PDBsum entry: 1axk

Fusion protein

PDB id: 1axk

Contents

Protein chains

393 a.a. *

Metals

_CA ×2

Waters ×312

* Residue conservation analysis

PDB id:

1axk

Name:

Fusion protein

Title:

Engineered bacillus bifunctional enzyme gluxyn-1

Structure:

Gluxyn-1. Chain: a, b. Fragment: fusion of 1,3-1,4-beta-glucanase domain and 1,4- beta-xylanase domain. Engineered: yes. Other_details: active as both a 1,3-1,4-beta-glucanase and a 1,4-beta-xylanase

Source:

Fragment: 1,4-beta-xylanase domain. Bacillus subtilis. Organism_taxid: 1423. Cell_line: dh5-alpha. Expressed in: escherichia coli. Expression_system_taxid: 562. Expression_system_cell_line: dh5-alpha.

Resolution:

2.10Å R-factor: 0.177 R-free: 0.224

Authors:

J.Ay,U.Heinemann

Key ref:

J.Aÿ et al. (1998). Structure and function of the Bacillus hybrid enzyme GluXyn-1: native-like jellyroll fold preserved after insertion of autonomous globular domain. Proc Natl Acad Sci U S A, 95, 6613-6618. PubMed id: 9618460 DOI: 10.1073/pnas.95.12.6613

Date:

16-Oct-97 Release date: 11-May-99

Protein chains

P18429  (XYNA_BACSU) -  Endo-1,4-beta-xylanase A

Seq: 213 a.a.

Struc: 393 a.a.*

Protein chains

P23904  (GUB_PAEMA) -  Beta-glucanase

Seq: 237 a.a.

Struc: 393 a.a.

* PDB and UniProt seqs differ at 26 residue positions (black crosses)

Enzyme reactions

• Enzyme class 1: E.C.3.2.1.8 - Endo-1,4-beta-xylanase.
• Reaction: Endohydrolysis of 1,4-beta-D-xylosidic linkages in xylans.
• Enzyme class 2: E.C.3.2.1.73 - Licheninase.
• Reaction: Hydrolysis of 1,4-beta-D-glycosidic linkages in beta-D-glucans containing 1,3- and 1,4-bonds.

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

 Gene Ontology (GO) functional annotation 

• Biological process: metabolic process (3 terms)
• Biochemical function: hydrolase activity (4 terms)

DOI no: 10.1073/pnas.95.12.6613

Proc Natl Acad Sci U S A 95:6613-6618 (1998)

PubMed id: 9618460

Structure and function of the Bacillus hybrid enzyme GluXyn-1: native-like jellyroll fold preserved after insertion of autonomous globular domain.

J.Aÿ, F.Götz, R.Borriss, U.Heinemann.

ABSTRACT

The 1,3-1,4-beta-glucanase from Bacillus macerans (wtGLU) and the 1, 4-beta-xylanase from Bacillus subtilis (wtXYN) are both single-domain jellyroll proteins catalyzing similar enzymatic reactions. In the fusion protein GluXyn-1, the two proteins are joined by insertion of the entire XYN domain into a surface loop of cpMAC-57, a circularly permuted variant of wtGLU. GluXyn-1 was generated by protein engineering methods, produced in Escherichia coli and shown to fold spontaneously and have both enzymatic activities at wild-type level. The crystal structure of GluXyn-1 was determined at 2.1 A resolution and refined to R = 17.7% and R(free) = 22.4%. It shows nearly ideal, native-like folding of both protein domains and a small, but significant hinge bending between the domains. The active sites are independent and accessible explaining the observed enzymatic activity. Because in GluXyn-1 the complete XYN domain is inserted into the compact folding unit of GLU, the wild-type-like activity and tertiary structure of the latter proves that the folding process of GLU does not depend on intramolecular interactions that are short-ranged in the sequence. Insertion fusions of the GluXyn-1 type may prove to be an easy route toward more stable bifunctional proteins in which the two parts are more closely associated than in linear end-to-end protein fusions.

Selected figure(s)

Figure 1.
Fig. 1. Design and construction of GluXyn-1. (A) Schematic structure of GluXyn-1 and the parental enzymes used for the construction of the bifunctional insertion fusion protein. The circularly permuted cpMAC-57 (9) starts with residue 57 of wtGLU whereas the original N and C termini are covalently linked leading to an enzyme whose C terminus is residue 56. cpMAC-57 and wtXYN (bold line) were linked by cutting the loop between residues 212 and 1 of cpMAC-57 (numbering according to GLU) and connecting those residues with the terminal residues of XYN. The resulting construct, GluXyn-1, starts with N57 and ends with C56 just as cpMAC-57, but contains in its central part the full-length sequence of mature XYN. For simplicity, only the sequences encoding the mature enzymes are shown. Amino acid residues in GluXyn-1 are numbered according to their sequence positions in the parent proteins, wtGLU and wtXYN, throughout this paper. (B) Construction of GluXyn-1 by PCR splicing of gene fragments. Fragment A covers the upstream regulatory sequence of the gene encoding the B. macerans 1,3-1,4- -glucanase (open box) and sequences encoding the signal peptide (SP) and the mature 1,3-1,4- -glucanase from residues 57 to 212 (solid box). Fragment B encodes the full-length mature XYN, and fragment C consists of the coding region for residues 1-56 of B. macerans -glucanase (solid box) and a short segment of the 3' noncoding region of cpMAC-57 (open box). Using sequence specific primers, the three fragments were amplified by splicing-by-overlap extension whereby terminal extensions complementary to the adjacent sequence in the resulting GluXyn-1-encoding construct (shaded box) were linked to the synthesized fragments. In the second (fragments B and C) and third step of amplification (fragments A and BC) the obtained fragments were used to amplify the full-length hybrid gene encoding GluXyn-1. Sequence specific primers PP15-PP18 are indicated by arrows. Direct (DIR) and reverse (REV) primers annealing with the flanking vector sequences also are shown.

Figure 2.
Fig. 2. Schematic drawing of the crystal structure of GluXyn-1. Both polypeptide chain termini are in the GLU domain (Upper) of the insertion fusion protein. In the MOLSCRIPT (40) diagram, the N-terminal part of the GLU domain is colored in green, the XYN domain (Lower) is in blue, and the C-terminal part of the GLU domain, after XYN in the sequence, is in yellow. -Sheets are shown as curved arrows, -helices are shown as red, wound ribbons, the calcium ion bound to the GLU domain is shown as a purple sphere half concealed by a -strand, and the disulfide bridge of the GLU domain is shown in ball-and-stick representation (gold).

Figures were selected by the author.