C. Fanutti et al., THE CONSERVED NONCATALYTIC 40-RESIDUE SEQUENCE IN CELLULASES AND HEMICELLULASES FROM ANAEROBIC FUNGI FUNCTIONS AS A PROTEIN DOCKING DOMAIN, The Journal of biological chemistry, 270(49), 1995, pp. 29314-29322
Two cDNAs, designated xynA and manA, encoding xylanase A (XYLA) and ma
nnanase A (MANA), respectively, were isolated from a cDNA Library deri
ved from mRNA extracted from the anaerobic fungus, Piromyces. XYLA and
MANA displayed properties typical of endo beta 1,4-xylanases and mann
anases, respectively. Neither enzyme hydrolyzed cellulosic substrates.
The nucleotide sequences of xynA and manA revealed open reading frame
s of 1875 and 1818 base pairs, respectively, coding for proteins of M(
r) 68,049 (XYLA) and 68,055 (MANA). The deduced primary structure of M
ANA revealed a 458-amino acid sequence that exhibited identity with Ba
cillus and Pseudomonas fluorescens subsp, cellulosa mannanases belongi
ng to glycosyl hydrolase Family 26. A 40-residue reiterated sequence,
which was homologous to duplicated noncatalytic domains previously obs
erved in Neocallimastix patriciarum xylanase A and endoglucanase B, wa
s located at the C terminus of MANA. XYLA contained two regions that e
xhibited sequence identity with the catalytic domains of glycosyl hydr
olase Family 11 xylanases and were separated by a duplicated 40-residu
e sequence that exhibited strong homology to the C terminus of MANA. A
nalysis of truncated derivatives of MANA confirmed that the N-terminal
458-residue sequence constituted the catalytic domain, while the C-te
rminal domain was not essential for the retention of catalytic activit
y. Similar deletion analysis of XYLA showed that the C-terminal cataly
tic domain homologue exhibited catalytic activity, but the correspondi
ng putative N-terminal catalytic domain did not function as a xylanase
. Fusion of the reiterated noncatalytic 40-residue sequence conserved
in XYLA and MANA to glutathione S-transferase, generated a hybrid prot
ein that did not associate with cellulose, but bound to 97- and 116-kD
a polypeptides that are components of the multienzyme cellulase-hemice
llulase complexes of Piromyces and Neocallimastix patriciarum, respect
ively. The role of this domain in the assembly of the enzyme complex i
s discussed.