Crystal Structure of a Bacterial Lipase fromChromobacterium viscosumATCC 6918 Refined at 1.6 Å Resolution

Lang, Dietmar A.; Hofmann, Birgit; Haalck, Lutz; Hecht, Hans‐Jürgen; Spener, Friedrich; Schmid, Rolf D.; Schomburg, Dietmar

doi:10.1006/jmbi.1996.0352

Cited by 133 publications

(130 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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“…A notable feature of the T. fusca cutinase model is that the enzyme does not contain a lid insertion commonly observed in true lipases (31)(32)(33)(34), and exposes its nucleophilic serine to the solvent (Fig. 6B).…”

Section: Table 3 Monomeric Products Released From Cutin Hydrolysis Bymentioning

confidence: 99%

Identification and Characterization of Bacterial Cutinase

Chen

Tong

Woodard

et al. 2008

Journal of Biological Chemistry

209

168

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Cutinase, which exists in both fungi and bacteria, catalyzes the cleavage of the ester bonds of cutin. Fungal cutinases have been extensively studied, however, reports on bacterial cutinases have been limited due to the lack of knowledge concerning the identity of their open reading frames. In the present study, the cutinase from Thermobifida fusca was induced by cutin and purified to homogeneity by following p-nitrophenyl butyrate hydrolyzing activity. Peptide mass fingerprinting analysis of the wild-type enzyme matched two proteins, Tfu_0883 and Tfu_0882, which are 93% identical in sequence. Both proteins were cloned and overexpressed in their mature form. Recombinant Tfu_0883 and Tfu_0882 display very similar enzymatic properties and were confirmed to be cutinases by their capability to hydrolyze the ester bonds of cutin. Comparative characterization of Fusarium solani pisi and T. fusca cutinases indicated that they have similar substrate specificity and catalytic properties except that the T. fusca enzymes are thermally more stable. Homology modeling revealed that T. fusca cutinases adopt an ␣/␤-hydrolase fold that exhibits both similarities and variations from the fungal cutinase structure. A serine hydrolase catalytic mechanism involving a Ser 170 -His 248 -Asp 216 (Tfu_0883 numbering) catalytic triad was supported by active site-directed inhibition studies and mutational analyses. This is the first report of cutinase encoding genes from bacterial sources.Cutin is a major component of the plant cuticle. It is an insoluble lipid-polyester formed primarily from C 16 and C 18 hydroxy and epoxy fatty acids (Fig.

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“…Many bacterial lipases for which structures are available contain a Ca 2ϩ -binding site. These include the enzymes from Chromobacterium viscosum (28), Pseudomonas aeruginosa (35), and Burkholderia glumae (basonym Pseudomonas glumae) (BGL) (37). Many lipases have been found to require certain metals for activity and/or to enhance activity and (thermo)stability.…”

Section: Fig 4 Effect Of Phmentioning

confidence: 99%

Purification and Characterization of Two Highly Thermophilic Alkaline Lipases fromThermosyntropha lipolytica

Salameh

Wiegel

2007

Appl Environ Microbiol

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Two thermostable lipases were isolated and characterized from Thermosyntropha lipolytica DSM 11003, an anaerobic, thermophilic, alkali-tolerant bacterium which grows syntrophically with methanogens on lipids such as olive oil, utilizing only the liberated fatty acid moieties but not the glycerol. Lipases LipA and LipB were purified from culture supernatants to gel electrophoretic homogeneity by ammonium sulfate precipitation and hydrophobic interaction column chromatography. The apparent molecular masses of LipA and LipB determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 50 and 57 kDa, respectively. The temperature for maximal activity of LipA and LipB was around 96°C, which is, so far as is known, the highest temperature for maximal activity among lipases, and the pH optima for growth determined at 25°C (pH 25°C optima) were 9.4 and 9.6, respectively. LipA and LipB at 100°C and pH 25°C 8.0 retained 50% activity after 6 and 2 h of incubation, respectively. Both enzymes exhibited high activity with long-chain fatty acid glycerides, yielding maximum activity with trioleate (C 18:1 ) and, among the p-nitrophenyl esters, with p-nitrophenyl laurate. Hydrolysis of glycerol ester bonds occurred at positions 1 and 3. The activities of both lipases were totally inhibited by 10 mM phenylmethylsulfonyl fluoride and 10 mM EDTA. Metal analysis indicated that both LipA and LipB contain 1 Ca 2؉ and one Mn 2؉ ion per monomeric enzyme unit. The addition of 1 mM MnCl 2 to dialyzed enzyme preparations enhanced the activities at 96°C of both LipA and LipB by threefold and increased the durations of their thermal stability at 60°C and 75°C, respectively, by 4 h.

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Crystal Structure of a Bacterial Lipase fromChromobacterium viscosumATCC 6918 Refined at 1.6 Å Resolution

Cited by 133 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

Identification and Characterization of Bacterial Cutinase

Purification and Characterization of Two Highly Thermophilic Alkaline Lipases fromThermosyntropha lipolytica

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