Biomedical subjects

K E Jaeger

Publications and source records attributed to K E Jaeger.

At least 19 recordsLinked to original sources

PubMed2001

Lipolytic enzymes LipA and LipB from Bacillus subtilis differ in regulation of gene expression, biochemical properties, and three-dimensional structure.

Bacillus subtilis secretes the lipolytic enzymes LipA and LipB. We show here that they are differentially expressed depending on the composition of the growth medium: LipA is produced in rich and in minimal medium, whereas LipB is present only in rich medium. A comparison of biochemical characteristics revealed that LipB is thermostable at pH 11 but becomes thermolabile at pH 5. However, construction of a variant carrying the substitution A76G in the conserved lipase pentapeptide reversed these effects. The atomic coordinates from the LipA crystal structure were used to build a three-dimensional structural model of LipB, which revealed that 43 out of 45 residues different from LipA are surface-located allowing to rationalize the differences observed in the substrate preferences of the two enzymes.

Bacillus subtilis↗

PubMed2001

The crystal structure of Bacillus subtilis lipase: a minimal alpha/beta hydrolase fold enzyme.

The X-ray structure of the lipase LipA from Bacillus subtilis has been determined at 1.5 A resolution. It is the first structure of a member of homology family 1.4 of bacterial lipases. The lipase shows a compact minimal alpha/beta hydrolase fold with a six-stranded parallel beta-sheet flanked by five alpha-helices, two on one side of the sheet and three on the other side. The catalytic triad residues, Ser77, Asp133 and His156, and the residues forming the oxyanion hole (backbone amide groups of Ile12 and Met78) are in positions very similar to those of other lipases of known structure. However, no lid domain is present and the active-site nucleophile Ser77 is solvent-exposed. A model of substrate binding is proposed on the basis of a comparison with other lipases with a covalently bound tetrahedral intermediate mimic. It explains the preference of the enzyme for substrates with C8 fatty acid chains.

Acyltransferases↗

PubMed2001

Directed evolution and the creation of enantioselective biocatalysts.

Directed evolution has emerged as a key technology to generate enzymes with new or improved properties that are of major importance to the biotechnology industry. A directed evolution approach starts with the identification of a target enzyme to be optimized and the cloning of the corresponding gene. An efficient expression system is needed before the target gene is subjected to random mutagenesis and/or in vitro recombination, thereby creating molecular diversity. Subsequently, improved enzyme variants are identified, preferably after being secreted into culture medium, by screening or selection for the desired property. The genes encoding the improved enzymes are then used to parent the next round of directed evolution. Enantioselectivity is a biocatalyst property of major biotechnological importance that is, however, difficult to deal with. We discuss recent examples of creating enantioselective biocatalysts by directed evolution.

Amidohydrolases↗

PubMed2001

DsbA and DsbC affect extracellular enzyme formation in Pseudomonas aeruginosa.

DsbA and DsbC proteins involved in the periplasmic formation of disulfide bonds in Pseudomonas aeruginosa were identified and shown to play an important role for the formation of extracellular enzymes. Mutants deficient in either dsbA or dsbC or both genes were constructed, and extracellular elastase, alkaline phosphatase, and lipase activities were determined. The dsbA mutant no longer produced these enzymes, whereas the lipase activity was doubled in the dsbC mutant. Also, extracellar lipase production was severely reduced in a P. aeruginosa dsbA mutant in which an inactive DsbA variant carrying the mutation C34S was expressed. Even when the lipase gene lipA was constitutively expressed in trans in a lipA dsbA double mutant, lipase activity in cell extracts and culture supernatants was still reduced to about 25%. Interestingly, the presence of dithiothreitol in the growth medium completely inhibited the formation of extracellular lipase whereas the addition of dithiothreitol to a cell-free culture supernatant did not affect lipase activity. We conclude that the correct formation of the disulfide bond catalyzed in vivo by DsbA is necessary to stabilize periplasmic lipase. Such a stabilization is the prerequisite for efficient secretion using the type II pathway.

Alkaline Phosphatase↗

PubMed2001

Disulfide bond in Pseudomonas aeruginosa lipase stabilizes the structure but is not required for interaction with its foldase.

Pseudomonas aeruginosa secretes a 29-kDa lipase which is dependent for folding on the presence of the lipase-specific foldase Lif. The lipase contains two cysteine residues which form an intramolecular disulfide bond. Variant lipases with either one or both cysteines replaced by serines showed severely reduced levels of extracellular lipase activity, indicating the importance of the disulfide bond for secretion of lipase through the outer membrane. Wild-type and variant lipase genes fused to the signal sequence of pectate lyase from Erwinia carotovora were expressed in Escherichia coli, denatured by treatment with urea, and subsequently refolded in vitro. Enzymatically active lipase was obtained irrespective of the presence or absence of the disulfide bond, suggesting that the disulfide bond is required neither for correct folding nor for the interaction with the lipase-specific foldase. However, cysteine-to-serine variants were more readily denatured by treatment at elevated temperatures and more susceptible to proteolytic degradation by cell lysates of P. aeruginosa. These results indicate a stabilizing function of the disulfide bond for the active conformation of lipase. This conclusion was supported by the finding that the disulfide bond function could partly be substituted by a salt bridge constructed by changing the two cysteine residues to arginine and aspartate, respectively.

Biological Transport↗

PubMed2000

Rapid gene inactivation in Pseudomonas aeruginosa.

A rapid and efficient method to inactivate genes in Pseudomonas aeruginosa has been developed. It is based on pKnockout vectors which carry either a gentamicin or a streptomycin/spectinomycin resistance cassette allowing for selection in P. aeruginosa where these vectors do not replicate. A PCR fragment of the gene of interest carrying 5'- and 3'-truncations is cloned into a pKnockout vector, mobilized into P. aeruginosa, and subsequently integrated into the chromosomal copy of the target gene. The orientation of the fragment determines whether (i) the target gene is disrupted without blocking the transcription of downstream genes or (ii) the insertion exerts a polar effect thereby leading to inactivation of a whole operon.

Conjugation, Genetic↗

PubMed2000

Crystal structure of pseudomonas aeruginosa lipase in the open conformation. The prototype for family I.1 of bacterial lipases.

The x-ray structure of the lipase from Pseudomonas aeruginosa PAO1 has been determined at 2.54 A resolution. It is the first structure of a member of homology family I.1 of bacterial lipases. The structure shows a variant of the alpha/beta hydrolase fold, with Ser(82), Asp(229), and His(251) as the catalytic triad residues. Compared with the "canonical" alpha/beta hydrolase fold, the first two beta-strands and one alpha-helix (alphaE) are not present. The absence of helix alphaE allows the formation of a stabilizing intramolecular disulfide bridge. The loop containing His(251) is stabilized by an octahedrally coordinated calcium ion. On top of the active site a lid subdomain is in an open conformation, making the catalytic cleft accessible from the solvent region. A triacylglycerol analogue is covalently bound to Ser(82) in the active site, demonstrating the position of the oxyanion hole and of the three pockets that accommodate the sn-1, sn-2, and sn-3 fatty acid chains. The inhibited enzyme can be thought to mimic the structure of the tetrahedral intermediate that occurs during the acylation step of the reaction. Analysis of the binding mode of the inhibitor suggests that the size of the acyl pocket and the size and interactions of the sn-2 binding pocket are the predominant determinants of the regio- and enantio-preference of the enzyme.

Amino Acid Sequence↗

PubMed2000

Enantioselective enzymes for organic synthesis created by directed evolution.

A new concept for the creation of enzymes displaying improved enantioselectivity in a given reaction is described; it is based on "evolution in the test tube". Accordingly, proper molecular biological methods for random mutagenesis, gene expression, and high-throughput screening systems for the rapid assay of enantioselectivity are combined. Several rounds of mutagenesis and screening are generally necessary in order to create mutant enzymes that show high degrees of enantioselectivity, as in the case of the lipase-catalyzed hydrolytic kinetic resolution of a chiral ester in which the original enantioselectivity of 2 % ee (E = 1) increases to > 90% ee (E = 25).

Catalysis↗

PubMed2000

Directed evolution of an enantioselective lipase.

BACKGROUND: The biocatalytic production of enantiopure compounds is of steadily increasing importance to the chemical and biotechnological industry. In most cases, however, it is impossible to identify an enzyme that possesses the desired enantioselectivity. Therefore, there is a strong need to create by molecular biological methods novel enzymes which display high enantioselectivity. RESULTS: A bacterial lipase from Pseudomonas aeruginosa (PAL) was evolved to catalyze with high enantioselectivity the hydrolysis of the chiral model substrate 2-methyldecanoic acid p-nitrophenyl ester. Successive rounds of random mutagenesis by ep-PCR and saturation mutagenesis resulted in an increase in enantioselectivity from E=1.1 for the wild-type enzyme to E=25.8 for the best variant which carried five amino acid substitutions. The recently solved three-dimensional structure of PAL allowed us to analyze the structural consequences of these substitutions. CONCLUSIONS: A highly enantioselective lipase was created by increasing the flexibility of distinct loops of the enzyme. Our results demonstrate that enantioselective enzymes can be created by directed evolution, thereby opening up a large area of novel applications in biotechnology.

Amino Acid Sequence↗

PubMed2000

Directed evolution of enantioselective enzymes for organic chemistry.

The production of enantiopure compounds is of steadily increasing importance to the chemical and biotechnological industries. In principal, the application of directed evolution in combination with newly developed screening methods enables the generation of enzymes with improved enantioselectivity. The first and most advanced example relates to a bacterial lipase from Pseudomonas aeruginosa. This enzyme was evolved towards a model substrate to yield in a lipase mutant showing > 90% enantiomeric excess as compared to 2% for the wild-type lipase. The creation of enantioselective enzymes by directed evolution will become an important technology in the near future.

Chemistry, Organic↗

PubMed2000

A novel extracellular esterase from Bacillus subtilis and its conversion to a monoacylglycerol hydrolase.

A novel gene lipB, which encodes an extracellular lipolytic enzyme, was identified in the Bacillus subtilis genomic DNA sequence. We have cloned and overexpressed lipB in B. subtilis and Escherichia coli and have also purified the enzyme from a B. subtilis culture supernatant to electrophoretic homogeneity. Four different lipase assays were used to determine its catalytic activity: pH-stat, spectrophotometry, fluorimetry and the monomolecular film technique. LipB preferentially hydrolysed triacylglycerol-esters and p-nitrophenyl-esters of fatty acids with short chain lengths of </= 10 carbon atoms. Triolein, which is a typical substrate for true lipases, was not hydrolysed at all. These results led us to classify LipB as an esterase rather than a lipase. The catalytic triad of LipB consists of residues Ser78, Asp134, and His157 as demonstrated by amino-acid sequence alignments and site-directed mutagenesis. The nucleophile Ser78 is located in a lipase-specific consensus sequence, which is Ala-X-Ser-X-Gly for most Bacillus lipases. All other bacterial lipases contain a glycine residue instead of the alanine at position-2 with respect to the catalytic serine. We have investigated the role of this alanine residue by constructing LipB variant A76G, thereby restoring the lipase-specific consensus motif. When compared with LipB this variant showed a markedly reduced thermostability but an increased stability at pH 5-7. Determination of the specific activities of wild-type LipB and variant A76G using a monomolecular film of the substrate monoolein revealed an interesting result: the A76G substitution had converted the esterase LipB into a monoacylglycerol hydrolase.

Amino Acid Sequence↗

PubMed1999

Bacterial lipolytic enzymes: classification and properties.

Knowledge of bacterial lipolytic enzymes is increasing at a rapid and exciting rate. To obtain an overview of this industrially very important class of enzymes and their characteristics, we have collected and classified the information available from protein and nucleotide databases. Here we propose an updated and extensive classification of bacterial esterases and lipases based mainly on a comparison of their amino acid sequences and some fundamental biological properties. These new insights result in the identification of eight different families with the largest being further divided into six subfamilies. Moreover, the classification enables us to predict (1) important structural features such as residues forming the catalytic site or the presence of disulphide bonds, (2) types of secretion mechanism and requirement for lipase-specific foldases, and (3) the potential relationship to other enzyme families. This work will therefore contribute to a faster identification and to an easier characterization of novel bacterial lipolytic enzymes.

Amino Acid Sequence↗

PubMed1999

Specificity of the lipase-specific foldases of gram-negative bacteria and the role of the membrane anchor.

Folding of lipases that are secreted by Pseudomonads and other gram-negative bacteria via the type II secretion pathway is facilitated by dedicated chaperones, called lipase-specific foldases (Lifs). Lifs are membrane-anchored proteins with a large periplasmic domain. The functional interaction between the Lif and its cognate lipase is specific, since the Pseudomonas aeruginosa Lif was found not to substitute for Lifs from Burkholderia glumae or Acinetobacter calcoaceticus. However, the P. aeruginosa Lif was able to activate the lipase from the closely related species P. alcaligenes. Hybrid proteins constructed from parts of the P. aeruginosa and B. glumae Lifs revealed that the C-terminal 138 amino acids of the B. glumae Lif determine the specificity of the interaction with the cognate lipase. Furthermore, the periplasmic domain of the B. glumae Lif was functional when cloned in frame with a cleavable signal sequence, which demonstrates that the membrane anchor is not essential for Lif function in vivo. However, the recombinant Lif was released into the medium, indicating that the function of the membrane anchor is to prevent secretion of the Lif together with the lipase.

Acinetobacter calcoaceticus↗

PubMed1999

A novel lipolytic enzyme located in the outer membrane of Pseudomonas aeruginosa.

A lipase-negative deletion mutant of Pseudomonas aeruginosa PAO1 still showed extracellular lipolytic activity toward short-chain p-nitrophenylesters. By screening a genomic DNA library of P. aeruginosa PAO1, an esterase gene, estA, was identified, cloned, and sequenced, revealing an open reading frame of 1,941 bp. The product of estA is a 69.5-kDa protein, which is probably processed by removal of an N-terminal signal peptide to yield a 67-kDa mature protein. A molecular mass of 66 kDa was determined for (35)S-labeled EstA by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The amino acid sequence of EstA indicated that the esterase is a member of a novel GDSL family of lipolytic enzymes. The estA gene showed high similarity to an open reading frame of unknown function located in the trpE-trpG region of P. putida and to a gene encoding an outer membrane esterase of Salmonella typhimurium. Amino acid sequence alignments led us to predict that this esterase is an autotransporter protein which possesses a carboxy-terminal beta-barrel domain, allowing the secretion of the amino-terminal passenger domain harboring the catalytic activity. Expression of estA in P. aeruginosa and Escherichia coli and subsequent cell fractionation revealed that the enzyme was associated with the cellular membranes. Trypsin treatment of whole cells released a significant amount of esterase, indicating that the enzyme was located in the outer membrane with the catalytic domain exposed to the surface. To our knowledge, this esterase is unique in that it exemplifies in P. aeruginosa (i) the first enzyme identified in the outer membrane and (ii) the first example of a type IV secretion mechanism.

Amino Acid Sequence↗

PubMed1999

Bacterial biocatalysts: molecular biology, three-dimensional structures, and biotechnological applications of lipases.

Bacteria produce and secrete lipases, which can catalyze both the hydrolysis and the synthesis of long-chain acylglycerols. These reactions usually proceed with high regioselectivity and enantioselectivity, and, therefore, lipases have become very important stereoselective biocatalysts used in organic chemistry. High-level production of these biocatalysts requires the understanding of the mechanisms underlying gene expression, folding, and secretion. Transcription of lipase genes may be regulated by quorum sensing and two-component systems; secretion can proceed either via the Sec-dependent general secretory pathway or via ABC transporters. In addition, some lipases need folding catalysts such as the lipase-specific foldases and disulfide-bond-forming proteins to achieve a secretion-competent conformation. Three-dimensional structures of bacterial lipases were solved to understand the catalytic mechanism of lipase reactions. Structural characteristics include an alpha/beta hydrolase fold, a catalytic triad consisting of a nucleophilic serine located in a highly conserved Gly-X-Ser-X-Gly pentapeptide, and an aspartate or glutamate residue that is hydrogen bonded to a histidine. Four substrate binding pockets were identified for triglycerides: an oxyanion hole and three pockets accommodating the fatty acids bound at position sn-1, sn-2, and sn-3. The differences in size and the hydrophilicity/hydrophobicity of these pockets determine the enantiopreference of a lipase. The understanding of structure-function relationships will enable researchers to tailor new lipases for biotechnological applications. At the same time, directed evolution in combination with appropriate screening systems will be used extensively as a novel approach to develop lipases with high stability and enantioselectivity.

Bacteria↗

PubMed1998

A novel heat-stable lipolytic enzyme from Sulfolobus acidocaldarius DSM 639 displaying similarity to polyhydroxyalkanoate depolymerases.

A fragment of genomic DNA from Sulfolobus acidocaldarius DSM 639 encoding a lipolytic enzyme was cloned and sequenced. The 314-amino acid polypeptide displays a maximum sequence similarity (43%) to a putative polyhydroxyalkanoate depolymerase from Pseudomonas oleovorans and contains the pentapeptide G-X1-S-X2-G which is typical of serine hydrolases. The protein is highly thermostable and is able to hydrolyse a variety of lipid substrates thus providing a promising tool for potential biotechnological applications.

Amino Acid Sequence↗

PubMed1998

Overexpression, immobilization and biotechnological application of Pseudomonas lipases.

Pseudomonas lipases play an important role in biotechnology both as hydrolases for detergent additives and as synthases catalyzing the kinetic resolution of racemic compounds. Large-scale production of Pseudomonas lipases requires correct folding and secretion through the bacterial membranes. Controllable expression of the gene lipH encoding a lipase-specific foldase proves to be important for overexpression in the homologous host Escherichia coli. Construction of appropriate His-tagged fusion proteins permitted overexpression, secretion and one-step purification of lipase from culture supernatants of the homologous host Pseudomonas aeruginosa. The immobilization of lipases in hydrophobic sol-gel materials derived from alkylsilane precursors of the type RSi(OCH3)3 or mixtures of RSi(OCH3)3 and Si(OCH3)4 provides highly active chemically and thermally stable heterogeneous biocatalysts. The entrapped lipases are excellent catalysts in a variety of synthetic organic transformations. Using directed evolution based on error prone PCR, the enantioselectivity of the hydrolysis of a chiral ester, catalyzed by the lipase from P. aeruginosa, can be increased from ee 2 to ee 81% in just four mutagenesis cycles.

Bacterial Proteins↗

PubMed1998

Microbial lipases form versatile tools for biotechnology.

Lipases are secreted into the culture medium by many bacteria and fungi. They catalyse not only the hydrolysis but also the synthesis of long-chain acylglycerols. Important uses in biotechnology include their addition to detergents, the manufacture of food ingredients, pitch control in the pulp and paper industry, and biocatalysis of stereoselective transformations. This makes them the most widely used class of enzymes in organic chemistry. Immobilization in hydrophobic sol-gel matrices and in vitro evolution are promising novel approaches to increasing the stability or enantioselectivity, respectively, of lipases.

Bacteria↗