B.S. Kim

Crystal Structure and Functional Characterization of an Esterase (EaEST) from Exiguobacterium antarcticum.

A novel microbial esterase, EaEST, from a psychrophilic bacterium Exiguobacterium antarcticum B7, was identified and characterized. To our knowledge, this is the first report describing structural analysis and biochemical characterization of an esterase isolated from the genus Exiguobacterium. Crystal structure of EaEST, determined at a resolution of 1.9 Å, showed that the enzyme has a canonical α/β hydrolase fold with an α-helical cap domain and a catalytic triad consisting of Ser96, Asp220, and His248. Interestingly, the active site of the structure of EaEST is occupied by a peracetate molecule, which is the product of perhydrolysis of acetate. This result suggests that EaEST may have perhydrolase activity. The activity assay showed that EaEST has significant perhydrolase and esterase activity with respect to short-chain p-nitrophenyl esters (≤C8), naphthyl derivatives, phenyl acetate, and glyceryl tributyrate. However, the S96A single mutant had low esterase and perhydrolase activity. Moreover, the L27A mutant showed low levels of protein expression and solubility as well as preference for different substrates. On conducting an enantioselectivity analysis using R- and S-methyl-3-hydroxy-2-methylpropionate, a preference for R-enantiomers was observed. Surprisingly, immobilized EaEST was found to not only retain 200% of its initial activity after incubation for 1 h at 80°C, but also retained more than 60% of its initial activity after 20 cycles of reutilization. This research will serve as basis for future engineering of this esterase for biotechnological and industrial applications.

Biochemical and Structural Analysis of a Novel Esterase from Caulobacter crescentus related to Penicillin-Binding Protein (PBP).

Considering that the prevalence of antibiotic-resistant pathogenic bacteria is largely increasing, a thorough understanding of penicillin-binding proteins (PBPs) is of great importance and crucial significance because this enzyme family is a main target of β-lactam-based antibiotics. In this work, combining biochemical and structural analysis, we present new findings that provide novel insights into PBPs. Here, a novel PBP homologue (CcEstA) from Caulobacter crescentus CB15 was characterized using native-PAGE, mass spectrometry, gel filtration, CD spectroscopy, fluorescence, reaction kinetics, and enzyme assays toward various substrates including nitrocefin. Furthermore, the crystal structure of CcEstA was determined at a 1.9 Å resolution. Structural analyses showed that CcEstA has two domains: a large α/β domain and a small α-helix domain. A nucleophilic serine (Ser68) residue is located in a hydrophobic groove between the two domains along with other catalytic residues (Lys71 and Try157). Two large flexible loops (UL and LL) of CcEstA are proposed to be involved in the binding of incoming substrates. In conclusion, CcEstA could be described as a paralog of the group that contains PBPs and β-lactamases. Therefore, this study could provide new structural and functional insights into the understanding this protein family.

Characterization of a novel SGNH-type esterase from Lactobacillus plantarum

Lactic acid bacteria (LAB) are sources of a large variety of microbial ester hydrolases because they can produce a wide range of short-chain esters, phenolic alcohols, and fatty acids. Here, a novel SGNH-type esterase (LpSGNH1) from Lactobacillus plantarum WCFS1 was identified, functionally characterized, and immobilized for biotechnological applications. Homologs of LpSGNH1 are also found in many lactic acid bacteria (LAB) species. Biochemical features of LpSGNH1 were investigated using mass spectrometry, gel filtration chromatography, enzyme kinetics, fluorescence, and circular dichroism (CD) spectroscopy. LpSGNH1 were retained its activity under conditions that would be encountered during fermentations. Interestingly, LpSGNH1 exhibited the ability to act on a broad range of substrates including ketoprofen acetate, cefotaxime (CTX), and 7-aminocephalosporanic acid (7-ACA) as well as glucose pentaacetate, acetylxylan, and acetylalginate, which make LpSGNH1 a great candidate for extensive industrial applications. Furthermore, cross-linked enzyme aggregates of LpSGNH1 (CLEA-LpSGNH1) displayed recycling ability and thermal stability compared to free LpSGNH1, which could be useful for industrial applications. This work highlights the importance of LpSGNH1 in the preparation of commercial compounds, and LpSGNH1 can be used as a model system of SGNH esterases in lactic acid bacteria.