Fig. 4: Modulation of HA length.
From: Structural insights into translocation and tailored synthesis of hyaluronan
a, XlHAS1 composite model showing mutagenized residues forming the active site (colored blue and teal for residues of the GT domain and gating loop, respectively) or the HA channel (colored light pink and pink for residues of TMHs and IFHs, respectively). b, Mutagenesis of active-site-lining residues. In vitro HA biosynthesis by the indicated XlHAS1 mutants for 1 h or 8 h (short or long reaction, respectively). Agarose gels were stained with Stains-all. The dashed line roughly indicates the WT’s highest-molecular-weight product. c, Catalytic rates of the mutants shown in b. Rates, normalized to WT, report substrate turnovers and were determined by quantifying UDP release in real time during HA synthesis (n = 8). d, Catalytic activity of gating loop mutants of CvHAS. Activity was determined by quantifying 3H-labeled HA by scintillation counting and is expressed relative to WT10. Numbers in parentheses indicate residue numbers in XlHAS1 (n = 3). e, Mutagenesis of charged channel and active-site-lining residues. In vitro HA biosynthesis by the indicated mutants, similar to c (n = 8). NEC, no-enzyme control. Error bars in c–e represent the s.d. from the mean. f, Similar to b but for charged residues. g, Comparison of HA products obtained from NC64A Chlorella algae infected with PBCV-1 virus and in vitro synthesized by purified XlHAS1 and CvHAS in LMNG + CHS micelle (det.) and proteoliposome (lip.). The boxed region indicates the hyaluronidase-sensitive isolated sample. The experiment was repeated three times with similar results.
