RNase H enzymes are sequence-nonspecific endonucleases that cleave RNA strands in RNA/DNA hybrid duplexes, an enzymatic process essential in DNA replication and repair in both prokaryotes and eukaryotes. Also, RNase H activity of the reverse transcriptase in human immunodeficiency viruses (HIV-1 and HIV-2) is indispensable for the viral replication cycle. RNase H enzymes play an central role in the development of gene therapies and are targets for novel antivirals. It is therefore of great importance to gain a detailed understanding of the RNase H catalytic mechanism to improve drug design. We utilized Bacillus halodurans RNase H1 ( Bh RNase H1) to shed light on its function and catalytic mechanism. Room-temperature neutron crystallography of the wild-type and inactive D132N mutant enzymes revealed that E109, belonging to the catalytic DEDD motif, can change its protonation state, allowing us to propose its role in the protonation of the leaving O3′ hydroxyl group of RNA. X-ray crystallography has demonstrated the ability of the RNA/DNA duplex to slide along the protein surface upon metal ion binding at site M A , transforming a product mimic into a Michelis-like complex, which confirms an essential role of the M A metal ion in catalysis. • Neutron structures of apo -wild-type and apo -D132N mutant Bh RNase H1 map protonation states in the enzyme active site. • Side chain of E109 from the catalytic DEDD motif changes its protonation state from being deprotonated in apo -wild-type enzyme to protonated in apo -D132N structure. • RNA/DNA hybrids bind to Bh RNase H1 to mimic product and Michaelis complexes depending on the number of metal ions occupying metal binding sites. • Protonation of the leaving O3′ group of cleaved RNA is proposed to occur from O2′ through the side chain of E109. X-ray and neutron structures of Bh RNase H1 in the apo -form and in complex with RNA/DNA hybrids are described.
Gerlits et al. (Sun,) studied this question.