Three Neocallimastix patriciarum esterases associated with the degradation of complex polysaccharides are members of a new family of hydrolases

Abstract

Acetylesterase and cinnamoyl ester hydrolase activities were demonstrated in culture supernatant of the anaerobic ruminal fungus Neocallimastix patriciarum. A cDNA expression library from N. patriciarum was screened for esterases using p-naphthyl acetate and a model cinnamoyl ester compound. cDNA clones representing four different esterase genes (bnaA-D) were isolated. None of the enzymes had cinnamoyl ester hydrolase activity, but two of the enzymes (BnaA and BnaC) had acetylxylan esterase activity. bnaA, bnaB and bnaC encode proteins with several distinct domains. Carboxy-terminal repeats in BnaA and BnaC are homologous to protein-docking domains in other enzymes from Neocallimastix species and another anaerobic fungus, a Piromyces sp. The catalytic domains of BnaB and BnaC are members of a recently described family of Ser/His active site hydrolases [Upton, C. & Buckley, J. T. (1995). Trends Biochem Sci 20, 178-1791. BnaB exhibits 40% amino acid identity to a domain of unknown function in the CelE cellulase from Clostridium thermocellum and BnaC exhibits 52% amino acid identity to a domain of unknown function in the XynB xylanase from Ruminococcus flavefaciens. BnaA, whilst exhibiting less than 10 O/ O overall amino acid identity to BnaB or BnaC, or to any other known protein, appears to be a member of the same family of hydrolases, having the three universally conserved amino acid sequence motifs. Several other previously described esterases are also shown to be members of this family, including a rhamnogalacturonan acetylesterase from Aspergillus aculeatus. However, none of the other previously described enzymes with acetylxylan esterase activity are members of this family of hydrolases.

Catalytic role of conserved HQGE motif in the CE6 carbohydrate esterase family

“…BLOCK V 30 sequences were found to be homologous to R.44 with two fungal AXEs from Neocallimastix patriciarum [12] and Orpinomyces sp. PC-2 [13], exhibiting the highest levels of sequence homology ($52% identity and $67% similarity).…”

Understanding the structural and functional properties of carbohydrate esterases with a special focus on hemicellulose deacetylating acetyl xylan esterases

“…The CE class-2 AcXEs are active on hexopyranosyl residues of the oligo- and polysaccharides with lower activity against acetylxylan and xylanoside residues. These enzymes are known for its transesterification reactions of hexose and hexosyl residues specifically on position 6, respectively (Dalrymple et al 1997; Montanier et al 2009b; Topakas et al 2010). In contrast with class-1 and 2 enzymes, CE class-3 AcXEs exhibit substrate specificity towards a wide range of acetylated carbohydrate residues.…”

Phylogeny, classification and metagenomic bioprospecting of microbial acetyl xylan esterases

“…There are currently no reported CE2 AcXE sequences from archaeal or protozoan sources. CE2 AcXEs, which were first described by Dalrymple, Cybinski [63] in the fungus Neocallimastix patriciarum, are active on acetylated birchwood xylan and α-naphthyl acetate. The CE2 AcXEs from Cellvibrio japonicus and Clostridium thermocellum have been best characterised and are reported to be generally active on aryl esters and selectively active on acetylated xylan, showing higher activities on acetylated konjac glucomannan than on acetylated birchwood xylan and diacetylated xylopyranosides [23,64].…”

“…Neocallimastix patriciarum [45,63], AxeA from Orpinomyces sp. [43] and AxeA from a bovine metagenome [12], all of which are of rumen origin.…”

“…A range of environments containing a diversity of lignocellulose-degrading organisms, such as forest soils, termite guts, animal rumens and compost have yielded a range of AcXE-expressing organisms, including the fungal species Aspergillus [58,59], Anoxybacillus [76], Trichoderma [98], Orpinomyces [43], Penicillium [57], Schizophyllum [98], Fusarium [99], Coprinopsis [100], Rhodotorula [41] and Neocallimastix [63] and bacteria such as Bacillus [101], Butyrivibrio [102], Caldicellulosiruptor [103], Caldocellum [104], Clostridium [72],…”