Clostridioides difficile specific DNA adenine methyltransferase CamA squeezes and flips adenine out of DNA helix

Jujun Zhou,Xing Zhang,Robert M Blumenthal,Xiaodong Cheng,John R Horton

doi:10.1038/s41467-021-23693-w

Abstract

Clostridioides difficile infections are an urgent medical problem. The newly discovered C.difficileadenine methyltransferase A (CamA) is specified by all C. difficile genomes sequenced to date (>300), but is rare among other bacteria. CamA is an orphan methyltransferase, unassociated with a restriction endonuclease. CamA-mediated methylation at CAAAAA is required for normal sporulation, biofilm formation, and intestinal colonization by C. difficile. We characterized CamA kinetic parameters, and determined its structure bound to DNA containing the recognition sequence. CamA contains an N-terminal domain for catalyzing methyl transfer, and a C-terminal DNA recognition domain. Major and minor groove DNA contacts in the recognition site involve base-specific hydrogen bonds, van der Waals contacts and the Watson-Crick pairing of a rearranged A:T base pair. These provide sufficient sequence discrimination to ensure high specificity. Finally, the surprisingly weak binding of the methyl donor S-adenosyl-l-methionine (SAM) might provide avenues for inhibiting CamA activity using SAM analogs.

Highlights

Clostridioides difficile infections are an urgent medical problem
There are bacterial “orphan” MTases—so named as they are not paired with a restriction endonuclease—that in many cases are involved in controlling chromosome replication, DNA repair, and gene expression[16,23]
The newly discovered C. difficile adenine methyltransferase A (CamA) enzyme is another orphan MTase, is present in all C. difficile genomes sequenced to date (>300), and is active in all C. difficile genomes subjected to PacBio singlemolecule real-time DNA sequencing, but is rarely found in other bacteria[24]

Summary

Introduction

Clostridioides difficile infections are an urgent medical problem. The newly discovered C. difficile adenine methyltransferase A (CamA) is specified by all C. difficile genomes sequenced to date (>300), but is rare among other bacteria. Major and minor groove DNA contacts in the recognition site involve base-specific hydrogen bonds, van der Waals contacts and the Watson-Crick pairing of a rearranged A:T base pair. These provide sufficient sequence discrimination to ensure high specificity. The newly discovered CamA enzyme (named for Clostridioides difficile adenine methyltransferase A) is another orphan MTase, is present in all C. difficile genomes sequenced to date (>300), and is active in all C. difficile genomes subjected to PacBio singlemolecule real-time DNA sequencing (which can detect N6methyladenine—N6mA), but is rarely found in other bacteria[24]. We show the kinetic parameters and structural features of CamA in complex with cognate substrate DNA and, given the critical consequences of C. difficile infection to human health, discuss the potential for CamA as a novel therapeutic target

Methods

Results

Conclusion