Abstract
Chitin is one of the most abundant renewable organic materials found on earth. The chitin utilization locus in Flavobacterium johnsoniae, which encodes necessary proteins for complete enzymatic depolymerization of crystalline chitin, has recently been characterized but no detailed structural information on the enzymes was provided. Here we present protein structures of the F. johnsoniae chitobiase (FjGH20) and chitinase B (FjChiB). FjGH20 is a multi-domain enzyme with a helical domain not before observed in other chitobiases and a domain organization reminiscent of GH84 (β-N-acetylglucosaminidase) family members. The structure of FjChiB reveals that the protein lacks loops and regions associated with exo-acting activity in other chitinases and instead has a more solvent accessible substrate binding cleft, which is consistent with its endo-chitinase activity. Additionally, small angle X-ray scattering data were collected for the internal 70 kDa region that connects the N- and C-terminal chitinase domains of the unique 158 kDa multi-domain chitinase A (FjChiA). The resulting model of the molecular envelope supports bioinformatic predictions of the region comprising six domains, each with similarities to either Fn3-like or Ig-like domains. Taken together, the results provide insights into chitin utilization by F. johnsoniae and reveal structural diversity in bacterial chitin metabolism.
Highlights
Chitin is one of the most abundant renewable organic materials found on earth
The chitinolytic machinery of the bacterium Serratia marcescens has served as a model system for enzymatic chitin turnover, and consists of five core enzymes: SmChiA and B, SmChiC, CBP21 (LPMO), and a chitobiase converting chitooligosaccharides (CHOs) into GlcNAc2
The predicted catalytic domain of FjGH20 shares 25 to 38% sequence identity with previously characterized GH20 family members, but the overall amino acid sequence identity is as low as 15% compared to certain studied enzymes
Summary
The chitin utilization locus in Flavobacterium johnsoniae, which encodes necessary proteins for complete enzymatic depolymerization of crystalline chitin, has recently been characterized but no detailed structural information on the enzymes was provided. An atypical feature of the ChiUL compared to other characterized PULs from gut bacteria is that the main chitinase, FjChiA, is not attached to the outer membrane but is instead solubly secreted into the extracellular milieu by the Type IX secretion s ystem[8,9,10] This possibly reflects the fact that the environment of F. johnsoniae is less nutrient dense compared to the gastrointestinal tracts of animals and may require both protein secretion and cellular motility for efficient scavenging for resources[11]. The fourth enzyme, the FjGH20 chitobiase/N-acetylglucosaminidase, was active only on CHOs
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