Structural and Functional Analysis of a Multimodular Hyperthermostable Xylanase-Glucuronoyl Esterase from Caldicellulosiruptor kristjansonii

Abstract

The hyperthermophilic bacterium Caldicellulosiruptor kristjansonii encodes an unusual enzyme, CkXyn10C-GE15A, which incorporates two catalytic domains, a xylanase and a glucuronoyl esterase, and five carbohydrate-binding modules (CBMs) from families 9 and 22. The xylanase and glucuronoyl esterase catalytic domains were recently biochemically characterized, as was the ability of the individual CBMs to bind insoluble polysaccharides. Here, we further probed the abilities of the different CBMs from CkXyn10C-GE15A to bind to soluble poly- and oligosaccharides using affinity gel electrophoresis, isothermal titration calorimetry, and differential scanning fluorimetry. The results revealed additional binding properties of the proteins compared to the former studies on insoluble polysaccharides. Collectively, the results show that all five CBMs have their own distinct binding preferences and appear to complement each other and the catalytic domains in targeting complex cell wall polysaccharides. Additionally, through renewed efforts, we have achieved partial structural characterization of this complex multidomain protein. We have determined the structures of the third CBM9 domain (CBM9.3) and the glucuronoyl esterase (GE15A) by X-ray crystallography. CBM9.3 is the second CBM9 structure determined to date and was shown to bind oligosaccharide ligands at the same site but in a different binding mode compared to that of the previously determined CBM9 structure from Thermotoga maritima. GE15A represents a unique intermediate between reported fungal and bacterial glucuronoyl esterase structures as it lacks two inserted loop regions typical of bacterial enzymes and a third loop has an atypical structure. We also report small-angle X-ray scattering measurements of the N-terminal CBM22.1–CBM22.2–Xyn10C construct, indicating a compact arrangement at room temperature.

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“…No binding data are currently available for these however, and it is an open question what part(s) of the cell wall these CBM2 modules target. One studied bacterial GE that is coupled to several CBMs and that has been thoroughly investigated is Ck GE15A [ 33 , 39 ]. The full-length Ck Xyn10C-GE15A enzyme does not comprise only the GE, but consists of two N-terminal CBM22 modules, linked to a glycoside hydrolase family 10 (GH10) xylanase, three additional CBM9 modules before the GE domain, which is followed by cadherin and surface-layer homology domains thought to anchor the large enzyme to the outer Gram-positive cell wall.…”

“…The full-length Ck Xyn10C-GE15A enzyme does not comprise only the GE, but consists of two N-terminal CBM22 modules, linked to a glycoside hydrolase family 10 (GH10) xylanase, three additional CBM9 modules before the GE domain, which is followed by cadherin and surface-layer homology domains thought to anchor the large enzyme to the outer Gram-positive cell wall. Interestingly, the CBMs display various binding properties, and collectively enable the protein to bind virtually all major plant cell wall glycans [ 33 , 39 ]. Likely, also other CBM-appended GEs benefit from CBMs to help guide them to their complex LCC target substrate, or close proximity thereof.…”

“…As already shown by initial crystallographic ligand binding studies ( Figure 4 ) [ 43 ], the GlcA moiety of the esters cleaved by GEs binds in a pocket located above the main parallel β-sheet of the ABH fold ( Figure 3 ), which is located on a fairly open surface in the structurally characterized fungal members of CE15 [ 38 , 43 , 44 ]. In structurally characterized bacterial enzymes, sequence insertions create high ridges on one or two sides of the substrate binding surface ( Figure 3 ), indicating that the enzymes might interact more intimately with their complex substrate than their fungal counterparts [ 28 , 31 , 39 , 42 , 45 , 46 ].…”

“…The interaction with the lignin portion of LCC substrates has been more difficult to assess, and thus far no structures of Michaelis complexes of GEs have been solved. However, the large inserts close to the active site in bacterial enzymes have been proposed to interact with lignin as they in several cases have a surface rich in aromatic residues facing the presumed ‘lignin side’ of the substrate ( Figure 6 ) [ 28 , 31 , 39 ]. Possibly, these regions could be involved in LCC interactions, both productively to access the complex lignin–carbohydrate substrate, or to help avoid unproductive binding to lignin as described for Tt CE15A, above [ 32 ].…”

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“…CesA, a bimodular acetyl xylan esterase-glucuronoyl esterase, studied by Aurilia, et al [ 101 ] as component of the cellulosome from Ruminococcus flavefaciens , revealed a C terminal domain whose GE function was subsequently demonstrated by Biely et al [ 102 ] after its expression in E. coli . A multiple domain glucuronoyl esterase (CkXyn10C-GE15A), containing a module with xylanase activity and others with carbohydrate binding function, was studied from the hyperthermophilic bacterium Caldicellulosiruptor kristjansonii [ 103 ]. CkXyn10C-GE15 is the most thermostable GE studied so far, with an optimal temperature around 72 °C.…”