Crystal structures and properties of de novo circularly permuted 1,3-1,4-β-glucanases

Abstract

The 1,3-1,4-beta-glucanases from Bacillus macerans and Bacillus licheniformis, as well as related hybrid enzymes, are stable proteins comprised of one compact jellyroll domain. Their structures are studied in an effort to reveal the degree of redundancy to which the three-dimensional structure of protein domains is encoded by the amino acid sequence. For the hybrid 1,3-1,4-beta-glucanase H(A16-M), it could be shown recently that a circular permutation of the sequence giving rise to the variant cpA16M-59 is compatible with wildtype-like enzymatic activity and tertiary structure (Hahn et al., Proc. Natl. Acad. Sci. USA 91:10417-10421, 1994). Since the circular permutation yielding cpA16M-59 mimicks that found in the homologous enzyme from Fibrobacter succinogenes, the question arose whether de novo circular permutations, not guided by molecular evolution of the 1,3-1,4-beta-glucanases, could also produce proteins with native-like fold. The circularly permuted variants cpA16M-84, cpA16M-127, and cpA16M-154 were generated by PCR mutagenesis of the gene encoding H(A16-M), synthesized in Escherichia coli and shown to be active in beta-glucan hydrolysis. CpA16M-84 and cpA16M-127 were crystallized in space groups P2(1) and P1, respectively, and their crystal structures were determined at 1.80 and 2.07 A resolution. In both proteins the main parts of the beta-sheet structure remain unaffected by the circular permutation as is evident from a root-mean-square deviation of main chain atoms from the reference structure within the experimental error. The only major structural perturbation occurs near the novel chain termini in a surface loop of cpA16M-84, which becomes destabilized and rearranged. The results of this study are interpreted to show that: (1) several circular permutations in the compact jellyroll domain of the 1,3-1,4-beta-glucanases are tolerated without radical change of enzymatic activity or tertiary structure, (2) the three-dimensional structures of simple domains are encoded by the amino acid sequence with sufficient redundancy to tolerate a change in the sequential order of secondary structure elements along the sequence, and (3) the native N-terminal region is not needed to guide the folding polypeptide chain toward its native conformation.

Structural Redesign of Lipase B from Candida antarctica by Circular Permutation and Incremental Truncation

“…Several earlier crystallographic studies of laboratory-permutated proteins have shown only minor deviations in those proteins’ tertiary structures except in two regions: the original termini and the newly created N and C-termini 28–32. The fusion of the original termini often creates structurally poorly defined loops, in particular in the presence of extra amino acids to link distant residues.…”

Intersubunit circular permutation of human hemoglobin

“…Several other monomeric proteins have since been circularly permuted through genetic manipulations, all of which maintain a nativelike function. [3][4][5][6][7][8][9][10][11] Single subunits of multimeric proteins can also withstand circularly permuted sequences. For example, circularly permuted catalytic chains of aspartate transcarbamoylase combined in vitro with regulatory chains produced a folded and functional multimeric protein.…”

Thermal Stabilization of an Endoglucanase by Cyclization

“…Furthermore, the increased activities of the circular enzyme variants LicA-C1, LicA-C2, and LicA-C5 compared to the linear LicA-L1 suggest that the introduced junction and loop do not result in activity loss of the enzyme. Earlier studies have shown that circular permutations in the compact jelly roll domain of LicA are tolerated and did not result in significant changes in the enzymatic activity or tertiary structure [37, 38]. Therein, it was shown that the direct peptide bond linking of the N- and C-termini, without the addition of extra residues, did not introduce strain into the molecule.…”