The open conformation of a Pseudomonas lipase

Schrag, Joseph D.; Li, Yunge; Cygler, Mirosław; Lang, Dietmar A.; Burgdorf, Tanja; Hecht, H.J.; Schmid, Rolf D.; Schomburg, Dietmar; Rydel, T.J.; Oliver, J. D.; Strickland, L. C.; Dunaway, C. M.; Larson, Steven B.; Day, John; McPherson, Alexander

doi:10.1016/s0969-2126(97)00178-0

Cited by 272 publications

(225 citation statements)

References 42 publications

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“…Topologically the bacterial lipases consist of a variant of the ␣-␤ hydrolase fold and a cap domain, which includes a lid subdomain (helices ␣4 and -5). The catalytic triad in bacterial lipases is buried (16,17); the substrate gains access into the active site when a large hinge motion displaces the lid subdomain sidewise (18,19). Bacterial lipases with a buried catalytic triad and a buried lipid binding site, on one hand, and PPT1 with an exposed catalytic triad and an exposed lipid binding site represent the extremes.…”

Section: Resultsmentioning

confidence: 99%

The Crystal Structure of Palmitoyl Protein Thioesterase-2 (PPT2) Reveals the Basis for Divergent Substrate Specificities of the Two Lysosomal Thioesterases, PPT1 and PPT2

Calero

Gupta

Nonato

et al. 2003

Journal of Biological Chemistry

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Mutations in palmitoyl protein thioesterase-1 (PPT1) have been found to cause the infantile form of neuronal ceroid lipofuscinosis, which is a lysosomal storage disorder characterized by impaired degradation of fatty acid-modified proteins with accumulation of amorphous granular deposits in cortical neurons, leading to mental retardation and death. Palmitoyl protein thioesterase-2 (PPT2) is a second lysosomal hydrolase that shares a 26% identity with PPT1. A previous study had suggested that palmitoyl-CoA was the preferred substrate of PPT2. Furthermore, PPT2 did not hydrolyze palmitate from the several S-palmitoylated protein substrates. Interestingly, PPT2 deficiency in a recent transgenic mouse model is associated with a form of neuronal ceroid lipofuscinosis, suggesting that PPT1 and -2 perform nonredundant roles in lysosomal thioester catabolism. In the current paper, we present the crystal structure of PPT2 at a resolution of 2.7 Å. Comparisons of the structures of PPT1 and -2 show very similar architectural features; however, conformational differences in helix ␣4 lead to a solvent-exposed lipid-binding groove in PPT1. The limited space between two parallel loops (␤3-␣A and ␤8-␣F) located immediately above the lipidbinding groove in PPT2 restricts the binding of fatty acids with bulky head groups, and this binding groove is significantly larger in PPT1. This structural difference accounts for the ability of PPT2 to hydrolyze an unbranched structure such as palmitoyl-CoA but not palmitoylcysteine or palmitoylated proteins. Furthermore, differences in fatty acid chain length specificity of PPT1 and -2, also reported here, are explained by the structure and may provide a biochemical basis for their non-redundant roles.

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Section: Resultsmentioning

confidence: 99%

The Crystal Structure of Palmitoyl Protein Thioesterase-2 (PPT2) Reveals the Basis for Divergent Substrate Specificities of the Two Lysosomal Thioesterases, PPT1 and PPT2

Calero

Gupta

Nonato

et al. 2003

Journal of Biological Chemistry

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“…An extensive search of the PDB shows that this motif is not characteristic for magnesium binding, but rather for calcium binding. Similar loops bind Ca 2+ cations in different bacterial lipases, such as lipases from Pseudomonas glumae (formerly Chromobacterium viscosum) (Noble et al 1993;Lang et al 1996) and Burkholderia cepacia (formerly Pseudomonas cepacia) (Shrag et al 1997;Luić et al 2001). The lipases are not the only enzymes that contain such a metal binding motif; it is also observed in human DNA polymerase b (Pelletier and Sawaya 1996).…”

Section: Metal Bindingmentioning

confidence: 99%

Crystal structures of TM0549 and NE1324—two orthologs of E. coli AHAS isozyme III small regulatory subunit

et al. 2007

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Crystal structures of two orthologs of the regulatory subunit of acetohydroxyacid synthase III (AHAS, EC 2.2.1.6) from Thermotoga maritima (TM0549) and Nitrosomonas europea (NE1324) were determined by single-wavelength anomalous diffraction methods with the use of selenomethionine derivatives at 2.3 Å and 2.5 Å , respectively. TM0549 and NE1324 share the same fold, and in both proteins the polypeptide chain contains two separate domains of a similar size. Each protein contains a C-terminal domain with ferredoxin-type fold and an N-terminal ACT domain, of which the latter is characteristic for several proteins involved in amino acid metabolism. The ferredoxin domain is stabilized by a calcium ion in the crystal structure of NE1324 and by a Mg(H 2 O) 6 2+ ion in TM0549. Both TM0549 and NE1324 form dimeric assemblies in the crystal lattice.Keywords: acetohydroxyacid synthase; actolactate synthase; regulatory subunit; ACT domain; AHAS; protein refolding Acetohydroxyacid synthase (AHAS, EC 2.2.1.6) catalyzes two physiologically significant reactions in the synthesis of isoleucine, valine, and leucine. In the first reaction, which is an initial step in the synthesis of isoleucine, 2-aceto-2-hydroxy butyrate is produced by condensation of 2-ketobutyrate with pyruvate. In the second reaction, 2-acetolactate is synthesized from two pyruvate molecules, and this provides substrates for synthesis of valine and leucine (Umbarger 1978(Umbarger , 1987Chipman et al. 1998). This pathway of branched-chain amino acid biosynthesis is characteristic for bacteria, fungi, algae, and higher plants, but not for animals. Accordingly, AHAS inhibitors have been developed as herbicides which have found broad application (Short and Colborn 1999). The inhibitors of branched-chain amino acids synthesis are also being tested as potential antituberculosis agents (Grandoni et al. 1998;Zohar et al. 2003;Choi et al. 2005).Three different FAD-dependent AHAS isozymes (I, II, and III) are found in enterobacteria (e.g., Escherichia ), but most other organisms from the bacteria encode only a single AHAS enzyme, similar to isozyme III from E. coli. The acetohydroxyacid synthases are multimeric proteins, and most frequently their biological unit is composed of two catalytic subunits (CSU) and two small regulatory subunits (SSU). In the absence of the SSU subunit, the CSU dimer is unstable (Vyazmensky et al. 1996), suggesting that in the holoenzyme the AHAS dimer is stabilized by a SSU dimer. The large catalytic subunits also show very weak activity without their associated regulatory subunits (Weinstock et al. 1992;Pang and Duggleby 1999). The E. coli AHAS enzyme can be reconstituted with SSU subunits from other organisms (Porat et al. 2004), and the reconstituted enzymes form stable heterotetramers. The properly associated holoenzymes show full catalytic activity, and are also susceptible to valine inhibition.Previous mutagenesis studies (Mendel et al. 2001;Kaplun et al. 2006) revealed that the valine binding sites are located at the dimerizatio...

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“…It should be noticed that at pH 7, lipase from B. cepacia is negatively charged (isoelectric point = 5.2) 29 while the superparamagnetic nanoparticles exhibit a positive charge due to the protonation of the aliphatic amines (pKa ca. 9).…”

Section: Resultsmentioning

confidence: 99%

Enzymatic kinetic resolution of (RS)-1-(Phenyl)ethanols by Burkholderia cepacia lipase immobilized on magnetic nanoparticles

Rebelo¹,

Netto²,

Toma³

et al. 2010

J. Braz. Chem. Soc.

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A lipase proveniente da Burkholderia cepacia imobilizada em nanopartículas superparamagnéticas usando diferentes metodologias de imobilização (adsorção e quimiosorção) foi eficientemente aplicada como biocatalisador reciclável na resolução cinética de (RS)-1-(fenil) etanols através de reações de transesterificação. Os (R)-ésteres e os (S)-alcoóis foram obtidos com excelente excesso enantiomérico (> 99%), o que corresponde a um perfeito processo de resolução cinética enzimática (conversão 50%, E > 200). As reações de transesterificação catalisadas pela lipase de B. cepacia imobilizada pela metodologia com glutaraldeído apresentaram os melhores resultados em termos de conversão após 8 ciclos de reação.Lipase from Burkholderia cepacia immobilized on superparamagnetic nanoparticles using adsorption and chemisorption methodologies was efficiently applied as recyclable biocatalyst in the enzymatic kinetic resolution of (RS)-1-(phenyl)ethanols via transesterification reactions. (R)-Esters and the remaining (S)-alcohols were obtained with excellent enantiomeric excess (> 99%), which corresponds to a perfect process of enzymatic kinetic resolution (conversion 50%, E > 200). The transesterification reactions catalysed with B. cepacia lipase immobilized by the glutaraldehyde method showed the best results in terms of reusability, preserving the enzyme activity (conversion 50%, E > 200) for at least 8 successive cycles.

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The open conformation of a Pseudomonas lipase

Cited by 272 publications

References 42 publications

The Crystal Structure of Palmitoyl Protein Thioesterase-2 (PPT2) Reveals the Basis for Divergent Substrate Specificities of the Two Lysosomal Thioesterases, PPT1 and PPT2

The Crystal Structure of Palmitoyl Protein Thioesterase-2 (PPT2) Reveals the Basis for Divergent Substrate Specificities of the Two Lysosomal Thioesterases, PPT1 and PPT2

Crystal structures of TM0549 and NE1324—two orthologs of E. coli AHAS isozyme III small regulatory subunit

Enzymatic kinetic resolution of (RS)-1-(Phenyl)ethanols by Burkholderia cepacia lipase immobilized on magnetic nanoparticles

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