Structural and Functional Analysis of a Multimodular Hyperthermostable Xylanase-Glucuronoyl Esterase from Caldicellulosiruptor kristjansonii
Artikel i vetenskaplig tidskrift, 2021

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.

Författare

Daniel Krska

Chalmers, Biologi och bioteknik, Industriell bioteknik

Scott Mazurkewich

Wallenberg Wood Science Center (WWSC)

Chalmers, Biologi och bioteknik, Industriell bioteknik

Haley A. Brown

University of Michigan

Yusuf Theibich

Köpenhamns universitet

J. C. N. Poulsen

Köpenhamns universitet

Adeline L. Morris

University of Michigan

Nicole M. Koropatkin

University of Michigan

L. Lo Leggio

Köpenhamns universitet

Johan Larsbrink

Chalmers, Biologi och bioteknik, Industriell bioteknik

Wallenberg Wood Science Center (WWSC)

Biochemistry

0006-2960 (ISSN) 1520-4995 (eISSN)

Vol. 60 27 2206-2220

Utveckling av värmetåliga enzymblandningar

Energimyndigheten (Dnr 2016‑011207), 2018-01-01 -- 2019-12-31.

Formas (Dnr 2016-01065), 2017-05-01 -- 2020-12-31.

Structure-based engineering of glucuronoyl esterases for separation of lignin and carbohydrates

Novo Nordisk Fonden (27698), 2018-01-01 -- 2020-12-31.

Ämneskategorier

Biokemi och molekylärbiologi

Strukturbiologi

Biokatalys och enzymteknik

DOI

10.1021/acs.biochem.1c00305

PubMed

34180241

Mer information

Senast uppdaterat

2021-08-21