was possible to obtain a β-fructosidase sequence for Rhodotorula mucilaginoasa from in silico search. The bioinformatics analyses suggest that the sequence of the β-fructosidase could be a functional enzyme because it contains the main features characterizing the GH32 family including the conserved motifs, catalytic triad, structure and molecular weight.

Enzyme Template Model

Predicted fructosidase from Rhodotorula mucilaginosa

PDB 4eqv.1.A (Invertase from S.

cerevisiae) Sequence identity

42 %

Table 21. Boxshade analysis of predicted β-fructosidase from Rhodotorula mucilaginosa (in red is highlighted the catalytic triad).

B.4. Cloning and expression of the new β-fructosidase from Rhodotorula mucilaginosa for