(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 > eudicotyledons: NE > Gunneridae: NE > Pentapetalae: NE > rosids: NE > fabids: NE > Malpighiales: NE > Euphorbiaceae: NE > Crotonoideae: NE > Codiaeae: NE > Baliospermum: NE > Baliospermum montanum: NE
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 MVSAHFILIHTICHGAWLWYKLIPLLQSAGHNATAIDLVASGIDPRQLEQ IGTWEQYSEPLFTLIESIPEGKKVILVGESGGGINIALAAEKYPEKVSAL VFHNALMPDIDHSPAFVYKKFSEVFTDWKDSIFSNYTYGNDTVTAVELGD RTLAENIFSNSPIEDVELAKHLVRKGSFFEQDLDTLPNFTSEGYGSIRRV YVYGEEDQIFSRDFQLWQINNYKPDKVYCVPSADHKIQISKVNELAQILQ EVANSASDLLAVA
References
Title: Immobilized Baliospermum montanum hydroxynitrile lyase catalyzed synthesis of chiral cyanohydrins Jangir N, Padhi SK Ref: Bioorg Chem, 84:32, 2019 : PubMed
Hydroxynitrile lyase (HNL) catalyzed enantioselective CC bond formation is an efficient approach to synthesize chiral cyanohydrins which are important building blocks in the synthesis of a number of fine chemicals, agrochemicals and pharmaceuticals. Immobilization of HNL is known to provide robustness, reusability and in some cases also enhances activity and selectivity. We optimized the preparation of immobilization of Baliospermium montanum HNL (BmHNL) by cross linking enzyme aggregate (CLEA) method and characterized it by SEM. Optimization of biocatalytic parameters was performed to obtain highest % conversion and ee of (S)-mandelonitrile from benzaldehyde using CLEA-BmHNL. The optimized reaction parameters were: 20min of reaction time, 7 U of CLEA-BmHNL, 1.2mM substrate, and 300mM citrate buffer pH 4.2, that synthesized (S)-mandelonitrile in approximately 99% ee and approximately 60% conversion. Addition of organic solvent in CLEA-BmHNL biocatalysis did not improve in % ee or conversion of product unlike other CLEA-HNLs. CLEA-BmHNL could be successfully reused for eight consecutive cycles without loss of conversion or product formation and five cycles with a little loss in enantioselectivity. Eleven different chiral cyanohydrins were synthesized under optimal biocatalytic conditions in up to 99% ee and 59% conversion, however the % conversion and ee varied for different products. CLEA-BmHNL has improved the enantioselectivity of (S)-mandelonitrile synthesis compared to the use of purified BmHNL. Nine aldehydes not tested earlier with BmHNL were converted into their corresponding (S)-cyanohydrins for the first time using CLEA-BmHNL. Among the eleven (S)-cyanohydrins syntheses reported here, eight of them have not been synthesized by any CLEA-HNL. Overall, this study showed preparation, characterization of a stable, robust and recyclable biocatalyst i.e. CLEA-BmHNL and its biocatalytic application in the synthesis of different (S)-aromatic cyanohydrins.
Title: Structural and functional analysis of hydroxynitrile lyase from Baliospermum montanum with crystal structure, molecular dynamics and enzyme kinetics Nakano S, Dadashipour M, Asano Y Ref: Biochimica & Biophysica Acta, 1844:2059, 2014 : PubMed
Hydroxynitrile lyases (HNLs) catalyze degradation of cyanohydrins to hydrogen cyanide and the corresponding ketone or aldehyde. HNLs can also catalyze the reverse reaction, i.e., synthesis of cyanohydrins. Although several crystal structures of S-selective hydroxynitrile lyases (S-HNLs) have been reported, it remains unknown whether and how dynamics at the active site of S-HNLs influence their broad substrate specificity and affinity. In this study, we analyzed the structure, dynamics and function of S-HNL from Baliospermum montanum (BmHNL), which has an alpha/beta hydrolase fold. Two crystal structures of BmHNL, apo1 and apo2, were determined at 2.55 and 1.9A, respectively. Structural comparison between BmHNL (apo2) and S-HNL from Hevea brasiliensis with (S)-mandelonitrile bound to the active site revealed that hydrophobic residues at the entrance region of BmHNL formed hydrophobic interactions with the benzene ring of the substrate. The flexible structures of these hydrophobic residues were confirmed by a 15ns molecular dynamics simulation. This flexibility regulated the size of the active site cavity, enabling binding of various substrates to BmHNL. The high affinity of BmHNL toward substrates containing a benzene ring was also confirmed by comparing the kinetics of BmHNL and S-HNL from Manihot esculenta. Taken together, the results indicated that the flexibility and placement of the residues are important for the broad substrate specificity of S-HNLs.
A novel S-hydroxynitrile lyase (HNL) was purified from leaves of a plant, Baliospermum montanum, by ammonium sulfate fractionation and column chromatographies. Full-length cDNA and genomic DNA were cloned and sequenced. The latter contained two introns and one ORF encoding a 263-residue protein (subunit: 29.5 kDa). The hnl gene was expressed in Escherichia coli and the enzyme was characterized including detailed kinetic studies of 20 substrates for (S)-cyanohydrin synthesis. The enzyme exhibited the highest specific activity (178 U/mg), k(cat) (98/s) and k(cat)/K(m) ratio for piperonal. k(cat)/K(m) ratio for aromatic aldehydes was much larger than those of aliphatic aldehydes and ketones. It was strongly inhibited by AgNO(3), PMSF, phenol and methyl ethyl ketone, showed an optimum at pH 5, while having activity at range of 4-6.5. It exhibited stability at wide pH range 2.4-11, the highest activity at 20 degrees C, being active at 0-65 degrees C. The enzyme showed variations in residues involved in substrate pocket and substrate entrance channel compared to other S-selective HNLs, based on a model was built. C-terminal short truncations provided more enzyme production. Gel filtration revealed a 60-65 kDa molecular mass for this non-FAD enzyme and its C-terminally truncated forms using three buffer compositions, indicating dimeric structures.
Baliospermum montanum. bmhnl (S)-hydroxynitrile lyase
