In Vitro Analysis of Roles of a Disulfide Bridge and a Calcium Binding Site in Activation of
            <i>Pseudomonas</i>
            sp. Strain KWI-56 Lipase

Yang, Jun; Kobayashi, Koei; Iwasaki, Yoshinobu; Nakano, Hideo; Yamané, Tsuneo

doi:10.1128/jb.182.2.295-302.2000

Cited by 44 publications

(21 citation statements)

References 41 publications

(49 reference statements)

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“…Site‐directed mutagenesis of the Ca 2+ binding site of Pseudomonas sp . lipase resulted in a reduction of the active enzyme (22). The known low dissociation constants for the binding of Ca 2+ that have been determined (e.g., thermitase (14)) do not exclude the possibility that this ion is already bound to the pre‐pro‐enzyme of these enzymes in the cell, where the free calcium ion concentration is less than 1 μM, i.e., a factor of 1000 lower than that outside the cell (23).…”

Section: Introductionmentioning

confidence: 99%

Ca2+ Is a Cofactor Required for Membrane Transport and Maturation and Is a Yield-Determining Factor in High Cell Density Penicillin Amidase Production

Kasche

Ignatova

Märkl

et al. 2008

Biotechnol Progress

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Penicillin amidases (PAs) from E. coli and A. faecalis are periplasmic enzymes that contain one tightly bound Ca(2+) per molecule that does not directly participate in the enzymatic function. This ion may, however, be required for the maturation of the pre-pro-enzyme. The pro-enzyme of homologous PAs are translocated through the Tat- (E. coli PA(EC)) and Sec- (A. faecalis PA(AF)) transport systems, respectively. Cell fractionation, electrophoresis, immunoblotting, and activity staining demonstrated that Ca(2+) binding is required for the membrane transport and maturation of the pro-enzyme to active enzyme. Pro-enzyme without Ca(2+) was targeted to the membrane but not translocated. Influence of Ca(2+) in medium and feed was studied for high cell density cultivations of E. coli expressing these enzymes. Without Ca(2+) in the feed the synthesis of the pre-pro-enzyme was hardly influenced. At optimal Ca(2+) content in the feed the active enzyme amount could be increased by 2 orders of magnitude up to 0.9 g/L (PA(EC)) and 2.3 g/L (PA(AF)) or 4% (PA(EC)) and 8% (PA(AF)) of the cell dry weight. The corresponding specific activities are 1700 U (PA(EC)) and 14000 U (PA(AF)) per gram cell dry weight, respectively. These values are higher than those published previously. Thus, for optimal yields of the studied and other extra- and periplasmic enzymes that require Ca(2+) or other ions as cofactors for membrane transport and maturation, sufficient cofactor must be added in the feed.

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Section: Introductionmentioning

confidence: 99%

Ca2+ Is a Cofactor Required for Membrane Transport and Maturation and Is a Yield-Determining Factor in High Cell Density Penicillin Amidase Production

Kasche

Ignatova

Märkl

et al. 2008

Biotechnol Progress

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“…An important role during the folding process was suggested for the cysteine residues of Burkholderia cepacia lipase, which could not be refolded in the presence of DTT or ␤-mercaptoethanol (17). Such a folding function was recently confirmed by investigation of cysteine-to-serine variants of the B. cepacia lipase for which only low activities were obtained in an in vitro transcription-translation system (48). Also the wildtype lipase from P. aeruginosa was refolded with lower efficiency in the presence of ␤-mercaptoethanol (37).…”

Section: Fig 4 Stability Of In Vitro Refolded Wild-type and Variantmentioning

confidence: 71%

Disulfide Bond in Pseudomonas aeruginosa Lipase Stabilizes the Structure but Is Not Required for Interaction with Its Foldase

2001

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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.The gram-negative bacterium Pseudomonas aeruginosa secretes an array of different enzymes via three distinct secretion pathways (40,42,43,46). Most of them are exported in two consecutive steps through the so-called type II secretory pathway into the extracellular medium (11), among them a lipase LipA with a molecular mass of 29 kDa (39). This lipase shows remarkable enzymatic characteristics, e.g., a pronounced stereoselectivity (32) and a broad substrate specificity (23), making it an interesting candidate for biotechnological applications (24).Enzymes being secreted via the type II secretion pathway contain a N-terminal signal sequence mediating the translocation across the cytoplasmic membrane by the Sec apparatus which has intensively been studied in Escherichia coli (8,31). An additional machinery consisting of at least 12 so-called Xcp proteins is required for the secretion of the periplasmic intermediates through the outer membrane of P. aeruginosa (11). Although much effort has been expended to identify a potential recognition signal for the Xcp machinery within the extracellular proteins which distinguishes them from periplasmic proteins, no common linear recognition sequence has so far been identified. There is increasing evidence that extracellular proteins fold into a transport-competent conformation before translocation across the outer membrane (3,5,(12)(13)(14)(15)30), suggesting that the formation necessary for secretion is a threedimensional structural element composed of noncontiguous amino acid residues or parts of the primary structure. Many of the proteins secreted via the type II pathway contain a disulfide bon...

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“…However, much difficulty has been encountered in expressing the lipases in E. coli, which is due mainly to their requirements for specific Lifs (13,17,35). We expect that the novel finding of this study and further characterization of LipK P112Q will be useful in studying the folding pathway of Pseudomonas lipases and provide an effective method for the expression of industrially important lipases.…”

Section: Vol 183 2001mentioning

confidence: 98%

“…Recently, Pseudomonas lipases were reclassified into six subfamilies among family I of bacterial lipases (4), since a large number of lipases were also isolated from other genera, and some Pseudomonas species have been reclassified as Burkholderia spp. (10,31,35). Subfamily I.1 includes the lipases from Pseudomonas aeruginosa (7,34) and Pseudomonas fragi (3), and subfamily I.2 includes those from Burkholderia cepacia and Burkholderia glumae (11,20).…”

mentioning

confidence: 99%

Lipase and Its Modulator from Pseudomonas sp. Strain KFCC 10818: Proline-to-Glutamine Substitution at Position 112 Induces Formation of Enzymatically Active Lipase in the Absence of the Modulator

Kim

Jang

et al. 2001

J Bacteriol

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In Vitro Analysis of Roles of a Disulfide Bridge and a Calcium Binding Site in Activation of Pseudomonas sp. Strain KWI-56 Lipase

Cited by 44 publications

References 41 publications

Ca2+ Is a Cofactor Required for Membrane Transport and Maturation and Is a Yield-Determining Factor in High Cell Density Penicillin Amidase Production

Ca2+ Is a Cofactor Required for Membrane Transport and Maturation and Is a Yield-Determining Factor in High Cell Density Penicillin Amidase Production

Disulfide Bond in Pseudomonas aeruginosa Lipase Stabilizes the Structure but Is Not Required for Interaction with Its Foldase

Lipase and Its Modulator from Pseudomonas sp. Strain KFCC 10818: Proline-to-Glutamine Substitution at Position 112 Induces Formation of Enzymatically Active Lipase in the Absence of the Modulator

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