Abstract
Polyamines are essential for cell growth and proliferation. In plants and many bacteria, including Helicobacter pylori, the parent polyamine putrescine is only produced through the metabolism of N-carbamoylputrescine by N-carbamoylputrescine amidase (CPA). Thus, CPA is a crucial intermediate enzyme. Moreover, the absence of CPA in humans makes its presence in H. pylori a potential target for the development of new therapeutics against this pathogen. Despite this enzyme's presence in plants and bacteria, its function is not completely explored. Using structure-guided biochemical and biophysical studies on H. pylori CPA, we discovered an aromatic cluster containing four conserved tryptophans near the catalytic site and elucidated its role. Mutational studies revealed that they are individually vital to enzyme function. Unlike wild-type, which forms a hexamer, the Trp to Ala mutants only formed dimers. Interestingly, two other conserved residues, Gln155 and Asp278, interact with the tryptophan cluster and perform similar roles. Our results indicate that aromatic-aromatic and H-bonding contacts between the residues (Trp156-Trp273, Trp196-Gln155, and Trp153-Asp278) play a crucial role in stimulating activity through hexamer formation. Additionally, Trp156 is essential to generating a catalytically efficient hexamer. These results suggest dual roles for the tryptophans; in hexamer formation and in generating its functionally active form, thereby providing a mechanistic understanding into the role of the cluster. We also elucidated the catalytic roles of Glu43, Lys115, and Cys152, which are present at the active site. Our findings highlight, for the first time, the importance of a tryptophan cluster in H. pylori CPA that can be exploited to design therapeutic inhibitors.