ABSTRACT
A novel esterase, EstD11, has been discovered in a hot spring metagenomic library. It is a thermophilic and thermostable esterase with an optimum temperature of 60°C. A detailed substrate preference analysis of EstD11 was done using a library of chromogenic ester substrate that revealed the broad substrate specificity of EstD11 with significant measurable activity against 16 substrates with varied chain length, steric hindrance, aromaticity and flexibility of the linker between the carboxyl and the alcohol moiety of the ester. The tridimensional structures of EstD11 and the inactive mutant have been determined at atomic resolutions. Structural and bioinformatic analysis, confirm that EstD11 belongs to the family IV, the hormone-sensitive lipase (HSL) family, from the α/ß-hydrolase superfamily. The canonical α/ß-hydrolase domain is completed by a cap domain, composed by two subdomains that can unmask of the active site to allow the substrate to enter. Eight crystallographic complexes were solved with different substrates and reaction products that allowed identification of the hot-spots in the active site underlying the specificity of the protein. Crystallization and/or incubation of EstD11 at high temperature provided unique information on cap dynamics and a first glimpse of enzymatic activity in vivo. Very interestingly, we have discovered a unique Met zipper lining the active site and the cap domains that could be essential in pivotal aspects as thermo-stability and substrate promiscuity in EstD11.
ABSTRACT
Carica papaya latex is one of the most studied sources of plant lipases. However, the complexity of the matrix composition makes it difficult to isolate and purify the lipolytic enzymes present in Carica papaya latex. Therefore, diverse strategies have been developed to study the catalytic properties of these enzymes.Recently the first lipase from Carica papaya latex (CpLip1) has been successfully cloned and expressed in order to study their catalytic properties. In order to improve the catalytic properties and increase the potential for its use at industrial scale.In this chapter, a practical protocol to recombinant CpLip1 lipase is given.
Subject(s)
Carica/enzymology , Gene Expression , Lipase/metabolism , Bile Acids and Salts/pharmacology , Carica/genetics , Enzyme Activation/drug effects , Lipase/geneticsABSTRACT
Cell wall recycling and ß-lactam antibiotic resistance are linked in Enterobacteriaceae and in Pseudomonas aeruginosa. This process involves a large number of murolytic enzymes, among them a cytoplasmic peptidoglycan amidase AmpD, which plays an essential role by cleaving the peptide stem from key intermediates en route to the ß-lactamase production (a resistance mechanism) and cell wall recycling. Uniquely, P. aeruginosa has two additional paralogues of AmpD, designated AmpDh2 and AmpDh3, which are periplasmic enzymes. Despite the fact that AmpDh2 and AmpDh3 share a common motif for their respective catalytic domains, they are each comprised of multidomain architectures and exhibit distinct oligomerization properties. We review herein the structural and biochemical properties of orthologous and paralogous AmpD proteins and discuss their implication in cell wall recycling and antibiotic resistance processes.
Subject(s)
Bacterial Proteins/chemistry , Cell Wall/enzymology , Enterobacteriaceae/enzymology , Metalloproteases/chemistry , N-Acetylmuramoyl-L-alanine Amidase/chemistry , Periplasm/enzymology , Pseudomonas aeruginosa/enzymology , Virulence Factors/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Catalytic Domain , Cell Wall/chemistry , Drug Resistance, Microbial/genetics , Enterobacteriaceae/genetics , Gene Expression , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/metabolism , Metalloproteases/genetics , Metalloproteases/metabolism , Models, Molecular , N-Acetylmuramoyl-L-alanine Amidase/genetics , N-Acetylmuramoyl-L-alanine Amidase/metabolism , Peptidoglycan/metabolism , Periplasm/chemistry , Protein Multimerization , Protein Structure, Secondary , Pseudomonas aeruginosa/genetics , Structural Homology, Protein , Virulence Factors/genetics , Virulence Factors/metabolismABSTRACT
Lipases from plants have very interesting features for application in different fields. This chapter provides an overview on some of the most important aspects of plant lipases, such as sources, applications, physiological functions, and specificities. Lipases from laticifers and particularly Carica papaya lipase (CPL) have emerged as a versatile autoimmobilized biocatalyst. However, to get a better understanding of CPL biocatalytic properties, the isolation and purification of individual C. papaya lipolytic enzymes become necessary. In this chapter, a practical protocol for partial purification of the latex-associated lipolytic activity from C. papaya is given.
