RESUMEN
Exo-rhamnogalacturonan lyase from Penicillium chrysogenum 31B (PcRGLX) was recently classified as a member of polysaccharide lyase (PL) family 26 along with hypothetical proteins derived from various organisms. In this study, we determined the crystal structure of PcRGLX as the first structure of a member of this family. Based on the substrate-binding orientation and substrate specificity, PcRGLX is an exo-type PL that cleaves rhamnogalacturonan from the reducing end. Analysis of PcRGLX-complex structures with reaction products indicate that the active site possesses an L-shaped cleft that can accommodate galactosyl side chains, suggesting side-chain-bypassing activity in PcRGLX. Furthermore, we determined the residues critical for catalysis by analyzing the enzyme activities of inactive variants.
Asunto(s)
Proteínas Fúngicas/química , Pectinas/química , Penicillium chrysogenum/enzimología , Polisacárido Liasas/química , Catálisis , Cristalografía por Rayos X , Relación Estructura-ActividadRESUMEN
We previously described an endo-acting rhamnogalacturonan (RG) lyase, termed PcRGL4A, of Penicillium chrysogenum 31B. Here, we describe a second RG lyase, called PcRGLX. We determined the cDNA sequence of the Pcrglx gene, which encodes PcRGLX. Based on analyses using a BLAST search and a conserved domain search, PcRGLX was found to be structurally distinct from known RG lyases and might belong to a new polysaccharide lyase family together with uncharacterized fungal proteins of Nectria haematococca, Aspergillus oryzae, and Fusarium oxysporum. The Pcrglx cDNA gene product (rPcRGLX) expressed in Escherichia coli demonstrated specific activity against RG but not against homogalacturonan. Divalent cations were not essential for the enzymatic activity of rPcRGLX. rPcRGLX mainly released unsaturated galacturonosyl rhamnose (ΔGR) from RG backbones used as the substrate from the initial stage of the reaction, indicating that the enzyme can be classified as an exo-acting RG lyase (EC 4.2.2.24). This is the first report of an RG lyase with this mode of action in Eukaryota. rPcRGLX acted synergistically with PcRGL4A to degrade soybean RG and released ΔGR. This ΔGR was partially decorated with galactose (Gal) residues, indicating that rPcRGLX preferred oligomeric RGs to polymeric RGs, that the enzyme did not require Gal decoration of RG backbones for degradation, and that the enzyme bypassed the Gal side chains of RG backbones. These characteristics of rPcRGLX might be useful in the determination of complex structures of pectins.
Asunto(s)
Penicillium chrysogenum/enzimología , Penicillium chrysogenum/genética , Polisacárido Liasas/genética , Polisacárido Liasas/metabolismo , Clonación Molecular , ADN de Hongos/química , ADN de Hongos/genética , Escherichia coli/genética , Expresión Génica , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADN , Especificidad por SustratoRESUMEN
Rhamnogalacturonan lyase (PcRGL4A) was purified from the culture supernatant of Penicillium chrysogenum 31B. PcRGL4A optimal activity occurred between pH 7-8 and at 40 °C. Conserved Domain Search analysis identified PcRGL4A as a member of Polysaccharide Lyase family 4. PcRGL4A contains two conserved catalytic and four conserved substrate-binding residues as determined by X-ray crystallography of the Aspergillus aculeatus RG lyase. Recombinant PcRGL4A (rPcRGL4A) expressed in Escherichia coli demonstrated specific activity against rhamnogalacturonan (RG) but not homogalacturonan. Analysis of the RG reaction products by high-performance anion-exchange chromatography revealed that rPcRGL4A cleaved the substrate in an endo-manner and that the major final product was an RG tetrasaccharide with 4-deoxy-4,5-unsaturated galacturonic acid at the nonreducing end. Based on these results, PcRGL4A was classified as an endo-acting RG lyase (EC 4.2.2.23). Divalent cations were not essential for the enzymatic activity of rPcRGL4A, but addition of calcium ions to the reaction mixture increased enzymatic activity. rPcRGL4A demonstrated a preference for RG lacking galactose decoration.
