Pectinolytic lyases: a comprehensive review of sources, category, property, structure, and catalytic mechanism of pectate lyases and pectin lyases
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ABSTRACT:
SUBMITTER: Zheng L
PROVIDER: S-EPMC10992409 | biostudies-literature | 2021 Aug
REPOSITORIES: biostudies-literature
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Project description:Two pectate lyases were identified from Paenibacillus amylolyticus 27C64; both enzymes demonstrated activity on methylated pectin in addition to polygalacturonic acid. PelA is in a subclass of the pectate lyase family III. PelB shows some features of pectate lyase family I but is highly divergent.
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Project description:Previous genetic studies have revealed that a pollen-specific calmodulin-binding protein, No Pollen Germination 1 (NPG1), is required for pollen germination. However, its mode of action is unknown. Here we report direct interaction of NPG1 with pectate lyase-like proteins (PLLs). A truncated form of AtNPG1 lacking the N-terminal tetratricopeptide repeat 1 (TPR1) failed to interact with PLLs, suggesting that it is essential for NPG1 interaction with PLLs. Localization studies with AtNPG1 fused to a fluorescent reporter driven by its native promoter revealed its presence in the cytosol and cell wall of the pollen grain and the growing pollen tube of plasmolyzed pollen. Together, our data suggest that the function of NPG1 in regulating pollen germination is mediated through its interaction with PLLs, which may modify the pollen cell wall and regulate pollen tube emergence and growth.
| S-EPMC4053719 | biostudies-literature
Project description:The unique active site of the Caldicellulosiruptor bescii family 3 pectate lyase (PL3) enzyme has been thoroughly characterized using a series of point mutations, X-ray crystallography, pK(a) calculations and biochemical assays. The X-ray structures of seven PL3 active-site mutants, five of them in complex with intact trigalacturonic acid, were solved and characterized structurally, biochemically and computationally. The results confirmed that Lys108 is the catalytic base, but there is no clear candidate for the catalytic acid. However, the reaction mechanism can also be explained by an antiperiplanar trans-elimination reaction, in which Lys108 abstracts a proton from the C5 atom without the help of simultaneous proton donation by an acidic residue. An acidified water molecule completes the anti β-elimination reaction by protonating the O4 atom of the substrate. Both the C5 hydrogen and C4 hydroxyl groups of the substrate must be orientated in axial configurations, as for galacturonic acid, for this to be possible. The wild-type C. bescii PL3 displays a pH optimum that is lower than that of Bacillus subtilis PL1 according to activity measurements, indicating that C. bescii PL3 has acquired a lower pH optimum by utilizing lysine instead of arginine as the catalytic base, as well as by lowering the pK(a) of the catalytic base in a unique active-site environment.
| S-EPMC4556314 | biostudies-literature
Project description:Pectin lyases are a group of enzymes that are thought to contribute to many biological processes, such as the degradation of pectin. However, until this study, no comprehensive study incorporating phylogeny, chromosomal location, gene duplication, gene organization, functional divergence, adaptive evolution, expression profiling and functional networks has been reported for Arabidopsis. Sixty-seven pectin lyase genes have been identified, and most of them possess signal sequences targeting the secretory pathway. Phylogenetic analyses identified five gene groups with considerable conservation among groups. Pectin lyase genes were non-randomly distributed across chromosomes and clustering was evident. Functional divergence and adaptive evolution analyses suggested that purifying selection was the main force driving pectin lyase evolution, although some critical sites responsible for functional divergence might be the consequence of positive selection. A stigma- and receptacle-specific expression promoter was identified, and it had increased expression in response to wounding. Two hundred and eighty-eight interactions were identified by functional network analyses, and most of these were involved in cellular metabolism, cellular transport and localization, and stimulus responses. This investigation contributes to an improved understanding of the complexity of the Arabidopsis pectin lyase gene family.
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Project description:Fungal genomes often contain several copies of genes that encode carbohydrate active enzymes having similar activity. The copies usually have slight sequence variability, and it has been suggested that the multigenecity represents distinct reaction optima versions of the enzyme. Whether the copies represent differences in substrate attack proficiencies of the enzyme have rarely been considered. The genomes of Aspergillus species encode several pectin lyases (EC 4.2.2.10), which all belong to polysaccharide lyase subfamily PL1_4 in the CAZy database. The enzymes differ in terms of sequence identity and phylogeny, and exhibit structural differences near the active site in their homology models. These enzymes catalyze pectin degradation via eliminative cleavage of the α-(1,4) glycosidic linkages in homogalacturonan with a preference for linkages between methyl-esterified galacturonate residues. This study examines four different pectin lyases (PelB, PelC, PelD, and PelF) encoded by the same Aspergillus sp. (namely A. luchuensis), and further compares two PelA pectin lyases from two related Aspergillus spp. (A. aculeatus and A. tubingensis). We report the phylogeny, enzyme kinetics, and enzymatic degradation profiles of the enzymes' action on apple pectin, citrus pectin, and sugar beet pectin. All the pectin lyases exerted highest reaction rate on apple pectin [degree of methoxylation (DM) 69%, degree of acetylation (DAc) 2%] and lowest reaction rate on sugar beet pectin (DM 56%, DAc 19%). Activity comparison at pH 5-5.5 produced the following ranking: PelB > PelA > PelD > PelF > PelC. The evolution of homogalacturonan-oligomer product profiles during reaction was analyzed by liquid chromatography with mass spectrometry (LC-MS) detection. This analyses revealed subtle differences in the product profiles indicating distinct substrate degradation preferences amongst the enzymes, notably with regard to acetyl substitutions. The LC-MS product profiling analysis thus disclosed that the multigenecity appears to provide the fungus with additional substrate degradation versatility. This product profiling furthermore represents a novel approach to functionally compare pectin-degrading enzymes, which can help explain structure-function relations and reaction properties of disparate copies of carbohydrate active enzymes. A better understanding of the product profiles generated by pectin modifying enzymes has significant implications for targeted pectin modification in food and biorefinery processes.
| S-EPMC7406575 | biostudies-literature
Project description:Catalytic promiscuity is a useful, but accidental, enzyme property, so finding catalytically promiscuous enzymes in nature is inefficient. Some ancestral enzymes were branch points in the evolution of new enzymes and are hypothesized to have been promiscuous. To test the hypothesis that ancestral enzymes were more promiscuous than their modern descendants, we reconstructed ancestral enzymes at four branch points in the divergence hydroxynitrile lyases (HNL's) from esterases ∼ 100 million years ago. Both enzyme types are α/β-hydrolase-fold enzymes and have the same catalytic triad, but differ in reaction type and mechanism. Esterases catalyze hydrolysis via an acyl enzyme intermediate, while lyases catalyze an elimination without an intermediate. Screening ancestral enzymes and their modern descendants with six esterase substrates and six lyase substrates found higher catalytic promiscuity among the ancestral enzymes (P < 0.01). Ancestral esterases were more likely to catalyze a lyase reaction than modern esterases, and the ancestral HNL was more likely to catalyze ester hydrolysis than modern HNL's. One ancestral enzyme (HNL1) along the path from esterase to hydroxynitrile lyases was especially promiscuous and catalyzed both hydrolysis and lyase reactions with many substrates. A broader screen tested mechanistically related reactions that were not selected for by evolution: decarboxylation, Michael addition, γ-lactam hydrolysis and 1,5-diketone hydrolysis. The ancestral enzymes were more promiscuous than their modern descendants (P = 0.04). Thus, these reconstructed ancestral enzymes are catalytically promiscuous, but HNL1 is especially so.
| S-EPMC5466365 | biostudies-literature
Project description:The host-specific plant pathogen Pseudomonas syringae elicits the hypersensitive response (HR) in nonhost plants and secretes the HrpZ harpin in culture via the Hrp (type III) secretion system. Previous genetic evidence suggested the existence of another harpin gene in the P. syringae genome. hrpW was found in a region adjacent to the hrp cluster in P. syringae pv. tomato DC3000. hrpW encodes a 42. 9-kDa protein with domains resembling harpins and pectate lyases (Pels), respectively. HrpW has key properties of harpins. It is heat stable and glycine rich, lacks cysteine, is secreted by the Hrp system, and is able to elicit the HR when infiltrated into tobacco leaf tissue. The harpin domain (amino acids 1 to 186) has six glycine-rich repeats of a repeated sequence found in HrpZ, and a purified HrpW harpin domain fragment possessed HR elicitor activity. In contrast, the HrpW Pel domain (amino acids 187 to 425) is similar to Pels from Nectria haematococca, Erwinia carotovora, Erwinia chrysanthemi, and Bacillus subtilis, and a purified Pel domain fragment did not elicit the HR. Neither this fragment nor the full-length HrpW showed Pel activity in A230 assays under a variety of reaction conditions, but the Pel fragment bound to calcium pectate, a major constituent of the plant cell wall. The DNA sequence of the P. syringae pv. syringae B728a hrpW was also determined. The Pel domains of the two predicted HrpW proteins were 85% identical, whereas the harpin domains were only 53% identical. Sequences hybridizing at high stringency with the P. syringae pv. tomato hrpW were found in other P. syringae pathovars, Pseudomonas viridiflava, Ralstonia (Pseudomonas) solanacearum, and Xanthomonas campestris. DeltahrpZ::nptII or hrpW::OmegaSpr P. syringae pv. tomato mutants were little reduced in HR elicitation activity in tobacco, whereas this activity was significantly reduced in a hrpZ hrpW double mutant. These features of hrpW and its product suggest that P. syringae produces multiple harpins and that the target of these proteins is in the plant cell wall.
| S-EPMC107559 | biostudies-literature
Project description:A dynamic mathematical model has been developed and validated to describe the synthesis of pectate lyases (Pels), the major virulence factors in Dickeya dadantii. This work focuses on the simultaneous modeling of the metabolic degradation of pectin by Pel enzymes and the genetic regulation of pel genes by 2-keto-3-deoxygluconate (KDG), a catabolite product of pectin that inactivates KdgR, one of the main repressors of pel genes. This modeling scheme takes into account the fact that the system is composed of two time-varying compartments: the extracellular medium, where Pel enzymes cleave pectin into oligomers, and the bacterial cytoplasm where, after internalization, oligomers are converted to KDG. Using the quasi-stationary state approximations, the model consists of some nonlinear differential equations for which most of the parameters could be estimated from the literature or from independent experiments. The few remaining unknown parameters were obtained by fitting the model equations against a set of Pel activity data. Model predictions were verified by measuring the time courses of bacterial growth, Pel production, pel mRNA accumulation, and pectin consumption under various growth conditions. This work reveals that pectin is almost totally consumed before the burst of Pel production. This paradoxical behavior can be interpreted as an evolutionary strategy to control the diffusion process so that as soon as a small amount of pectin is detected by the bacteria in its surroundings, it anticipates more pectin to come. The model also predicts the possibility of bistable steady states in the presence of constant pectin compounds.
| S-EPMC2937882 | biostudies-literature
Project description:Harpins, such as HrpN of Erwinia amylovora, are extracellular glycine-rich proteins that elicit the hypersensitive reaction (HR). We identified hrpW of E. amylovora, which encodes a protein similar to known harpins in that it is acidic, rich in glycine and serine, and lacks cysteine. A putative HrpL-dependent promoter was identified upstream of hrpW, and Western blot analysis of hrpL mutants indicated that the production of HrpW is regulated by hrpL. HrpW is secreted via the Hrp (type III) pathway based on analysis of wild-type strains and hrp secretion mutants. When infiltrated into plants, HrpW induced rapid tissue collapse, which required active plant metabolism. The HR-eliciting activity was heat stable and protease sensitive. Thus, we concluded that HrpW is a new harpin. HrpW of E. amylovora consists of two domains connected by a Pro and Ser-rich sequence. A fragment containing the N-terminal domain was sufficient to elicit the HR. Although no pectate lyase activity was detected, the C-terminal region of HrpW is homologous to pectate lyases of a unique class, suggesting that HrpW may be targeted to the plant cell wall. Southern analysis indicated that hrpW is conserved among several Erwinia species, and hrpW, provided in trans, enhanced the HR-inducing ability of a hrpN mutant. However, HrpW did not increase the virulence of a hrpN mutant in host tissue, and hrpW mutants retained the wild-type ability to elicit the HR in nonhosts and to cause disease in hosts.
| S-EPMC107558 | biostudies-literature
Project description:Phenylalanine ammonia-lyases (PALs) are attractive biocatalysts for the stereoselective synthesis of non-natural phenylalanines. The rational design of PALs with extended substrate scope, highlighted the substrate specificity-modulator role of residue I460 of Petroselinum crispum PAL. Herein, saturation mutagenesis at key residue I460 was performed in order to identify PcPAL variants of enhanced activity or to validate the superior catalytic properties of the rationally explored I460V PcPAL compared with the other possible mutant variants. After optimizations, the saturation mutagenesis employing the NNK-degeneracy generated a high-quality transformant library. For high-throughput enzyme-activity screens of the mutant library, a PAL-activity assay was developed, allowing the identification of hits showing activity in the reaction of non-natural substrate, p-MeO-phenylalanine. Among the hits, besides the known I460V PcPAL, several mutants were identified, and their increased catalytic efficiency was confirmed by biotransformations using whole-cells or purified PAL-biocatalysts. Variants I460T and I460S were superior to I460V-PcPAL in terms of catalytic efficiency within the reaction of p-MeO-Phe. Moreover, I460T PcPAL maintained the high specificity constant of the wild-type enzyme for the natural substrate, l-Phe. Molecular docking supported the favorable substrate orientation of p-MeO-cinnamic acid within the active site of I460T variant, similarly as shown earlier for I460V PcPAL (PDB ID: 6RGS).
| S-EPMC7355458 | biostudies-literature