Site-directed mutagenesis of bacterial cellulose synthase highlights sulfur-arene interaction as key to catalysis.
Cellulose is one of the most abundant biological polymers on Earth, and is synthesized by the cellulose synthase complex in cell membranes. Although many cellulose synthase genes have been identified over the past 25 years, functional studies of cellulose synthase using recombinant proteins have rarely been conducted. In this study, we conducted a functional analysis of cellulose synthase with site-directed mutagenesis, by using recombinant cellulose synthase reconstituted in living Escherichia coli cells that we recently constructed (cellulose-synthesizing E. coli, CESEC). We demonstrated that inactivating mutations at an important amino acid residue reduced cellulose production. In this study, an interesting loss-of-function mutation occurred on Cys308, whose main chain carbonyl plays an important role for locating the cellulose terminus. Mutating this cysteine to serine, thus changing sulfur to oxygen in the side chain, abolished cellulose production in addition to other apparent detrimental mutations. This unexpected result highlights that the thiol side-chain of this cysteine plays an active role in catalysis, and additional mutation experiments indicated that the sulfur-arene interaction around Cys308 is a key in cellulose-synthesizing activity. Data obtained by CESEC shed light on the function of cellulose synthase in living cells, and will deepen our understanding of the mechanism of cellulose synthase.
Research Institute for Sustainable Humanosphere (RISH), Kyoto University, Uji, Kyoto 611-0011, Japan.
Shi-Jing
SJ
Sun
Research Institute for Sustainable Humanosphere (RISH), Kyoto University, Uji, Kyoto 611-0011, Japan.
Yoshiki
Y
Horikawa
Research Institute for Sustainable Humanosphere (RISH), Kyoto University, Uji, Kyoto 611-0011, Japan.
Masahisa
M
Wada
Research Institute for Sustainable Humanosphere (RISH), Kyoto University, Uji, Kyoto 611-0011, Japan.
Junji
J
Sugiyama
Research Institute for Sustainable Humanosphere (RISH), Kyoto University, Uji, Kyoto 611-0011, Japan. Electronic address: timai@rish.kyoto-u.ac.jp.
Tomoya
T
Imai
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D002482:Cellulose / Q000096:biosynthesis; D003545:Cysteine / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013455:Sulfur / Q000737:chemistry
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D013455:Sulfur; D002482:Cellulose; D005964:Glucosyltransferases; C478648:cellulose synthase; D003545:Cysteine
Cellulose synthase; CesA; Gluconacetobacter xylinus; Site-directed mutagenesis; Sulfur–arene interaction