The virulence-associated protein HsvA from the fire blight pathogen Erwinia amylovora is a polyamine amidinotransferase

Abstract

Studies of virulence determinants in the bacterial phytopathogen , the cause of devastating fire blight disease in apple and pear, have shown that HsvA, a putative amidinotransferase enzyme located in the Hrp pathogenicity island, is required for systemic infection in apple. However, the mechanism by which HsvA contributes to virulence is unclear. To investigate the role of HsvA in virulence, we carried out a series of biochemical and structural studies to characterize the amidinotransferase activity of HsvA. We found that HsvA displays a preference for linear aliphatic polyamines as the amidino acceptor substrate, especially for spermidine and putrescine ( values of 33 μm and 3.9 mm, respectively). The three-dimensional structure, determined at 2.30 Å resolution using X-ray crystallography, revealed that the overall architecture of HsvA is similar to that of the human arginine-glycine amidinotransferase in the creatine biosynthesis pathway. The active site is located in the core of the protein at the base of a long, narrow substrate access channel. Specific amino acids near the entrance of the channel may serve as major determinants of the substrate specificity, including a glutamate residue at the rim of the channel entrance that appears to be positioned to interact with the distal primary amine in the putrescine substrate as well as the internal and distal amines in the spermidine substrate. These results suggest potential functions for HsvA as a virulence factor in fire blight and may also provide a basis for strategies to control fire blight by inhibiting HsvA activity.

Structural and functional characterization of proteins from the fire blight pathogen Erwinia amylovora. A review on the state of the art

“…The reaction mechanism of amidinotransferases involves the action of three key residues in the active site, cysteine, histidine and aspartic acid, in a double displacement (ping-pong) mechanism as the access to the active site does not allow simultaneous access to both amidino donor and acceptor during catalysis (Humm et al 1997a, b). A biochemical characterization and protein structure determination was carried out in 2017 with the aim of understanding the mechanism of action, the substrate specificity and suggest a possible role in virulence (Shanker et al 2017). The amidinotransferase activity was investigated on a panel of 16 candidate acceptors using arginine as an amidino donor and detecting the amount of ornithine released during a 30 min course reaction.…”

“…5). The role of HsvA in E. amylovora pathogenicity was proposed to be that of regulating the level of polyamines, possibly enhancing the fitness of the bacterium during infection, or by supporting the production of virulence factors (Shanker et al 2017).…”

Substrate Promiscuity of a Paralytic Shellfish Toxin Amidinotransferase

“…Understanding the reactivity and substrate promiscuity of SxtG provides the opportunity to identify a biocatalyst with a complementary substrate scope to known amidinotransferases. [20][21][22] We sought to assess the native function of SxtG in the cyanobacterial biosynthesis of paralytic shellfish toxins (PSTs) using purified protein from M. wollei overexpressed in Escherichia coli (see Supporting Information). Initial SxtG activity assays were performed using L-Arg (2) as an amidino group donor and the proposed native substrate, ethyl ketone 4, as an amidino group acceptor.…”

“…Similar to HsvA, the exterior of the substrate entrance pore in SxtG is surrounded with negatively charged Glu residues, Glu58, Glu59, Glu255 and Glu276, which are not conserved in StrB1 or AT and are replaced with Lys, Asn, Gly, and Ala, respectively. 20 The presence of negatively charged residues around the entrance to SxtG's substrate binding cavity could potentially select positively charged substrates over negatively charged or hydrophobic substrates. Taken together, the combination of an active site pocket lined with uniquely placed polar residues and a negatively charged entrance to the binding pocket likely play a significant role in determining the substrate specificity of SxtG.…”

“…Intermediate 5 is anticipated to undergo spontaneous cyclization to deliver aminoimidazole 6 , which is a molecule that has been previously identified in both PST-producing cyanobacteria and dinoflagellates. − Amidinotransferases are well-characterized for their activity on amino acid substrates; however, reactions of amidinotransferases on other classes of substrates are less well-studied. Understanding the reactivity and substrate promiscuity of SxtG provides the opportunity to identify a biocatalyst with a complementary substrate scope to known amidinotransferases. − …”