Crystallization and crystal manipulation of a steric chaperone in complex with its lipase substrate

Pauwels, Kris; Loris, Remy; Vandenbussche, Guy; Ruysschaert, Jean Marie; Wyns, Lode; Gelder, Patrick Van

doi:10.1107/s1744309105023055

Cited by 10 publications

(5 citation statements)

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“…The co-expression strategy that we used has the advantage of being simpler than in vitro strategies. For instance, Pauwels et al [ 52 ],[ 68 ] expressed the lipase of B. glumae and its foldase separately, in homologous and heterologous hosts, respectively, and then combined them during the protein purification step, in which the Ni 2+ affinity column was loaded with separate extracts from each protein. Additionally, in vitro folding is not always particularly effective.…”

Section: Discussionmentioning

confidence: 99%

First co-expression of a lipase and its specific foldase obtained by metagenomics

et al. 2014

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BackgroundMetagenomics is a useful tool in the search for new lipases that might have characteristics that make them suitable for application in biocatalysis. This paper reports the cloning, co-expression, purification and characterization of a new lipase, denominated LipG9, and its specific foldase, LifG9, from a metagenomic library derived from a fat-contaminated soil.ResultsWithin the metagenomic library, the gene lipg9 was cloned jointly with the gene of the foldase, lifg9. LipG9 and LifG9 have 96% and 84% identity, respectively, with the corresponding proteins of Aeromonas veronii B565. LipG9 and LifG9 were co-expressed, both in N-truncated form, in Escherichia coli BL21(DE3), using the vectors pET28a(+) and pT7-7, respectively, and then purified by affinity chromatography using a Ni2+ column (HiTrap Chelating HP). The purified enzyme eluted from the column complexed with its foldase. The molecular masses of the N-truncated proteins were 32 kDa for LipG9, including the N-terminal His-tag with 6 residues, and 23 kDa for LifG9, which did not have a His-tag. The biochemical and kinetic characteristics of the purified lipase-foldase preparation were investigated. This preparation was active and stable over a wide range of pH values (6.5-9.5) and temperatures (10-40°C), with the highest specific activity, of 1500 U mg−1, being obtained at pH 7.5 at 30°C. It also had high specific activities against tributyrin, tricaprylin and triolein, with values of 1852, 1566 and 817 U mg−1, respectively. A phylogenetic analysis placed LipG9 in the lipase subfamily I.1. A comparison of the sequence of LipG9 with those of other bacterial lipases in the Protein Data Bank showed that LipG9 contains not only the classic catalytic triad (Ser103, Asp250, His272), with the catalytic Ser occurring within a conserved pentapeptide, Gly-His-Ser-His-Gly, but also a conserved disulfide bridge and a conserved calcium binding site. The homology-modeled structure presents a canonical α/β hydrolase folding type I.ConclusionsThis paper is the first to report the successful co-expression of a lipase and its associated foldase from a metagenomic library. The high activity and stability of Lip-LifG9 suggest that it has a good potential for use in biocatalysis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-014-0171-7) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

First co-expression of a lipase and its specific foldase obtained by metagenomics

et al. 2014

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“…B. glumae LipA and Lif were produced and purified as described previously [31] , [32] . Urea (>99.8% purity) was purchased from Rose Chemicals, guHCl (>99.5% purity) was obtained from Fluka, Tris (PlusOne) from Pharmacia Biotech.…”

Section: Methodsmentioning

confidence: 99%

Decoding the Folding of Burkholderia glumae Lipase: Folding Intermediates En Route to Kinetic Stability

et al. 2012

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The lipase produced by Burkholderia glumae folds spontaneously into an inactive near-native state and requires a periplasmic chaperone to reach its final active and secretion-competent fold. The B. glumae lipase-specific foldase (Lif) is classified as a member of the steric-chaperone family of which the propeptides of α-lytic protease and subtilisin are the best known representatives. Steric chaperones play a key role in conferring kinetic stability to proteins. However, until present there was no solid experimental evidence that Lif-dependent lipases are kinetically trapped enzymes. By combining thermal denaturation studies with proteolytic resistance experiments and the description of distinct folding intermediates, we demonstrate that the native lipase has a kinetically stable conformation. We show that a newly discovered molten globule-like conformation has distinct properties that clearly differ from those of the near-native intermediate state. The folding fingerprint of Lif-dependent lipases is put in the context of the protease-prodomain system and the comparison reveals clear differences that render the lipase-Lif systems unique. Limited proteolysis unveils structural differences between the near-native intermediate and the native conformation and sets the stage to shed light onto the nature of the kinetic barrier.

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“…Usually, they are encoded in a bicistronic operon together with the cognate lipase suggesting highly specific recognition of their target enzymes (Shibata et al 1998;El Khattabi et al 1999). The protein complex formed by the lipase and the Lif of B. glumae was crystallized and its structure solved (Pauwels et al 2005(Pauwels et al , 2006. In this complex, the foldase consists of a mainly α-helical motif comprised of 11 α-helices with the N-and C-terminal regions forming minidomains built up by 3 helices each.…”

Section: Family I: Triacylglycerol Lipasesmentioning

confidence: 99%

Classification of Lipolytic Enzymes from Bacteria

Kovačić

Babić

Krauß

et al. 2018

Aerobic Utilization of Hydrocarbons, Oils and Lipids

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Lipolytic enzymes including lipases and esterases comprise a versatile group of enzymes with diverse amino acid sequences but related three-dimensional structures. Despite the large number of bacterial lipolytic enzymes so far identified (~5000), only a small portion (<10%) was cloned, expressed, and experimentally studied. Twenty years ago, Arpigny and Jaeger published a seminal study which systematically grouped bacterial lipolytic enzymes into eight families according to similarity of their amino acid sequences and physiological properties (Arpigny and Jaeger, Biochem J 343:177-183, 1999). Here, we present a comprehensive overview as an extension of the original classification covering all 19 presently known families of lipolytic enzymes. The conserved features of sequences and structures are described for all families in order to simplify the assignment of newly discovered lipolytic enzymes to the respective family. Furthermore, we have correlated the biochemical properties of some enzymes with the nature of the often extremophilic microorganism from which the respective enzyme was isolated. This may help to identify lipase families with potential as biocatalysts in industrial applications. As an example, family XV enzymes are stable and active at elevated temperatures; thus, enzymes of this family represent a potential source for novel biocatalysts.

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Crystallization and crystal manipulation of a steric chaperone in complex with its lipase substrate

Cited by 10 publications

References 23 publications

First co-expression of a lipase and its specific foldase obtained by metagenomics

First co-expression of a lipase and its specific foldase obtained by metagenomics

Decoding the Folding of Burkholderia glumae Lipase: Folding Intermediates En Route to Kinetic Stability

Classification of Lipolytic Enzymes from Bacteria

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