Sorghum bicolor mRNA for p-(S)-hydroxymandelonitrile lyase (SbHNL)
Comment
a deletion of two aa adjacent to active serine is responsible in part of a dramatically different active site and catalytic mechanism compared to wheat carboxypeptidase ( the structure of the cristallized isozyme II has some amino acid differences Leu11 Ala79 Gly112 Ser203 Thr408 Val409 Arg 410)
(Below N is a link to NCBI taxonomic web page and E link to ESTHER at designed phylum.) > cellular organisms: NE > Eukaryota: NE > Viridiplantae: NE > Streptophyta: NE > Streptophytina: NE > Embryophyta: NE > Tracheophyta: NE > Euphyllophyta: NE > Spermatophyta: NE > Magnoliophyta: NE > Mesangiospermae: NE > Liliopsida: NE > Petrosaviidae: NE > commelinids: NE > Poales: NE > Poaceae: NE > PACMAD clade: NE > Panicoideae: NE > Andropogonodae: NE > Andropogoneae: NE > Sorghinae: NE > Sorghum: NE > Sorghum bicolor: NE
No mutation 1 structure: 1GXS: Crystal Structure of Hydroxynitrile Lyase from Sorghum bicolor in Complex with Inhibitor Benzoic Acid: a novel cyanogenic enzyme No kinetic
LegendThis sequence has been compared to family alignement (MSA) red => minority aminoacid blue => majority aminoacid color intensity => conservation rate title => sequence position(MSA position)aminoacid rate Catalytic site Catalytic site in the MSA MAVFISSSGSPGRATATTTTTTTLLLAVLAAAAAAGLLLAPVAARGSPPE HDKQLQLQQQEDDRIPGLPGQPNGVAFGMYGGYVTIDDNNGRALYYWFQE ADTADPAAAPLVLWLNGGPGCSSIGLGAMQELGPFRVHTNGESLLLNEYA WNKAANILFAESPAGVVFSYSNTSSDLSMGDDKMAQDTYTFLVKWFERFP HYNYREFYIAGESGHFIPQLSQVVYRNRNNSPFINFQGLLVSSGLTNDHE DMIGMFELWWHHGLISDETRDSGLKVCPGTSFMHPTPECTEVWNKALAEQ GNINPYTIYTPTCDREPSPYQRRFWAPHGRAAPPPLMLPPYDPCAVFNSI NYLNLPEVQTALHANVSGIVEYPWTVCSNTIFDQWGQAADDLLPVYRELI QAGLRVWVYSGDTDSVVPVSSTRRSLAALELPVKTSWYPWYMAPTEREVG GWSVQYEGLTYVSPSGAGHLVPVHRPAQAFLLFKQFLKGEPMPAEEKNDI LLPSEKAPFY
References
Title: Crystal structure of hydroxynitrile lyase from Sorghum bicolor in complex with the inhibitor benzoic acid: a novel cyanogenic enzyme Lauble H, Miehlich B, Forster S, Wajant H, Effenberger F Ref: Biochemistry, 41:12043, 2002 : PubMed
The crystal structure of the hydroxynitrile lyase from Sorghum bicolor (SbHNL) in complex with the inhibitor benzoic acid has been determined at 2.3 A resolution and refined to a crystallographic R-factor of 16.5%. The SbHNL sequence places the enzyme in the alpha/beta hydrolase family where the active site nucleophile is predicted to be organized in a characteristic pentapeptide motif which is part of the active site strand-turn-helix motif. In SbHNL, however, a unique two-amino acid deletion is next to the putative active site Ser158, removing thereby the putative oxyanion hole-forming Tyr residue. The presented X-ray structure shows that the overall folding pattern of SbHNL is similar to that of the closely related wheat serine carboxypeptidase (CPD-WII); however, the deletion in SbHNL is forcing the putative active site residues away from the expected hydrolase binding site toward a small hydrophobic cleft, which also contains the inhibitor benzoic acid, defining thereby a completely different SbHNL active site architecture where the traditional view of a classic triad is not given any more. Rather, we propose a mechanism involving general base catalysis by the carboxy-terminal Trp270 carboxyl group and proton transfer toward the leaving nitrile group by an active site water molecule. The unexpected interactions of the inhibitor with the new SbHNL active site also reveal the structural basis for the enzyme's limited substrate specificity. The implications of this structure on the evolution of catalysis in the hydroxynitrile lyase superfamily are discussed.
Title: Molecular cloning of hydroxynitrile lyase from Sorghum bicolor (L.). Homologies to serine carboxypeptidases Wajant H, Mundry KW, Pfizenmaier K Ref: Plant Mol Biol, 26:735, 1994 : PubMed
The heterotetrameric enzyme hydroxynitrile lyase (HNL) from sorghum (EC 4.1.2.11) is involved in the catabolism of the cyanogenic glycoside dhurrin. We have isolated a cDNA clone comprising about 90% of the COOH terminal sequence of a precursor which encodes both subunit of HNL from Sorghum bicolor L. (SbHNL). Hence the subunits of SbHNL must be the result of post-translational processing. The deduced amino acid sequence of HNL shares significant sequence homology with members of the serine carboxypeptidase family. In particular, HNL from sorghum shares the catalytical triad Asp. His, and Ser with these enzymes which evolved in 3 groups of enzymes (carboxypeptidase, chymotrypsin, and subtilisin) by convergent evolution. Moreover, like serine carboxypeptidases, HNL from sorghum consists of two pairs of glycosylated cysteine linked A and B chains forming a heterotetramer of a molecular weight of 105,000 (carboxypeptidases 120,000). Thus, HNL from sorghum closely resembles to serine carboxypeptidases but differs from all other HNLs described so far. Western blotting experiments revealed cross reaction between carboxypeptidase from wheat and anti SbHNL antisera. Therefore, convergent evolution of HNLs from various ancestoral enzymes is conceivable. Hybridization of SbHNL cDNA to northern blots of total RNAs isolated from various organs of young sorghum seedlings shows the same expression pattern of HNL as found by means of western blotting or enzyme assays. Using PCR and Southern blot analysis, we demonstrated that the gene of SbHNL is free of introns. Further sequence analysis of cDNA clones and genomic DNA revealed a stretch of 23 adenine residues in the 3'-untranslated part of the gene. Both, intronless organisation of the gene and a genomic stretch of oligo A suggests that SbHNL may have evolved by a reverse transcription event.
Sorghum bicolor mRNA for p-(S)-hydroxymandelonitrile lyase (SbHNL)
