Effect of mutations in the T1.5 loop of pectate lyase A from<i>Erwinia chrysanthemi</i>EC16

Dehdashti, Seameen; Doan, C.N.; Chao, Kinlin; Yoder, Marilyn D.

doi:10.1107/s0907444903011491

Cited by 8 publications

(3 citation statements)

References 20 publications

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“…This linkage creates an "accordion-like" conformation between neighboring residues due to its signature axial C-4 configuration. The overall fiber is extended and flexible and can adopt a 2 1 or 3 1 helix, depending on the degree of hydration and presence of cations (14,(21)(22)(23). Variation in the potential three-dimensional structure of pectic fibers was supported by the galacturonate pentasaccharide-Pel cocrystal structure, which revealed that the substrate was a mixture of both 2 1 and 3 2 helical conformations (59).…”

Section: Pectin Structurementioning

confidence: 94%

Structural Biology of Pectin Degradation byEnterobacteriaceae

Abbott

Boraston

2008

Microbiol Mol Biol Rev

155

105

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SUMMARY Pectin is a structural polysaccharide that is integral for the stability of plant cell walls. During soft rot infection, secreted virulence factors from pectinolytic bacteria such as Erwinia spp. degrade pectin, resulting in characteristic plant cell necrosis and tissue maceration. Catabolism of pectin and its breakdown products by pectinolytic bacteria occurs within distinct cellular environments. This process initiates outside the cell, continues within the periplasmic space, and culminates in the cytoplasm. Although pectin utilization is well understood at the genetic and biochemical levels, an inclusive structural description of pectinases and pectin binding proteins by both extracellular and periplasmic enzymes has been lacking, especially following the recent characterization of several periplasmic components and protein-oligogalacturonide complexes. Here we provide a comprehensive analysis of the protein folds and mechanisms of pectate lyases, polygalacturonases, and carbohydrate esterases and the binding specificities of two periplasmic pectic binding proteins from Enterobacteriaceae. This review provides a structural understanding of the molecular determinants of pectin utilization and the mechanisms driving catabolite selectivity and flow through the pathway.

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Section: Pectin Structurementioning

confidence: 94%

Structural Biology of Pectin Degradation byEnterobacteriaceae

Abbott

Boraston

2008

Microbiol Mol Biol Rev

155

105

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“…S2, the supplementary note, and Table S3 in the supplemental material). Residue R236 is partially solvent exposed and is located in the T1.6 loop (the 6th loop of turn T1), which stacks against the T1.5 loop, containing a helical structure previously shown to enhance the catalytic activity of related pectate lyases (10). Its guanidinium The R236F substitution caused no structural change in the enzyme.…”

Section: Vol 74 2008 Thermostabilization Of Pectate Lyases 1185mentioning

confidence: 99%

Improvement of the Thermostability and Activity of a Pectate Lyase by Single Amino Acid Substitutions, Using a Strategy Based on Melting-Temperature-Guided Sequence Alignment

Xiao

Bergeron

Große

et al. 2008

Appl Environ Microbiol

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In the vast number of random mutagenesis experiments that have targeted protein thermostability, single amino acid substitutions that increase the apparent melting temperature (T m ) of the enzyme more than 1 to 2°C are rare and often require the creation of a large library of mutated genes. Here we present a case where a single beneficial mutation (R236F) of a hemp fiber-processing pectate lyase of Xanthomonas campestris origin (PL Xc ) produced a 6°C increase in T m and a 23-fold increase in the half-life at 45°C without compromising the enzyme's catalytic efficiency. This success was based on a variation of sequence alignment strategy where a mesophilic amino acid sequence is matched with the sequences of its thermophilic counterparts that have established T m values. Altogether, two-thirds of the nine targeted single amino acid substitutions were found to have effects either on the thermostability or on the catalytic activity of the enzyme, evidence of a high success rate of mutation without the creation of a large gene library and subsequent screening of clones. Combination of R236F with another beneficial mutation (A31G) resulted in at least a twofold increase in specific activity while preserving the improved T m value. To understand the structural basis for the increased thermal stability or activity, the variant R236F and A31G R236F proteins and wild-type PL Xc were purified and crystallized. By structure analysis and computational methods, hydrophobic desolvation was found to be the driving force for the increased stability with R236F.Improving the thermostability of a protein or enzyme is desirable for commercially viable bioprocesses that prolong product shelf life, increase energy efficiency, and save costs (30). Unfortunately, there are no simple rules for improving thermostability, the mechanisms of which include the formation of disulfide bridges, hydrophobic or aromatic interactions, contact order, hydrogen bonding, ion pairing, dimer-dimer interaction, and a preponderance of glutamine usage (32,36,41). A critical and challenging task in thermostability engineering is, therefore, choosing a method whereby promising results can be obtained quickly and efficiently. Both rational (structurebased, including molecular modeling) and randomized or irrational (directed evolution or random and combinatorial engineering) approaches to protein thermostability improvement have been applied with varying degrees of success (11,19,30). Notable single amino acid substitutions that have led to dramatic increases in melting temperatures (T m ; the temperature at which 50% of the protein is unfolded), by 20°C and 24°C, respectively, were identified in the Drosophila protein drk SH3 domain (24), and in a bacterial malate dehydrogenase (4). However, these successes were made possible only through a good understanding of the thermodynamics and kinetics of folding, or through knowledge of the electrostatic interactions in the dimer-dimer interface of the tetrameric protein. Otherwise, single amino acid substitutions t...

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“…Pels usually exist as multiple independently regulated isozymes that have 25-80% identity in amino acid sequence (Tamaki et al 1988;Hinton et al 1989). Comparison of the amino acid sequences of five major endo-Pels of E. chrysanthemi shows 60-84% identity (Bekri et al 1999;Dehdashti et al 2003). Shevchik et al (1997) cloned the pelI gene, of E. chrysanthemi 3937.…”

Section: Amino Acid Sequence Homologymentioning

confidence: 98%

Microbial pectate lyases: characterization and enzymological properties

Payasi

Sanwal

2008

World J Microbiol Biotechnol

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Pectate lyase plays an important role in plant pathogenesis. The enzyme is widely distributed in diverse families of microorganisms. The current knowledge including biochemical studies on microbial pectate lyases, such as isozymes, structure, reaction mechanism, purification and properties like molecular mass, pI, optimum pH and temperature, substrate specificity, metal ion requirement, inhibitors and activators, and kinetic parameters of the enzyme are reviewed.

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Effect of mutations in the T1.5 loop of pectate lyase A fromErwinia chrysanthemiEC16

Cited by 8 publications

References 20 publications

Structural Biology of Pectin Degradation byEnterobacteriaceae

Structural Biology of Pectin Degradation byEnterobacteriaceae

Improvement of the Thermostability and Activity of a Pectate Lyase by Single Amino Acid Substitutions, Using a Strategy Based on Melting-Temperature-Guided Sequence Alignment

Microbial pectate lyases: characterization and enzymological properties

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