THE CONSERVED NONCATALYTIC 40-RESIDUE SEQUENCE IN CELLULASES AND HEMICELLULASES FROM ANAEROBIC FUNGI FUNCTIONS AS A PROTEIN DOCKING DOMAIN

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.