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Novel protein fold discovered in the PabI family of restriction enzymes.

ABSTRACT: Although structures of many DNA-binding proteins have been solved, they fall into a limited number of folds. Here, we describe an approach that led to the finding of a novel DNA-binding fold. Based on the behavior of Type II restriction-modification gene complexes as mobile elements, our earlier work identified a restriction enzyme, R.PabI, and its cognate modification enzyme in Pyrococcus abyssi through comparison of closely related genomes. While the modification methyltransferase was easily recognized, R.PabI was predicted to have a novel 3D structure. We expressed cytotoxic R.PabI in a wheat-germ-based cell-free translation system and determined its crystal structure. R.PabI turned out to adopt a novel protein fold. Homodimeric R.PabI has a curved anti-parallel beta-sheet that forms a 'half pipe'. Mutational and in silico DNA-binding analyses have assigned it as the double-strand DNA-binding site. Unlike most restriction enzymes analyzed, R.PabI is able to cleave DNA in the absence of Mg(2+). These results demonstrate the value of genome comparison and the wheat-germ-based system in finding a novel DNA-binding motif in mobile DNases and, in general, a novel protein fold in horizontally transferred genes.

SUBMITTER: Miyazono K

PROVIDER: S-EPMC1874622 | biostudies-literature | 2007

REPOSITORIES: biostudies-literature

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Novel protein fold discovered in the PabI family of restriction enzymes.

Miyazono Ken-ichi K Watanabe Miki M Kosinski Jan J Ishikawa Ken K Kamo Masayuki M Sawasaki Tatsuya T Nagata Koji K Bujnicki Janusz M JM Endo Yaeta Y Tanokura Masaru M Kobayashi Ichizo I

Nucleic acids research 20070301 6

Although structures of many DNA-binding proteins have been solved, they fall into a limited number of folds. Here, we describe an approach that led to the finding of a novel DNA-binding fold. Based on the behavior of Type II restriction-modification gene complexes as mobile elements, our earlier work identified a restriction enzyme, R.PabI, and its cognate modification enzyme in Pyrococcus abyssi through comparison of closely related genomes. While the modification methyltransferase was easily r ...[more]

PMID: 17332011

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Project description:BACKGROUND: Bacterial populations are highly successful at colonizing new habitats and adapting to changing environmental conditions, partly due to their capacity to evolve novel virulence and metabolic pathways in response to stress conditions and to shuffle them by horizontal gene transfer (HGT). A common theme in the evolution of new functions consists of gene duplication followed by functional divergence. UlaG, a unique manganese-dependent metallo-?-lactamase (MBL) enzyme involved in L-ascorbate metabolism by commensal and symbiotic enterobacteria, provides a model for the study of the emergence of new catalytic activities from the modification of an ancient fold. Furthermore, UlaG is the founding member of the so-called UlaG-like (UlaGL) protein family, a recently established and poorly characterized family comprising divalent (and perhaps trivalent) metal-binding MBLs that catalyze transformations on phosphorylated sugars and nucleotides. RESULTS: Here we combined protein structure-guided and sequence-only molecular phylogenetic analyses to dissect the molecular evolution of UlaG and to study its phylogenomic distribution, its relatedness with present-day UlaGL protein sequences and functional conservation. Phylogenetic analyses indicate that UlaGL sequences are present in Bacteria and Archaea, with bona fide orthologs found mainly in mammalian and plant-associated Gram-negative and Gram-positive bacteria. The incongruence between the UlaGL tree and known species trees indicates exchange by HGT and suggests that the UlaGL-encoding genes provided a growth advantage under changing conditions. Our search for more distantly related protein sequences aided by structural homology has uncovered that UlaGL sequences have a common evolutionary origin with present-day RNA processing and metabolizing MBL enzymes widespread in Bacteria, Archaea, and Eukarya. This observation suggests an ancient origin for the UlaGL family within the broader trunk of the MBL superfamily by duplication, neofunctionalization and fixation. CONCLUSIONS: Our results suggest that the forerunner of UlaG was present as an RNA metabolizing enzyme in the last common ancestor, and that the modern descendants of that ancestral gene have a wide phylogenetic distribution and functional roles. We propose that the UlaGL family evolved new metabolic roles among bacterial and possibly archeal phyla in the setting of a close association with metazoans, such as in the mammalian gastrointestinal tract or in animal and plant pathogens, as well as in environmental settings. Accordingly, the major evolutionary forces shaping the UlaGL family include vertical inheritance and lineage-specific duplication and acquisition of novel metabolic functions, followed by HGT and numerous lineage-specific gene loss events.

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Novel protein fold discovered in the PabI family of restriction enzymes.

Publications

Novel protein fold discovered in the PabI family of restriction enzymes.

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