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 β-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 Mg2+. 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.

Functional Analysis of MmeI from Methanol UtilizerMethylophilus methylotrophus, a Subtype IIC Restriction-Modification Enzyme Related to Type I Enzymes

“…Nonetheless, bioinformatics and biochemical analyses revealed that some type II REases possess unrelated catalytic domains from different nuclease superfamilies, including PLD, HNH, GIY-YIG, and half-pipe (reviewed in reference 8). These findings have been recently highlighted by crystal structures of Mg 2ϩ -independent enzymes: PLD-like R.BfiI (20) and half-pipe R.PabI (38).…”

Diverse Functions of Restriction-Modification Systems in Addition to Cellular Defense

“…A vast number of REases, with the exception of BfiI and PabI, show an absolute requirement for Mg 2ϩ for DNA cleavage (8,9). Other divalent ions with similar atomic radii are a poor replacement at the catalytic center.…”

“…Generally, type II REases are homodimeric or homotetrameric and cleave DNA within or near their target site. These enzymes are highly diverse and are known to utilize at least five types of folds: PD-(D/E)XK, PLD, HNH, GIY-YIG, and halfpipe, e.g., R.EcoRI, R.BfiI, R.KpnI, R.Eco29kI, and R.PabI enzymes, respectively (2,(7)(8)(9)(10). Type II enzymes are the most widely studied and are also extensively utilized nucleases in genetic engineering.…”

A sequence-specific DNA glycosylase mediates restriction-modification in Pyrococcus abyssi

“…Other type II restriction enzymes belong to the phospholipase D (PLD) superfamily, the HNH superfamily and the GIY-YIG superfamily [6][7][8][9][10] . In addition to these restriction enzyme superfamilies, we found a half-pipe superfamily for which R.PabI from the hyperthermophilic archaea Pyrococcus abyssi is representative 11,12 . There is no protein with a structure similar to the overall fold of R.PabI 12 .…”

“…In addition to these restriction enzyme superfamilies, we found a half-pipe superfamily for which R.PabI from the hyperthermophilic archaea Pyrococcus abyssi is representative 11,12 . There is no protein with a structure similar to the overall fold of R.PabI 12 .…”