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Glycoside hydrolase (GH) family 12 comprises enzymes with a wide range of activities critical for the degradation of lignocellulose. However, the important roles of the loop regions of GH12 enzymes in substrate specificity and catalytic efficiency remain poorly understood. This study examined how the loop 3 region affects the enzymatic properties of GH12 glucanases using Nf EG12A from Neosartorya fischeri P1 and EG (PDB 1KS4 ) from Aspergillus niger . Acidophilic and thermophilic Nf EG12A had the highest catalytic efficiency ( k cat / K m , 3,001 and 263 ml/mg/s toward lichenin and carboxymethyl cellulose sodium [CMC-Na], respectively) known so far. Based on the multiple-sequence alignment and homology modeling, two specific sequences (FN and STTQA) were identified in the loop 3 region of GH12 endoglucanases from fungi. To determine their functions, these sequences were introduced into Nf EG12A, or the counterpart sequence STTQA was removed from EG. These modifications had no effects on the optimal pH and temperature or substrate specificity but changed the catalytic efficiency ( k cat / K m ) of these enzymes (in descending order, Nf EG12A [100%], Nf EG12A-FN [140%], and Nf EG12A-STTQA [190%]; EG [100%] and EGΔSTTQA [41%]). Molecular docking and dynamic simulation analyses revealed that the longer loop 3 in GH12 may strengthen the hydrogen-bond interactions between the substrate and protein, thereby increasing the turnover rate ( k cat ). This study provides a new insight to understand the vital roles of loop 3 for GH12 endoglucanases in catalysis. IMPORTANCE Loop structures play critical roles in the substrate specificity and catalytic hydrolysis of GH12 enzymes. Three typical loops exist in these enzymes. Loops 1 and 2 are recognized as the catalytic loops and are closely related to the substrate specificity and catalytic efficiency. Loop 3 locates in the −1 or +1 subsite and varies a lot in amino acid composition, which may play a role in catalysis. In this study, two GH12 glucanases, Nf EG12A and EG, which were mutated by introducing or deleting partial loop 3 sequences FN and/or STTQA, were selected to identify the function of loop 3. It revealed that the longer loop 3 of GH12 glucanases may strengthen the hydrogen network interactions between the substrate and protein, consequently increasing the turnover rate ( k cat ). This study proposes a strategy to increase the catalytic efficiency of GH12 glucanases by improving the hydrogen network between substrates and catalytic loops.

Isavuconazole is an extended-spectrum triazole with in vitro activity against a wide variety of fungal pathogens. Clinical isolates of molds Aspergillus lentulus and Neosartorya udagawae and yeast Cryptococcus gattii VGII (implicated in the outbreak in the Pacific Northwest, North America) exhibit reduced susceptibilities to several azoles but higher susceptibilities to isavuconazole.

A recent report on several cases of invasive aspergillosis caused by Neosartorya udagawae suggested distinctive patterns of disease progression between N. udagawae and Aspergillus fumigatus . This prompted us to characterize N. udagawae in comparison to A. fumigatus . Our findings showed that both species exist in two mating types at similar ratios and produce gliotoxin. However, the thermotolerance of the two species differs: while A. fumigatus is able to grow at 55°C but not at 10°C, N. udagawae is able to grow at 10°C but fails to grow at >42°C. Furthermore, compared to A. fumigatus , the conidia of N. udagawae require longer incubation periods to germinate at 37°C and are more susceptible to neutrophil attack as well as hydrogen peroxide; N. udagawae is also less virulent in gp91 phox − / − mice. These findings suggest that growth and susceptibility to the host response might account for the reduced virulence of N. udagawae and the subtle distinction in the progression of the disease caused by the two species.

Invasive fungal infections caused by Neosartorya pseudofischeri S. W. Peterson [anamorph Aspergillus thermomutatus (Paden) S. W. Peterson] are extremely rare. Phenotypically, the anamorphic state of N. pseudofischeri resembles Aspergillus fumigatus , the predominant agent of invasive aspergillosis in immunocompromised hosts. We report the recovery of three clinical isolates of N. pseudofischeri , all initially misidentified by morphological characteristics as A. fumigatus . All three isolates were correctly identified by sequencing portions of the β-tubulin and the rodlet A genes. Only one of the three isolates produced the confirmatory fruiting bodies and was thus classified as N. pseudofischeri ; the other isolates did not produce asci and were therefore identified as A. thermomutatus . All three isolates had higher MICs to voriconazole in vitro compared to A. fumigatus Af293. This report emphasizes that phenotypic identification of filamentous fungi may not identify morphologically similar, but genetically distinct, members of the genus Aspergillus section Fumigati . Accurate identification of these organisms may be clinically meaningful, given their potential differences in antifungal susceptibilities.

Nucleotide sequences of 426 bp from the mitochondrial (mt) cytochrome b genes of six anamorph species and two species of Neosartorya teleomophs of Aspergillus section Fumigati were determined. These sequences were used to build nucleotide- and amino acid-based trees for phylogenetic analysis. Thirteen strains of A. fumigatus including 10 clinical isolates of A. fumigatus , 1 type culture of A. fumigatus var. fumigatus , 1 type culture of A. fumigatus var. ellipticus , and 1 strain of A. fumigatus var. albus , had the same nucleotide sequences. One strain of A. fumisynnematus , two strains labeled A. neoellipticus , two strains of A. viridinutans , and one strain of A. duricaulis had distinct nucleotide and amino acid sequences. Two strains of A. brevipes were divided into two types. One produced a 1,500-bp fragment that included an intron. The nucleotide sequences of its two exons were similar to those of the A. fumigatus , and the derived amino acid sequence was the same as that for A. fumigatus . The other produced a 426-bp fragment and had the same nucleotide and amino acid sequences as A. unilateralis. Neosartorya fischeri var. fischeri and N. stramenia had nucleotide sequences that differed from that of A. fumigatus . These species possessed their own characteristic nucleotide sequences that differed from each other. In comparisons of homologous sequences from four other pathogenic species of Aspergillus , regions specific to section Fumigati were found. The mt cytochrome b gene analysis was valuable for the identification, classification, and phylogenetic analysis of isolates of section Fumigati .