Molecular cloning and characterization of a rhamnogalacturonan acetylesterase from Aspergillus aculeatus. Synergism between rhamnogalacturonan degrading enzymes.
A rhamnogalacturonan acetylesterase (RGAE) was purified to homogeneity from the filamentous fungus Aspergillus aculeatus, and the NH2-terminal amino acid sequence was determined. Full-length cDNAs encoding the enzyme were isolated from an A. aculeatus cDNA library using a polymerase chain reaction-generated product as a probe. The 936-base pair rha1 cDNA encodes a 250-residue precursor protein of 26,350 Da, including a 17-amino acid signal peptide. The rha1 cDNA was overexpressed in Aspergillus oryzae, a filamentous fungus that does not possess RGAE activity, and the recombinant enzyme was purified and characterized. Mass spectrometry of the native and recombinant RGAE revealed that the enzymes are heterogeneously glycosylated. In addition, the observed differences in their molecular masses, lectin binding patterns, and monosaccharide compositions indicate that the glycan moieties on the two enzymes are structurally different. The RGAE was shown to act in synergy with rhamnogalacturonase A as well as rhamnogalacturonase B from A. aculeatus in the degradation of apple pectin rhamnogalacturonan. RNA gel blot analyses indicate that the expression of rhamnogalacturonan degrading enzymes by A. acculeatus is regulated at the level of transcription and is subjected to carbon catabolite repression by glucose.
Kauppinen
S
S
GeneSearch, Novo Nordisk A/S, Novo Allé, Bagsvaerd, Denmark.
Christgau
S
S
Kofod
L V
LV
Halkier
T
T
Dörreich
K
K
Dalbøge
H
H
D000115:Acetylesterase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D005947:Glucose / Q000378:metabolism; D006031:Glycosylation; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010368:Pectins / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D018076:DNA, Complementary; D004271:DNA, Fungal; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D010368:Pectins; C096859:rhamnogalacturonan acetylesterase; D000115:Acetylesterase; D005947:Glucose