Insertion of a xylanase in xylose binding protein results in a xylose-stimulated xylanase.
BACKGROUND
Product inhibition can reduce catalytic performance of enzymes used for biofuel production. Different mechanisms can cause this inhibition and, in most cases, the use of classical enzymology approach is not sufficient to overcome this problem. Here we have used a semi-rational protein fusion strategy to create a product-stimulated enzyme.


RESULTS
A semi-rational protein fusion strategy was used to create a protein fusion library where the Bacillus subtilis GH11 xylanase A (XynA) was inserted at 144 surface positions of the Escherichia coli xylose binding protein (XBP). Two XynA insertions at XBP positions 209 ([209]XBP-Xyn-XBP) and 262 ([262]XBP-Xyn-XBP) showed a 20% increased xylanolytic activity in the presence of xylose, conditions where native XynA is inhibited. Random linkers of 1-4 Gly/Ala residues were inserted at the XynA N- and C-termini in the [209]XBP and [262]XBP, and the chimeras 2091A and 2621B were isolated, showing a twofold increased xylanolytic activity in the presence of xylose and k cat values of 200 and 240 s(-1) in the 2091A and 2621B, respectively, as compared to 70 s(-1) in the native XynA. The xylose affinity of the XBP was unchanged in the chimeras, showing that the ~3- to 3.5-fold stimulation of catalytic efficiency by xylose was the result of allosteric coupling between the XBP and XynA domains. Molecular dynamics simulations of the chimeras suggested conformation alterations in the XynA on xylose binding to the XBP resulted in exposure of the catalytic cavity and increased mobility of catalytic site residues as compared to the native XynA.


CONCLUSIONS
These results are the first report of engineered glycosyl hydrolase showing allosteric product stimulation and suggest that the strategy may be more widely employed to overcome enzyme product inhibition and to improve catalytic performance. Graphical abstractProtein fusion of a GH11 xylanase (in red) and a xylose binding protein (XBP, in blue) results in a xylanase-XBP chimera that presents allosteric activation of the xylanase activity by xylose (shown as a space-filled molecule bound to the xylanase-XBP chimera).
Johns Hopkins University, Baltimore, MD USA ; Departamento de Bioquímica e Imunologia, FMRP, Universidade de São Paulo-USP, Ribeirão Preto, SP Brazil.
Lucas Ferreira
LF
Ribeiro
Johns Hopkins University, Baltimore, MD USA.
Nathan
N
Nicholes
Institute for Bioscience and Biotechnology Research, Rockville, MD USA.
Jennifer
J
Tullman
Departamento de Bioquímica e Imunologia, FMRP, Universidade de São Paulo-USP, Ribeirão Preto, SP Brazil ; University of Maryland Baltimore County-UMBC, Baltimore, MD USA.
Liliane Fraga Costa
LF
Ribeiro
Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo-USP, Av. Bandeirantes, 3900, Ribeirão Preto, SP 14040-901 Brazil.
Carlos Alessandro
CA
Fuzo
Universidade Federal do Rio Grande do Norte, Natal, Brazil.
Davi Serradella
DS
Vieira
Departamento de Bioquímica e Imunologia, FMRP, Universidade de São Paulo-USP, Ribeirão Preto, SP Brazil.
Gilvan Pessoa
GP
Furtado
Johns Hopkins University, Baltimore, MD USA.
Marc
M
Ostermeier
Brazilian Bioethanol Science and Technology Laboratory CTBE/CNPEM, Campinas, Brazil ; Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo-USP, Av. Bandeirantes, 3900, Ribeirão Preto, SP 14040-901 Brazil.
Richard John
RJ
Ward
Enzyme engineering; Heterotropic allosteric regulation; Non-homologous genes; Semi-rational design