Homologous catalytic domains in a rumen fungal xylanase: evidence for gene duplication and prokaryotic origin

Abstract

A cDNA (xynA), encoding xylanase A (XYLA), was isolated from a cDNA library, derived from mRNA extracted from the rumen anaerobic fungus, Neocallimastix patriciarum. Recombinant XYLA, purified from Escherichia coli harbouring xynA, had a M(r) of 53,000 and hydrolysed oat-spelt xylan to xylobiose and xylose. The enzyme did not hydrolyse any cellulosic substrates. The nucleotide sequence of xynA revealed a single open reading frame of 1821 bp coding for a protein of M(r) 66,192. The predicted primary structure of XYLA comprised an N-terminal signal peptide followed by a 225-amino-acid repeated sequence, which was separated from a tandem 40-residue C-terminal repeat by a threonine/proline linker sequence. The large N-terminal reiterated regions consisted of distinct catalytic domains which displayed similar substrate specificities to the full-length enzyme. The reiterated structure of XYLA suggests that the enzyme was derived from an ancestral gene which underwent two discrete duplications. Sequence comparison analysis revealed significant homology between XYLA and bacterial xylanases belonging to cellulase/xylanase family G. One of these homologous enzymes is derived from the rumen bacterium Ruminococcus flavefaciens. The homology observed between XYLA and a rumen prokaryote xylanase could be a consequence of the horizontal transfer of genes between rumen prokaryotes and lower eukaryotes, either when the organisms were resident in the rumen, or prior to their colonization of the ruminant. It should also be noted that Neocallimastix XYLA is the first example of a xylanase which consists of reiterated sequences. It remains to be established whether this is a common phenomenon in other rumen fungal plant cell wall hydrolases.

Absence of branches from xylan in Arabidopsis gux mutants reveals potential for simplification of lignocellulosic biomass

“…AIR was prepared and analyzed by PACE, MS, and high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) as described elsewhere (10,(40)(41)(42) and in SI Materials and Methods.…”

Promiscuity in ligand-binding: The three-dimensional structure of a Piromyces carbohydrate-binding module, CBM29-2, in complex with cello- and mannohexaose

“…The catalytic components of the Piromyces equi cellulosome do not contain CBMs (9,10), suggesting that the capacity of this enzyme complex to attach to the plant cell wall is mediated by one or a small number of noncatalytic proteins. Recently, the first of these noncatalytic components was identified.…”

“…The plant cell wall degrading capacity of gut anaerobic fungi is vested in a multienzyme complex, termed the ''cellulosome'' (9), that binds to plant polysaccharides. The catalytic components of the Piromyces equi cellulosome do not contain CBMs (9,10), suggesting that the capacity of this enzyme complex to attach to the plant cell wall is mediated by one or a small number of noncatalytic proteins.…”

Cloning of a xylanase gene xyn2A from rumen fungus Neocallimastix sp. GMLF2 in Escherichia coli and its partial characterization

“…The xynA and xyn11A possess two homologous catalytic domains and also two dockerin domains, whereas xyn11B has only one catalytic domain and two dockerin domains (Gilbert et al 1992;Huang et al 2005). In order to investigate the effects of dockerin domains on the enzyme activity, Huang et al (2005) deleted the dockerin domains from the xyn11A and xyn11B.…”

“…Xylanase genes of xynA (Gilbert et al 1992), xynB , xyn3 (Durand et al 1996), xynC (Liu et al 1999), xyn11A and xyn11B (Huang et al 2005) from Neocallimastix sp. were studied extensively.…”