Functional genomics-based identification and characterization of α-glucoside- processing ORFs in the hyperthermophilic archaeon Pyrococcus furiosus
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چکیده
Running title: α-glucoside-processing enzymes in Pyrococcus furiosus 20 ABSTRACT Bioinformatics analysis and transcriptional response information for Pyroccoccus furiosus grown on α-glucans led to identification of a novel isomaltase (PF0132) representing a new GH family, a novel GH57 β-amylase (PF0870), and an extracellular starch-binding protein (1,141 amino 5 acids) (PF1109-1110), in addition to several other putative α-glucan processing enzymes. 3 Pyrococcus furiosus is a heterotrophic, hyperthermophilic archaeon that uses a range of glucans as carbon/energy sources (5). α-Glucan utilization in P. furiosus has been examined using transcriptional response analysis, providing the basis for proposed mechanisms of starch utilization (8). Bioinformatic analysis, based on domain homology (9) and domain families (12, 14) in the P. furiosus genome, identified additional ORFs that had putative functions related to 5 α-glucan utilization. P. furiosus was grown anaerobically at 80ºC in a sea salts-based medium (SSM) supplemented with 3.3 g/l of the either glycogen, maltose, pullulan, or starch (13). RNA was harvested and subjected to cDNA microarray analysis, as described previously (8, 16). Based on the leads identified from these complementary approaches, genes encoding candidate enzymes/proteins were cloned and expressed in E. coli (see Supplementary Table 1). 10 While no activity or function could be assigned to several of the ORFs identified, biochemical analysis of PF0132, PF0870, and PF1109/1110 provided insights into the roles that these proteins play in α-glucan processing (see Table 1). Recombinant PF0870 was specific for short α-glucans (Table 1). Indeed, bioinformatic analysis identified a domain in PF0870, related to glycoside hydrolase family 57 (GH57) (17). 15 Transcriptional analysis of PF0870 by qPCR (10) showed similar responses to maltose, glycogen, and starch, but not to pullulan. PF0870 was most active on pNP-α-maltopyranoside (k cat and K M were 962 s-1 and 82.7 µM, respectively, at 90°C, pH 7.0), less active on pNP-α-galactoside, and not active on other substrates. The presence or absence of various divalent cations or EDTA had minimal effect on enzyme activity. No transglycosylation activity was 20 noted, nor did PF0870 hydrolyze starch, glycogen, pullulan or large maltooligosaccharides. Biochemical properties of PF0870 most closely resemble β-amylases found in plants, fungi, and some bacteria; these progressively cleave maltose from the non-reducing end of starch, amylase, and maltodextrins. PF0870 is the most thermostable β-amylase known (T opt = 110°C). PF0870 primarily hydrolyzes maltotriose into glucose and maltose (data not shown), likely 4 functioning in vivo as the final processing step prior to intracellular …
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Defining genes in the genome of the hyperthermophilic archaeon Pyrococcus furiosus: implications for all microbial genomes.
The original genome annotation of the hyperthermophilic archaeon Pyrococcus furiosus contained 2,065 open reading frames (ORFs). The genome was subsequently automatically annotated in two public databases by the Institute for Genomic Research (TIGR) and the National Center for Biotechnology Information (NCBI). Remarkably, more than 500 of the originally annotated ORFs differ in size in the two ...
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