Efficient enzymatic conversion of crystalline polysaccharides is crucial for an economically and environmentally sustainable bioeconomy but remains unfavorably inefficient. We describe an enzyme that acts on the surface of crystalline chitin, where it introduces chain breaks and generates oxidized chain ends, thus promoting further degradation by chitinases. This enzymatic activity was discovered and further characterized by using mass spectrometry and chromatographic separation methods to detect oxidized products generated in the absence or presence of H(2)(18)O or (18)O(2). There are strong indications that similar enzymes exist that work on cellulose. Our findings not only demonstrate the existence of a hitherto unknown enzyme activity but also provide new avenues toward more efficient enzymatic conversion of biomass.
Tailoring steps in aromatic polyketide antibiotic biosynthesis are an important source of structural diversity and, consequently, an intriguing focal point for enzymological studies. PgaE and PgaM from Streptomyces sp. PGA64 are representatives of flavoenzymes catalyzing early post-PKS reactions in angucycline biosynthesis. This in vitro study illustrates that the chemoenzymatic conversion of UWM6 into the metabolite, gaudimycin C, requires multiple closely coupled reactions to prevent intermediate degradation. The NMR structure of gaudimycin C confirms that the reaction cascade involves C12- and C12b-hydroxylation, C2,3-dehydration, and stereospecific ketoreduction at C6. Enzymatic 18O incorporation studies verify that the oxygens at C12 and C12b derive from O2 and H2O, respectively. The results indicate that PgaM deviates mechanistically from flavoprotein monooxygenases, and suggest an alternative catalytic mechanism involving a quinone methide intermediate.
Enzymatic conversion of chitin, a β-1,4 linked polymer of N-acetylglucosamine, is of major interest in areas varying from the biorefining of chitin-rich waste streams to understanding how medically relevant fungi remodel their chitin-containing cell walls. Although numerous chitinolytic enzymes have been studied in detail, relatively little is known about enzymes capable of deacetylating chitin. We describe the structural and functional characterization of a 237 residue deacetylase (AnCDA) from Aspergillus nidulans FGSC A4. AnCDA acts on chito-oligomers, crystalline chitin, chitosan, and acetylxylan, but not on peptidoglycan. The K
m and k
cat of AnCDA for the first deacetylation of penta-N-acetyl-chitopentaose are 72 µM and 1.4 s−1, respectively. Combining mass spectrometry and analyses of acetate release, it was shown that AnCDA catalyses mono-deacetylation of (GlcNAc)2 and full deacetylation of (GlcNAc)3–6 in a non-processive manner. Deacetylation of the reducing end sugar was much slower than deacetylation of the other sugars in chito-oligomers. These enzymatic characteristics are discussed in the light of the crystal structure of AnCDA, providing insight into how the chitin deacetylase may interact with its substrates. Interestingly, AnCDA activity on crystalline chitin was enhanced by a lytic polysaccharide monooxygenase that increases substrate accessibility by oxidative cleavage of the chitin chains.
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