Family Report for: LipSM54-like_FamXVI

Bacterial lipases are an important group of enzymes that offer enormous potential in organic synthesis, and there is considerable interest in identifying and developing novel bacterial lipases. In previous studies, strains of the genus Stenotrophomonas were proved to be potential source of lipases, but there is little genetic information describing lipase from the genus Stenotrophomonas. We have cloned and characterized a novel lipase (LipSM54), the first lipase described from the genus Stenotrophomonas. Enzymatic study showed that LipSM54 was a cold-active, solvent-tolerant and alkaline lipase. Using bioinformatics tools, LipSM54 was found to be related only to several putative lipases from different bacterial origins, none of which could be assigned to any previously described bacterial lipase family. LipSM54 and these related putative lipases share four conserved motifs around the catalytic residues. These motifs clearly distinguish them from the known bacterial lipase families. Consequently, LipSM54 is the first characterized member of the novel bacterial lipase family.

In this work, a lipase from Pseudomonas alcaligenes CGMCC4405 (PaL) was cloned and expressed. It was very attractive that the recombinant PaL exhibited excellent enantioselectivity (E > 200) in the resolution of racemic d,l-menthyl propionate to produce l-menthol. The structure basis of enantiopreference is a fundamental scientific problem which needs to be resolved. In our research, molecular dynamic simulation (MD) research was employed to research the different binding modes of d and l-menthyl propionate. The results showed that when bound with slow-reacting enantiomer (d-menthyl propionate), the steric requirements of the large substituent (isopropyl) of the d-menthyl propionate force a rotation of the imidazole ring of catalytic residue His271 and further pushed the active site His271 away from its proper orientation. Moreover, the average distance between alcohol oxygen (Oalc) and HNe of catalytic His271 increased to 3.7 A, which was too far to form an essential hydrogen bond and further prevented efficient catalysis of slow enantiomer. This correlation of the distance between alcohol oxygen (Oalc) and HNe of catalytic His271 and the enantioselectivity was also confirmed by the result of site-directed mutagenesis.

The resolutions of racemic diastereomeric mixtures of menthyl propionate was performed by Pseudomonas alcaligenes lipase (PaL) to produce (2S, 5R) L-menthol. Because of the inherently low diastereopreference of PaL, covalent docking and molecular dynamic (MD) simulations were used to investigate possible avenues of improvement. Rational site-directed mutagenesis of PaL revealed residues V180 and A272 to be the hotspots for diastereopreference. The double V180L/A272F mutant exhibited the highest degree of diastereopreference, as the diastereomeric ratio of (2S, 5R) L-menthol increased towards both (2R, 5S) L-neomenthol (dr1) and (2R, 5R) D-isoneomenthol (dr2) (diastereomeric ratios dr1 and dr2 increased to 4.65 and 2.13 times that of wild-type PaL). MD simulation analysis indicated that these mutations decrease the flexibility of the surrounding protein regions. The combination of increased steric exclusion and decreased flexibility results in less favorable binding of the non-target substrates, (2R, 5S) L-neomenthyl propionate and (2R, 5R) D-isoneomenthyl propionate, to the V180L/A272F mutant. These results confirmed and further improved our previously proposed model of the diastereomer recognition mechanism based on the combined effect of steric exclusion and regional flexibility.

A novel esterase, MtEst45, was isolated from a fosmid genomic library of Microbulbifer thermotolerans DAU221. The encoding gene is predicted to have a mass of 45,564 Da and encodes 495 amino acids, excluding a 21 amino acid signal peptide. MtEst45 showed a low amino acid identity (approximately 23-24%) compared with other lipolytic enzymes belonging to Family III, a closely related bacterial lipolytic enzyme family. MtEst45 also showed a conserved GXSXG motif, G131IS133YG135, which was reported as active site of known lipolytic enzymes, and the putative catalytic triad composed of D237 and H265. Because these mutants of MtEst45, which was S133A, D237N, and H265L, had no activity, these catalytic triad is deemed essential for the enzyme catalysis. MtEst45 was overexpressed in Escherichia coli BL21 (DE3) and purified via His-tag affinity chromatography. The optimal pH and temperature of MtEst45 were estimated to be 8.17 and 46.27 degrees C by response surface methodology, respectively. Additionally, MtEst45 was also active between 1 and 15 degrees C. The optimal hydrolysis substrate for MtEst45 among p-nitrophenyl esters (C2-C18) was p-nitrophenyl butyrate, and the K m and V max values were 0.0998 mM and 550 mumol/min/mg of protein, respectively. MtEst45 was strongly inhibited by Hg(2+), Zn(2+), and Cu(2+) ions; by phenylmethanesulfonyl fluoride; and by beta-mercaptoethanol. Ca(2+) did not affect the enzyme's activity. These biochemical properties, sequence identity, and phylogenetic analysis suggest that MtEst45 represents a novel and valuable bacterial lipolytic enzyme family and is useful for biotechnological applications.

Bacterial lipases are an important group of enzymes that offer enormous potential in organic synthesis, and there is considerable interest in identifying and developing novel bacterial lipases. In previous studies, strains of the genus Stenotrophomonas were proved to be potential source of lipases, but there is little genetic information describing lipase from the genus Stenotrophomonas. We have cloned and characterized a novel lipase (LipSM54), the first lipase described from the genus Stenotrophomonas. Enzymatic study showed that LipSM54 was a cold-active, solvent-tolerant and alkaline lipase. Using bioinformatics tools, LipSM54 was found to be related only to several putative lipases from different bacterial origins, none of which could be assigned to any previously described bacterial lipase family. LipSM54 and these related putative lipases share four conserved motifs around the catalytic residues. These motifs clearly distinguish them from the known bacterial lipase families. Consequently, LipSM54 is the first characterized member of the novel bacterial lipase family.

In this work, a lipase from Pseudomonas alcaligenes CGMCC4405 (PaL) was cloned and expressed. It was very attractive that the recombinant PaL exhibited excellent enantioselectivity (E > 200) in the resolution of racemic d,l-menthyl propionate to produce l-menthol. The structure basis of enantiopreference is a fundamental scientific problem which needs to be resolved. In our research, molecular dynamic simulation (MD) research was employed to research the different binding modes of d and l-menthyl propionate. The results showed that when bound with slow-reacting enantiomer (d-menthyl propionate), the steric requirements of the large substituent (isopropyl) of the d-menthyl propionate force a rotation of the imidazole ring of catalytic residue His271 and further pushed the active site His271 away from its proper orientation. Moreover, the average distance between alcohol oxygen (Oalc) and HNe of catalytic His271 increased to 3.7 A, which was too far to form an essential hydrogen bond and further prevented efficient catalysis of slow enantiomer. This correlation of the distance between alcohol oxygen (Oalc) and HNe of catalytic His271 and the enantioselectivity was also confirmed by the result of site-directed mutagenesis.

The resolutions of racemic diastereomeric mixtures of menthyl propionate was performed by Pseudomonas alcaligenes lipase (PaL) to produce (2S, 5R) L-menthol. Because of the inherently low diastereopreference of PaL, covalent docking and molecular dynamic (MD) simulations were used to investigate possible avenues of improvement. Rational site-directed mutagenesis of PaL revealed residues V180 and A272 to be the hotspots for diastereopreference. The double V180L/A272F mutant exhibited the highest degree of diastereopreference, as the diastereomeric ratio of (2S, 5R) L-menthol increased towards both (2R, 5S) L-neomenthol (dr1) and (2R, 5R) D-isoneomenthol (dr2) (diastereomeric ratios dr1 and dr2 increased to 4.65 and 2.13 times that of wild-type PaL). MD simulation analysis indicated that these mutations decrease the flexibility of the surrounding protein regions. The combination of increased steric exclusion and decreased flexibility results in less favorable binding of the non-target substrates, (2R, 5S) L-neomenthyl propionate and (2R, 5R) D-isoneomenthyl propionate, to the V180L/A272F mutant. These results confirmed and further improved our previously proposed model of the diastereomer recognition mechanism based on the combined effect of steric exclusion and regional flexibility.