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 ß-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/ß 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 ß-lactamases. Therefore, this study could provide new structural and functional insights into the understanding this protein family.

Structural and Biochemical Characterization of an Octameric Carbohydrate Acetylesterase from Sinorhizobium meliloti.

Carbohydrate acetylesterases, which have a highly specific role among plant-interacting bacterial species, remove the acetyl groups from plant carbohydrates. Here, we determined the crystal structure of Est24, an octameric carbohydrate acetylesterase from Sinorhizobium meliloti, at 1.45 Å resolution and investigated its biochemical properties. The structure of Est24 consisted of five parallel ß strands flanked by α helices, which formed an octameric assembly with two distinct interfaces. The deacetylation activity of Est24 and its mutants around the substrate-binding pocket was investigated using several substrates, including glucose pentaacetate and acetyl alginate. Elucidation of the structure-function relationships of Est24 could provide valuable opportunities for biotechnological explorations.

Structural basis for the substrate selectivity of a HAD phosphatase from Thermococcus onnurineus NA1.

Proteins in the haloalkaloic acid dehalogenase (HAD) superfamily, which is one of the largest enzyme families, is generally composed of a catalytic core domain and a cap domain. Although proteins in this family show broad substrate specificities, the mechanisms of their substrate recognition are not well understood. In this study, we identified a new substrate binding motif of HAD proteins from structural and functional analyses, and propose that this motif might be crucial for interacting with hydrophobic rings of substrates. The crystal structure of TON_0338, one of the 17 putative HAD proteins identified in a hyperthermophilic archaeon, Thermococcus onnurineus NA1, was determined as an apo-form at 2.0 Å resolution. In addition, we determined the crystal structure TON_0338 in complex with Mg(2+) or N-cyclohexyl-2-aminoethanesulfonic acid (CHES) at 1.7 Å resolution. Examination of the apo-form and CHES-bound structures revealed that CHES is sandwiched between Trp58 and Trp61, suggesting that this Trp sandwich might function as a substrate recognition motif. In the phosphatase assay, TON_0338 was shown to have high activity for flavin mononucleotide (FMN), and the docking analysis suggested that the flavin of FMN may interact with Trp58 and Trp61 in a way similar to that observed in the crystal structure. Moreover, the replacement of these tryptophan residues significantly reduced the phosphatase activity for FMN. Our results suggest that WxxW may function as a substrate binding motif in HAD proteins, and expand the diversity of their substrate recognition mode.

Structural and biochemical characterization of a carbohydrate acetylesterase from Sinorhizobium meliloti 1021.

In many microorganisms, carbohydrate acetylesterases remove the acetyl groups from various types of carbohydrates. Sm23 from Sinorhizobium meliloti is a putative member of carbohydrate esterase family 3 (CE3) in the CAZy classification system. Here, we determined the crystal structure of Sm23 at 1.75 Å resolution and investigated functional properties using biochemical methods. Furthermore, immobilized Sm23 exhibited improved stability compared with soluble Sm23, which can be used for the design of plant cell wall degrading-systems.

Crystallographic analysis and biochemical applications of a novel penicillin-binding protein/ß-lactamase homologue from a metagenomic library.

Interest in penicillin-binding proteins and ß-lactamases (the PBP-ßL family) is increasing owing to their biological and clinical significance. In this study, the crystal structure of Est-Y29, a metagenomic homologue of the PBP-ßL family, was determined at 1.7 Šresolution. In addition, complex structures of Est-Y29 with 4-nitrophenyl phosphate (4NP) and with diethyl phosphonate (DEP) at 2.0 Šresolution were also elucidated. Structural analyses showed that Est-Y29 is composed of two domains: a ß-lactamase fold and an insertion domain. A deep hydrophobic patch between these domains defines a wide active site, and a nucleophilic serine (Ser58) residue is located in a groove defined primarily by hydrophobic residues between the two domains. In addition, three hydrophobic motifs, which make up the substrate-binding site, allow this enzyme to hydrolyze a wide variety of hydrophobic compounds, including fish and olive oils. Furthermore, cross-linked Est-Y29 aggregates (CLEA-Est-Y29) significantly increase the stability of the enzyme as well as its potential for extensive reuse in various deactivating conditions. The structural features of Est-Y29, together with biochemical and biophysical studies, could provide a molecular basis for understanding the properties and regulatory mechanisms of the PBP-ßL family and their potential for use in industrial biocatalysts.

Interaction of TIF-90 and filamin A in the regulation of rRNA synthesis in leukemic cells.

The transcription initiation factor I (TIF-IA) is an important regulator of the synthesis of ribosomal RNA (rRNA) through its facilitation of the recruitment of RNA polymerase I (Pol I) to the ribosomal DNA promoter. Activation of the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway, which occurs commonly in acute myelogenous leukemia, enhances rRNA synthesis through TIF-IA stabilization and phosphorylation. We have discovered that TIF-IA coexists with a splicing isoform, TIF-90, which is expressed preferentially in the nucleolus and at higher levels in proliferating and transformed hematopoietic cells. TIF-90 interacts directly with Pol I to increase rRNA synthesis as a consequence of Akt activation. Furthermore, TIF-90 binds preferentially to a 90-kDa cleavage product of the actin binding protein filamin A (FLNA) that inhibits rRNA synthesis. Increased expression of TIF-90 overcomes the inhibitory effect of this cleavage product and stimulates rRNA synthesis. Because activated Akt also reduces FLNA cleavage, these results indicate that activated Akt and TIF-90 function in parallel to increase rRNA synthesis and, as a consequence, cell proliferation in leukemic cells. These results provide evidence that the direct targeting of Akt would be an effective therapy in acute leukemias in which Akt is activated.

Crystallization and preliminary X-ray analysis of a highly stable novel SGNH hydrolase (Est24) from Sinorhizobium meliloti.

The SGNH hydrolase family includes enzymes that catalyze the hydrolysis of a broad range of substrates. Here, the crystallization and preliminary X-ray crystallographic studies of a novel SGNH hydrolase (Est24) from Sinorhizobium meliloti were performed. Recombinant Est24 protein containing an N-terminal His tag was expressed in Escherichia coli and purified to homogeneity. Est24 was then crystallized using a solution consisting of 0.2 M ammonium phosphate pH 4.6, 20% polyethylene glycol 3350. X-ray diffraction data were collected to a resolution of 1.45 Å with an R(merge) of 9.4%. The Est24 crystals belonged to space group C2, with unit-cell parameters a = 129.09, b = 88.63, c = 86.15 Å, α = 90.00, ß = 114.30, γ = 90.00°. A molecular-replacement solution was obtained using the crystal structure of Mycobacterium smegmatis arylesterase as a template and structure refinement of Est24 is in progress.

Structural and functional analyses of a bacterial homologue of hormone-sensitive lipase from a metagenomic library.

Intracellular mobilization of fatty acids from triacylglycerols in mammalian adipose tissues proceeds through a series of lipolytic reactions. Among the enzymes involved, hormone-sensitive lipase (HSL) is noteworthy for its central role in energy homeostasis and the pathogenic role played by its dysregulation. By virtue of its broad substrate specificity, HSL may also serve as an industrial biocatalyst. In a previous report, Est25, a bacterial homologue of HSL, was identified from a metagenomic library by functional screening. Here, the crystal structure of Est25 is reported at 1.49 Šresolution; it exhibits an α/ß-hydrolase fold consisting of a central ß-sheet enclosed by α-helices on both sides. The structural features of the cap domain, the substrate-binding pocket and the dimeric interface of Est25, together with biochemical and biophysical studies including native PAGE, mass spectrometry, dynamic light scattering, gel filtration and enzyme assays, could provide a basis for understanding the properties and regulation of hormone-sensitive lipase (HSL). The increased stability of cross-linked Est25 aggregates (CLEA-Est25) and their potential for extensive reuse support the application of this preparation as a biocatalyst in biotransformation processes.

Characterization, crystallization and preliminary X-ray diffraction analysis of an (S)-specific esterase (pfEstA) from Pseudomonas fluorescens KCTC 1767: enantioselectivity for potential industrial applications.

The structures and reaction mechanisms of enantioselective hydrolases, which can be used in industrial applications such as biotransformations, are largely unknown. Here, the X-ray crystallographic study of a novel (S)-specific esterase (pfEstA) from Pseudomonas fluorescens KCTC 1767, which can be used in the production of (S)-ketoprofen, is described. Multiple sequence alignments with other hydrolases revealed that pfEstA contains a conserved Ser67 within the S-X-X-K motif as well as a highly conserved Tyr156. Recombinant protein containing an N-terminal His tag was expressed in Escherichia coli, purified to homogeneity and characterized using SDS-PAGE, MALDI-TOF MS and enantioselective analysis. pfEstA was crystallized using a solution consisting of 1 M sodium citrate, 0.1 M CHES pH 9.5, and X-ray diffraction data were collected to a resolution of 1.9 Šwith an Rmerge of 7.9%. The crystals of pfEstA belonged to space group P2(1)2(1)2(1), with unit-cell parameters a=65.31, b=82.13, c=100.41 Å, α=ß=γ=90°.

The crystal structure of the periplasmic domain of Vibrio parahaemolyticus CpxA.

The Cpx two-component system of Gram-negative bacteria senses extracytoplasmic stresses using the histidine kinase CpxA, a membrane-bound sensor, and controls the transcription of the genes involved in stress response by the cytosolic response regulator CpxR, which is activated by the phosphorelay from CpxA. CpxP, a CpxA-associated protein, also plays an important role in the regulation of the Cpx system by inhibiting the autophosphorylation of CpxA. Although the stress signals and physiological roles of the Cpx system have been extensively studied, the lack of structural information has limited the understanding of the detailed mechanism of ligand binding and regulation of CpxA. In this study, we solved the crystal structure of the periplasmic domain of Vibrio parahaemolyticus CpxA (VpCpxA-peri) to a resolution of 2.1 Å and investigated its interaction with CpxP. VpCpxA-peri has a globular Per-ARNT-SIM (PAS) domain and a protruded C-terminal tail, which may be required for ligand sensing and CpxP binding, respectively. The direct interaction of the PAS core of VpCpxA-peri with VpCpxP was not detected by NMR, suggesting that the C-terminal tail or other factors, such as the membrane environment, are necessary for the binding of CpxA to CpxP.

Identification, crystallization and preliminary X-ray diffraction analysis of esterase A from Caulobacter crescentus CB15, a family VIII lipolytic enzyme.

The structures and functions of family VIII lipolytic enzymes, which have moderate sequence identity to class C ß-lactamases and penicillin-binding proteins, are largely unknown. Here, the X-ray crystallographic study of a family VIII esterase from Caulobacter crescentus CB15 (CcEstA) is described. Sequence analysis revealed that CcEstA has a conserved serine residue within the S-X-X-K motif which acts as a catalytic nucleophile. Recombinant protein containing an N-terminal His tag was expressed in Escherichia coli and purified to homogeneity. Functional studies showed that CcEstA acts on α- and ß-naphthyl acetate as substrates. In addition, it can catalyze the hydrolysis of ketoprofen ethyl ester, a highly useful product in industrial applications. CcEstA was crystallized using a solution consisting of 1.0 M potassium/sodium tartrate, 0.1 M imidazole pH 8.0, 0.2 M NaCl, and X-ray diffraction data were collected to a resolution of 1.62 Šwith an R(merge) of 9.4%. The crystals of CcEstA belonged to space group C222(1), with unit-cell parameters a = 172.23, b = 176.68, c = 47.93 Å. Structure determination is in progress.

Characterization, amyloid formation, and immobilization of a novel SGNH hydrolase from Listeria innocua 11262.

A novel oligomeric hydrolase (LI22) from Listeria innocua CLIP 11262 was identified, characterized, and immobilized for industrial application. Sequence analysis of LI22 revealed a putative catalytic triad (Ser(10)-Asp(176)-His(179)), and a conserved sequence motif Ser(S)(10)-Gly(G)(77)-Asn(N)(79)-His(H)(179) with moderate identities (<30%) with other members of the SGNH-hydrolase superfamily. LI22 was able to hydrolyze p-nitrophenyl acetate, α- and ß-naphthyl acetate, while the S10A mutant completely lost its activity. Structural properties of LI22 were investigated using gel filtration, circular dichroism (CD), fluorescence, molecular modeling, and gel filtration. We have shown that upon incubation in 30% TFE or 50% ethanol solution, LI22 was transformed into curly amyloid fibrils. Cross-linked enzyme aggregates of LI22 were prepared by precipitating the enzyme with ammonium sulfate and subsequent cross-linking with glutaraldehyde. Higher thermal and chemical stability, as well as good durability after repeated use of the LI22-CLEA, highlight its potential applicability as a biocatalyst in the pharmaceutical and chemical industries.

Crystallization and diffraction analysis of Sm23: an SGNH-family arylesterase from Sinorhizobium meliloti 1021.

Industrial demand for active biocatalysts with desirable biochemical properties is constantly increasing and the discovery and characterization of novel esterases is potentially useful for industrial processes. Here, X-ray crystallographic studies of an (R)-specific SGNH arylesterase (Sm23) from Sinorhizobium meliloti 1021 are reported. The recombinant protein was expressed in Escherichia coli with a His tag and purified to homogeneity. Sm23 was crystallized using 0.2 M magnesium formate as a precipitant and X-ray diffraction data were collected to a resolution of 2.2 Å with an R(merge) of 6.9%. The crystals of SM23 belonged to the I-centred tetragonal space group I4(1)22, with unit-cell parameters a = b = 126.6, c = 190.9 Å. A molecular-replacement solution was obtained using the crystal structure of arylesterase from Mycobacterium smegmatis as a template.