Biserka Kojić-Prodić

Probing Enzyme Promiscuity of SGNH Hydrolases

Several hydrolases of the SGNH superfamily, including the lipase SrLip from Streptomyces rimosus (Q93MW7), the acyl‐CoA thioesterase I TesA from Pseudomonas aeruginosa (Q9HZY8) and the two lipolytic enzymes EstA (from P. aeruginosa, O33407) and EstP (from Pseudomonas putida, Q88QS0), were examined for promiscuity. These enzymes were tested against four chemically different classes of a total of 34 substrates known to be hydrolysed by esterases, thioesterases, lipases, phospholipases, Tweenases and proteases. Furthermore, they were also analysed with respect to their amino acid sequences and structural homology, and their phylogenetic relationship was determined. The Pseudomonas esterases EstA and EstP each have an N‐terminal domain with catalytic activity together with a C‐terminal autotransporter domain, and so the hybrid enzymes EstAN–EstPC and EstPN–EstAC were constructed by swapping the corresponding N‐ and C‐terminal domains, and their hydrolytic activities were compared. Interestingly, substrate specificity and kinetic measurements indicated a significant influence of the autotransporter domains on the catalytic activities of these enzymes in solution. TesA, EstA and EstP were shown to function as esterases with different affinities and catalytic efficacies towards p‐nitrophenyl butyrate. Of all the enzymes tested, only SrLip revealed lipase, phospholipase, esterase, thioesterase and Tweenase activities.

Structural and Functional Characterisation of TesA - A Novel Lysophospholipase A from Pseudomonas aeruginosa

TesA from Pseudomonas aeruginosa belongs to the GDSL hydrolase family of serine esterases and lipases that possess a broad substrate- and regiospecificity. It shows high sequence homology to TAP, a multifunctional enzyme from Escherichia coli exhibiting thioesterase, lysophospholipase A, protease and arylesterase activities. Recently, we demonstrated high arylesterase activity for TesA, but only minor thioesterase and no protease activity. Here, we present a comparative analysis of TesA and TAP at the structural, biochemical and physiological levels. The crystal structure of TesA was determined at 1.9 Å and structural differences were identified, providing a possible explanation for the differences in substrate specificities. The comparison of TesA with other GDSL-hydrolase structures revealed that the flexibility of active-site loops significantly affects their substrate specificity. This assumption was tested using a rational approach: we have engineered the putative coenzyme A thioester binding site of E. coli TAP into TesA of P. aeruginosa by introducing mutations D17S and L162R. This TesA variant showed increased thioesterase activity comparable to that of TAP. TesA is the first lysophospholipase A described for the opportunistic human pathogen P. aeruginosa. The enzyme is localized in the periplasm and may exert important functions in the homeostasis of phospholipids or detoxification of lysophospholipids.

Structural characterization of extracellular lipase from Streptomyces rimosus: assignment of disulfide bridge pattern by mass spectrometry

Abstract The cloning, sequencing and high-level expression of the gene encoding extracellular lipase from Streptomyces rimosus R6-554W have been recently described, and the primary structure of this gene product was deduced using a bioinformatic approach. In this study, capillary electrophoresis-on-the-chip and mass spectrometry were used to characterize native and overexpressed extracellular lipase protein from S. rimosus. The exact molecular mass of the wild-type and the overexpressed lipase, determined by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, were in excellent agreement (Δm=0.11 Da and Δm=0.26 Da, respectively) with a value of 24165.76 Da calculated from the structure deduced from the nucleotide sequence, considering the mature enzyme with all six cysteines forming disulfide bridges. The primary structure derived from the nucleotide sequence was completely verified using a combination of tryptic digestion and formic acid cleavage of the protein, followed by peptide mass fingerprinting. Selected peptides were further investigated by MALDI low-energy collision-induced dissociation hybrid tandem mass spectrometry, allowing the unambiguous determination of their predicted amino acid sequence. No post-translational modifications of mature S. rimosus lipase were detected. Comparison of the peptide mass fingerprints from the reduced and non-reduced overexpressed enzyme unequivocally revealed three intramolecular disulfide bonds with the following linkages: C27-C52, C93-C101 and C151-C198.

Crystal and molecular structures of substituted β-D-glucofuranosidurono-6,3-lactones

Abstract The crystal and molecular structures of methyl β- d -glucofuranosidurono-6,3-lactone (1), 5-O-pivaloyl-β- d -glucofuranurono-6,3-lactone (2), and methyl 2-O-acetyl-5-O-pivaloyl-β- d -glucofuranosidurono-6,3-l (3) were determined by X-ray analysis. Crystals of compound 1 are orthorhombic, space group P212121 with the unit cell a = 7.074(1), b = 7.448(1), c = 14.995(2) A, Z = 4. Crystals of 2 and 3 are monoclinic, space group P21. The unit-cell parameters are a = 6.315(1), b = 9.307(2), c = 10.612(2) A, β = 98.68(1)°, Z = 2 for 2; and a = 13.337(1), b = 10.296(1), c = 13.544(1) A, β = 118.24(1)°, Z = 4 for 3. The structures were solved by MULTAN-80 and refined by a full-matrix procedure to final values of R = 0.042(1), 0.048(2), and 0.093(3). The furanoid rings in 1, 2, and conformer B of 3 appear in twisted. 1T2 conformation, and the lactone rings adopt conformations between T and E. The lactone ring of conformer A of 3 adopts a T conformation and that of the furanoid ring is E2. The molecular packing is through the hydrogen bonds involving hydroxyl groups HO-2 · ⋯ O-1 [2.812(2) A] in 1. A hydrogen bond between a hydroxyl group and the pivaloyl carbonyl group, HO-1 · ⋯ O-7 [2.855(3) A], connects the molecules of 2. There are no free hydroxyl groups in 3 and molecular packing reflects van der Waals interactions only.

Horseradish esterases: detection, purification and identification

Our goal is to characterize esterases from horseradish tissues and assign their physiological roles. In the present study we focused on isolation, purification and identification of esterases from different horseradish tissues: plantlets and two tumor tissue lines. Horizontal IEF system enabled separation of six esterase isoforms with quite different pI values as well as with pronounced differences in expression levels among analyzed tissues. Esterases were extracted, fractionated by means of cation exchange chromatography, and analyzed by planar gel electrophoresis (SDS–PAGE) and isoelectrical focusing (IEF), UV/Vis spectroscopy, MALDI mass spectrometry (MS) and MALDI-MS/MS. Several chromatographic strategies were applied for esterase purification and characterization. Two subsequent cation exchange chromatographic steps based on SP-Sepharose FF material, followed by in-solution digestion combined with MALDI-MS and MS/MS proved to be the best strategy for identification of two esterase proteins, namely Pectinesterase/pectinesterase inhibitor 18 and GDSL esterase/lipase ESM1.

Crystal and molecular structures of substituted α-d-glucopyranosides

Abstract The crystal and molecular structures of methyl 2,4,6-tri- O -pivaloyl-α- d -glucopyranoside ( 1 ), methyl 4,6- O -( R )-benzylidene-2- O -pivaloyl-α- d -glucopyranoside ( 2 ), and methyl 4,6- O -( R )-benzylidene-2,3-di- O -pivaloyl-α- d -glucopyranoside ( 3 ) were determined by X-ray analysis. Crystals of 1 are orthorhombic, space group P 2 1 2 1 2 1 with the unit cell a = 13.026(2), b = 16.832, c = 11.929(2) A, Z = 4. Crystals of 2 are monoclinic, space group P 2 1 . The unit-cell parameters are a = 6.519(1), b = 14.664(4), c = 10.635(4) A, β = 93.18(1)°, Z = 2. Crystals of 3 are orthorhombic, space group P 2 1 2 1 2 1 with a = 10.006(3), b = 13.874(3), c = 18.527(5) A, Z = 4. The structures were solved by MULTAN and refined by a full-matrix procedure to final values of R = 0.084 ( 1 ), 0.048 ( 2 ), and 0.069 ( 3 ). The pyranose ring in each compound adopts the 4 C 1 conformation. The 1,3-dioxane rings in 2 and 3 show a chair conformation. The molecular packing in 1 is through the hydrogen bonds involving HO-3 and the 6- O -pivaloyl carbonyl group [HO-3 ⋯ O-9, 2.855(8) A], which connect the molecules into a chain along . The endocyclic oxygen atom is involved in an intermolecular hydrogen-bond with HO-3 [2.848(4) A], joining molecules of 2 into the chains along . There are no free hydroxyl groups in 3 and molecular packing reflects van der Waals interactions only.