Staphylopine and pseudopaline dehydrogenase from bacterial pathogens catalyze reversible reactions and produce stereospecific metallophores

Abstract

Edited by Ruma Banerjee Pseudopaline and staphylopine are opine metallophores biosynthesized by Pseudomonas aeruginosa and Staphylococcus aureus, respectively. The final step in opine metallophore biosynthesis is the condensation of the product of a nicotianamine (NA) synthase reaction (i.e. L-HisNA for pseudopaline and D-HisNA for staphylopine) with an ␣-keto acid (␣-ketoglutarate for pseudopaline and pyruvate for staphylopine), which is performed by an opine dehydrogenase. We hypothesized that the opine dehydrogenase reaction would be reversible only for the opine metallophore product with (R)-stereochemistry at carbon C2 of the ␣-keto acid (prochiral prior to catalysis). A kinetic analysis using stopped-flow spectrometry with (R)-or (S)-staphylopine and kinetic and structural analysis with (R)-and (S)-pseudopaline confirmed catalysis in the reverse direction for only (R)-staphylopine and (R)-pseudopaline, verifying the stereochemistry of these two opine metallophores. Structural analysis at 1.57-1.85 Å resolution captured the hydrolysis of (R)-pseudopaline and allowed identification of a binding pocket for the L-histidine moiety of pseudopaline formed through a repositioning of Phe-340 and Tyr-289 during the catalytic cycle. Transient-state kinetic analysis revealed an ordered release of NADP ؉ followed by staphylopine, with staphylopine release being the rate-limiting step in catalysis. Knowledge of the stereochemistry for opine metallophores has implications for future studies involving kinetic analysis, as well as opine metallophore transport, metal coordination, and the generation of chiral amines for pharmaceutical development.

The ancient roots of nicotianamine: diversity, role, regulation and evolution of nicotianamine-like metallophores

“…37 However, in a highly chelating environment, the single mutant lacking cntL revealed such a compromised zinc accumulation, highlighting the importance of this pathway in these conditions. 38 Most of these studies have benefited and will continue to benefit from the input of chemists who not only produce nicotianamine-like molecules 35,54,46,[55][56][57] , but also contribute to their characterization and possible modifications to better understand their mode of action in vivo, as was recently reported for pseudopaline. 56 Recently, the complete export pathway of pseudopaline toward the extracellular space was determined and shown to be mediated by CntI for the crossing of the inner membrane, and also required the efflux pump MexAB, which exports pseudopaline from the periplasm to the extracellular space.…”

“…This study was however recently challenged, arguing that metal regulation was due to metal binding to xNA substrate solely and not to the enzyme. 55 Further studies are needed to clarify this issue, and, in particular, efforts should be made to try to evaluate the effect of buffers used by different authors, with high concentrations of citrate and Tris buffers in some studies. It is possible that the chelating properties of citrate and Tris could indeed mask subtle effects on this key step in staphylopine biosynthesis.…”

Structural insights into the ligand recognition and catalysis of the key aminobutanoyltransferase CntL in staphylopine biosynthesis

“…Recently, a possible negative feedback mechanism regulating the biosynthesis of staphylopine (StP, the metallophore utilized by the Cnt system in S. aureus ) was discovered: the binding of transition metals to an allosteric site of CntM, an enzyme in StP biosynthesis, strongly inhibited its enzyme activity 24 . Another study disproved the theory of an inhibitory effect of metals on the activity of CntM; the previously reported inhibition is caused by substrate limitation due to the formation of a noncatalytic substrate‐metal complex that CntM cannot recognize as substrate 25 …”

A General Stereocontrolled Synthesis of Opines through Asymmetric Pd‐Catalyzed N‐Allylation of Amino Acid Esters

“…[4] As another interesting biological mechanism, certain gram-negative bacteria such as pathogenic Pseudomonas aeruginosa and Staphylococcus aureus exploit the outstanding metallophoric properties of staphylopine (3) and pseudopalin (4) to acquire essential transition metals within a host cell. [5] These opines are released by the bacteria to form metal complexes which are then transferred back into the bacteria by specific transporter proteins. [5] Therefore, such opines are also of interest for the development of new antibiotics acting as molecular "trojan horses".…”

“…[5] These opines are released by the bacteria to form metal complexes which are then transferred back into the bacteria by specific transporter proteins. [5] Therefore, such opines are also of interest for the development of new antibiotics acting as molecular "trojan horses". [6] Due to their fascinating biological function and potential for the development of new pharmaceuticals, [7] opines represent an interesting challenge for stereoselective chemical synthesis.…”