Zinc in lipase L1 from<i>Geobacillus stearothermophilus</i>L1 and structural implications on thermal stability

Choi, Won-Chan; Kim, Myung Hee; Ro, Hyeon-Su; Ryu, Sang Ryeol; Oh, Tae-Kwang; Lee, Jung-Kee

doi:10.1016/j.febslet.2005.05.016

Cited by 52 publications

(51 citation statements)

References 21 publications

(22 reference statements)

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“…As such, it was assumed that the substitution of less hydrophobic residue (Arg157) to more hydrophobic residue (Ser) increased the internal hydrophobicity of R157S lipase to maintain the structural stability at a high temperature. Choi et al [38] reported that the wild-type enzyme which have Zn 2+ elevated the T m value from 51.1 to 69.3°C compared to its mutant enzymes (H87A, D61A/H87A and D61A/H81A/H87A/D238A). It indicated the importance of Zn 2+ for maintaining structural stability at high temperature.…”

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confidence: 99%

The Role of Arg157Ser in Improving the Compactness and Stability of ARM Lipase

Salleh¹

2012

JCSB

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Consensus approach is an efficient strategy to identify hot residue important for compactness and stability of protein. Structure of ARM lipase was modeled to explore the possible effect of critical point mutation towards structure and function. The significant difference of amino acid at position 157 between ARM lipase (Arg157) and other thermostable lipases (Ser157) was targeted as a critical residue. Using YASARA software, Arg157 was substituted to Ser and subsequently the energy minimized. Both ARM and R157S lipases were analyzed by MD simulations at different temperatures (50ºC,60ºC, and 70ºC). MD simulation result showed that R157S lipase had lower value of RMSD, RMSF, solvent accessible surface area (SASA) and radius of gyration than native ARM lipase. It indicated that R157S lipase had higher compactness in the structure leading to enhanced stability. To validate the computational data, the substitution of Arg157 to Ser has been conducted using site-directed mutagenesis experimentally. The catalytic efficiency (k cat /K M ) of R157S lipase was refold better than ARM lipase of 70°C. Circular dichorism study revealed that R157S lipase had increased thermostability with higher T m value (71.6°C) than its wildtype (63.9°C) indicating a better compactness as revealed by spectrofluorocence study. Thus the rational design of substituting Arg157 with Ser improved the protein folding of mutant lipase as shown in MD simulations and subsequently increased the catalytic effectiveness and thermodynamic stability. during evolution. The substitution of critical residue was analyzed using in silico to avoid unnecessary conformational distortion in engineered lipase and validate with kinetic study and thermodynamic study. Methods Preparation of 3D structures of ARM and R157S lipasesThe 3D structure of ARM lipase was predicted by comparative modeling. The first step in comparative modeling is the assignment of the unknown ARM lipase structure to a protein family with thousands of sequences already stored in protein databases (PDB). The structure of ARM lipase was modeled using available software packages such as HOMOLOGY and MODELLER, Accelrys, San Diego (http://www. accelrys.com). The unknown ARM lipase structure (target protein) was built from the known 3D structure of T1 lipase (2DSN) as a template. The final model of ARM lipase was evaluated using Ramachandran plot. Using YASARA, the three-dimensional structure of the mutant (R157S lipase) was modeled by substituting Arg157 to Ser and energy minimization was used to release bad contact. MD simulations of ARM and R157S lipasesMD simulations were performed on ARM lipase and R157S lipase structures using Amber03 force field in YASARA package. The periodic box was filled with explicit water molecules to a density of 0.98320 g/l. The whole system was neutralized by adding counter ions and all ionizable protein groups were protonated according to their tabulated pKa values at pH 7 of the medium. Water molecules were relaxed by simulated annealing procedure. Minimiza...

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confidence: 99%

The Role of Arg157Ser in Improving the Compactness and Stability of ARM Lipase

Salleh¹

2012

JCSB

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“…Additional information from our data analysis is listed in Table 1. Comparing L42 with other lipases with solved structures, we found sequence identities of 97% to BTL from Geobacillus thermocatenulatus and 98% and 94% to P1 and L1 from G. stearothermophilus, respectively (Tyndall et al, 2002;Choi et al, 2005;Carrasco-Ló pez et al, 2009). BTL has the highest optimum temperature for enzymatic activity (348 K); L42 has an optimum temperature of 343 K, followed by L1 (333 K) and P1 (328 K Kamini et al, 2000;Ishimoto et al, 2001), that Bacillus thermoleovorans CCR11 is stable in 70%(v/v) acetone, methanol, ethanol or propanol (CastroOchoa et al, 2005) and that Pseudomonas aeruginosa LST-03 retains its activity in 25%(v/v) DMSO or methanol (Ogino et al, 1999(Ogino et al, , 2000.…”

Section: Resultsmentioning

confidence: 96%

Crystallization and preliminary X-ray crystallographic analysis of a thermostable organic solvent-tolerant lipase fromBacillussp. strain 42

Khusaini¹,

Rahman²,

Ali³

et al. 2011

Acta Cryst Sect F

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An organic solvent-tolerant lipase from Bacillus sp. strain 42 was crystallized using the capillary-tube method. The purpose of studying this enzyme was in order to better understand its folding and to characterize its properties in organic solvents. By initially solving its structure in the native state, further studies on protein-solvent interactions could be performed. X-ray data were collected at 2.0 Å resolution using an in-house diffractometer. The estimated crystal dimensions were 0.09 Â 0.19 Â 0.08 mm. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 117. 41, b = 80.85, c = 99.44 Å , = 96.40 .

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“…Most genes affected by zur encode putative proteinases, of which 23 were upregulated and 12 were downregulated (Table 6). They included some Zn 2ϩ -dependent members (e.g., NADPH:quinone reductase and lipase) involved in the basic metabolism of carbohydrates, nucleic acids, and fatty acids (10). There were more than 20 unknown genes which are influenced by gene 310.…”

Section: Discussionmentioning

confidence: 99%

“…It plays critical roles in various cellular processes and physiological functions by either serving as a catalytic cofactor for numerous enzymes or maintaining the structure scaffold of metal-proteins (4,10). However, an excess of zinc ion is toxic to normal physiological processes because it can trigger the formation of hydroxyl radicals, resulting in severe damage to DNA, proteins, and lipids (53).…”

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confidence: 99%

Functional Definition and Global Regulation of Zur, a Zinc Uptake Regulator in a Streptococcus suis Serotype 2 Strain Causing Streptococcal Toxic Shock Syndrome

Feng

Zhang

et al. 2008

J Bacteriol

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Zinc is an essential trace element for all living organisms and plays pivotal roles in various cellular processes. However, an excess of zinc is extremely deleterious to cells. Bacteria have evolved complex machineries (such as efflux/influx systems) to control the concentration at levels appropriate for the maintenance of zinc homeostasis in cells and adaptation to the environment. The Zur (zinc uptake regulator) protein is one of these functional members involved in the precise control of zinc homeostasis. Here we identified a zur homologue designated 310 from Streptococcus suis serotype 2, strain 05ZYH33, a highly invasive isolate causing streptococcal toxic shock syndrome. Biochemical analysis revealed that the protein product of gene 310 exists as a dimer form and carries zinc ions. An isogenic gene replacement mutant of gene 310, the ⌬310 mutant, was obtained by homologous recombination. Physiological tests demonstrated that the ⌬310 mutant is specifically sensitive to Zn 2؉ , while functional complementation of the ⌬310 mutant can restore its duration capability, suggesting that 310 is a functional member of the Zur family. Two-dimensional electrophoresis indicated that nine proteins in the ⌬310 mutant are overexpressed in comparison with those in the wild type. DNA microarray analyses suggested that 121 genes in the ⌬310 mutant are affected, of which 72 genes are upregulated and 49 are downregulated. The transcriptome of S. suis serotype 2 with high Zn 2؉ concentrations also showed 117 differentially expressed genes, with 71 upregulated and 46 downregulated. Surprisingly, more than 70% of the genes differentially expressed in the ⌬310 mutant were the same as those in S. suis serotype 2 that were differentially expressed in response to high Zn 2؉ concentration, consistent with the notion that 310 is involved in zinc homeostasis. We thus report for the first time a novel zinc-responsive regulator, Zur, from Streptococcus suis serotype 2.Streptococcus suis 2 is a gram-positive pathogenic bacterium capable of infecting both piglets and humans and causing serious diseases such as arthritis, meningitis, and septicemia (58). It has spread to nearly 20 countries, resulting in more than 400 human cases of severe infection worldwide (38). In particular, two recent outbreaks (in 1998 and 2005) of severe human S. suis serotype 2 infections in China were characterized by streptococcal toxic shock syndrome, implying that highly invasive variants of S. suis serotype 2 might be emerging in Asia (63,72). Virulence factors related to the pathogenesis of S. suis serotype 2 have been partially elucidated and include capsular polysaccharide (56), suilysin (37), muraminidase-released protein (42), and extracellular factor (59). Very recently, our group (8) reported the whole genome sequences of two virulent S. suis serotype 2 isolates (05ZYH33 and 98HAH12).Similar to other transition metals such as iron, manganese,

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Zinc in lipase L1 fromGeobacillus stearothermophilusL1 and structural implications on thermal stability

Cited by 52 publications

References 21 publications

The Role of Arg157Ser in Improving the Compactness and Stability of ARM Lipase

The Role of Arg157Ser in Improving the Compactness and Stability of ARM Lipase

Crystallization and preliminary X-ray crystallographic analysis of a thermostable organic solvent-tolerant lipase fromBacillussp. strain 42

Functional Definition and Global Regulation of Zur, a Zinc Uptake Regulator in a Streptococcus suis Serotype 2 Strain Causing Streptococcal Toxic Shock Syndrome

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