Role of lysine, tryptophan and calcium in the β-elimination activity of a low-molecular-mass pectate lyase from Fusarium moniliformae

Abstract

An extracellular pectate lyase from Fusarium moniliformae was purified to homogeneity by affinity chromatography followed by gel filtration, with a yield of 76.5%. Laser desorption MS of the enzyme gave a molecular mass of 12,133.5 +/- 2.5 Da. The pectate lyase was a glycoprotein with a 5% carbohydrate content and had a pl value of 9.1. Atomic-emission spectrometry showed that Ca2+ was a part of the holoenzyme held by carboxy groups of the protein. These results support the hypothesis of a putative Ca2+ site suggested by Yodder, Keen and Jurnak [(1993) Science 260, 1503-1507] in the crystal structure of pectate lyase C of Erwinia chrysanthemi. Loss of Ca2+ was observed by treatment with EGTA or carboxy-modifying Woodward's reagent K, with subsequent loss of enzyme activity. Tryptophan fluorescence quenching showed that Ca2+ does not affect binding of substrate to enzyme. Chemical-modification and substrate-protection studies showed the presence of lysine and tryptophan at or near the active site of the pectate lyase. Chemically modified enzyme showed no major structural changes as determined by CD. Amino acid analyses of native, trinitrobenzenesulphonate (TBNS)-treated and substrate-protected TNBS-treated enzyme showed that a single essential residue of lysine is present at or near the active-site. Substrate-affinity studies showed that tryptophan could be essential for substrate binding, whereas lysine could be involved in the catalysis. Fluorescence quenching further confirmed the involvement of tryptophan in substrate binding. The reaction mechanism involving beta-elimination by this enzyme is discussed.

Rhizobium etli HrpW is a pectin-degrading enzyme and differs from phytopathogenic homologues in enzymically crucial tryptophan and glycine residues

“…W105V and W105A, respectively) did not result in enzymically active proteins. A role for a single, unspecified tryptophan residue in pectate lyase activity was suggested previously by Rao et al (1996), while aromatic amino acid residues such as tryptophan and phenylalanine are also found in the substrate-binding cleft of pectin lyases (Benen & Visser, 2003). Recently, aromatic residues were shown to be involved in the recognition of oligogalacturonides by a periplasmic binding protein involved in pectin metabolism of Yersinia enterocolitica (Abbott & Boraston, 2007).…”

Cloning of the Trichoderma reesei cDNA Encoding a Glucuronan Lyase Belonging to a Novel Polysaccharide Lyase Family

“…Jenkins et al (22) argued that the role of Ca 2ϩ is to withdraw electrons from the C-6 carboxylate of the substrate. This effect acidifies the C-5 proton, making it more susceptible to attack by a base (14,36). Ca 2ϩ in TrGL may play a similar role in substrate binding and catalysis.…”

Microbial pectate lyases: characterization and enzymological properties

“…The MM of Pel generally varies from 20 to 70 kDa (Table 1). However, the MM of Pel from F. moniliformae is reported to be 11 kDa by gel filtration, 10.6 kDa by ultracentrifugation and 17.5 kDa by SDS-PAGE (Rao et al 1996).…”

“…PelZ activity of E. chrysanthemi is inhibited by epicatechin, salicyclic acid and indole acetic acid with K i values of 0.20, 0.38 and 0.30 mM, respectively (Pissavin et al 1998). The five Shevchik et al (1997) F. moniliformae 0.6 1.2 Rao et al (1996) F. solani 1.0 0.13 Crawford and Kolattukudy (1987) P. cellulosa 2.0-4.0 0.104 Brown et al (2001) P. fluorescens 0.5 1.28 Liao et al (1997) P. marginalis 1.0 Hayashi et al (1997) P. viridiflava 0.5 1.11 Liao et al (1997) Thermoanaerobacter italicus -0.5 Kozianowski et al (1997) major Pels from E. chrysanthemi are also inhibited by epicatechin with a K i ranging between 0.1 and 0.2 mM and salicylic acid with a K i of 0.2-0.4 mM (Tardy et al 1997).…”

“…Form this strain, PelA was purified to homogeneity employing two steps, namely chromatofocusing and hydrophobic interaction chromatography to yield specific activity of 2,300 U mg -1 dry weight. A Pel from F. moniliformae has been purified to homogeneity by affinity chromatography followed by gel filtration (Rao et al 1996). The enzyme is a glycoprotein with a 5% carbohydrate content.…”