Transition State Interactions in a Promiscuous Enzyme: Sulfate and Phosphate Monoester Hydrolysis by<i>Pseudomonas aeruginosa</i>Arylsulfatase

Loo, Bert van; Berry, Ryan D.; Boonyuen, Usa; Mohamed, Mark F.; Goličnik, Marko; Hengge, Alvan C.; Hollfelder, Florian

doi:10.1101/327643

Cited by 2 publications

(1 citation statement)

References 91 publications

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“…Interestingly, the sequence of the YejM periplasmic domain is more closely related to LtaS in Gram-positive bacteria than pEtN transferases EptA and MCR-1 of Gramnegative bacteria (Figure 1b), suggesting YejM and LtaS may be distant orthologs. Members of the larger alkaline phosphatase family balance specificity and promiscuity in their evolution around the active site resulting in multidimensional activity transitions that may also hold true for the sub-family YejM belongs to [34][35][36] .…”

Section: Discussionmentioning

confidence: 99%

New functional identity of the essential inner membrane protein YejM: the cardiolipin translocator is also a metalloenzyme

Gabale

Palomino

Kim

et al. 2020

Preprint

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Recent recurrent outbreaks of Gram-negative bacteria show the critical need to target essential bacterial mechanisms to fight the increase of antibiotic resistance. Pathogenic Gram-negative bacteria have developed several strategies to protect themselves against the host immune response and antibiotics. One strategy is to remodel the outer membrane where several genes are involved. yejM was discovered as an essential gene in E. coli and S. typhimurium that plays a critical role in their virulence by changing the outer membrane permeability by translocating and increasing the cardiolipin lipid concentration. How the inner membrane protein YejM with its periplasmic domain acts as a cardiolipin translocator remains unknown. Despite overwhelming structural similarity of the periplasmic domains of two YejM homologues with hydrolases like arylsulfatases, no enzymatic activity has been reported for YejM. Our studies reveal an intact active site with bound metal ions in the structure of YejM periplasmic domain. Furthermore, we show that YejM has a phosphatase activity that is dependent on the presence of magnesium ions and is linked to its cardiolipin translocation properties. Understanding the molecular mechanism by which YejM is involved in OM remodeling will help to identify a new drug target in the fight against the increased antibiotic resistance.

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Section: Discussionmentioning

confidence: 99%

New functional identity of the essential inner membrane protein YejM: the cardiolipin translocator is also a metalloenzyme

Gabale

Palomino

Kim

et al. 2020

Preprint

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Evolutionary repurposing of a sulfatase: A new Michaelis complex leads to efficient transition state charge offset

Miton

Jonas

Fischer

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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The recruitment and evolutionary optimization of promiscuous enzymes is key to the rapid adaptation of organisms to changing environments. Our understanding of the precise mechanisms underlying enzyme repurposing is, however, limited: What are the active-site features that enable the molecular recognition of multiple substrates with contrasting catalytic requirements? To gain insights into the molecular determinants of adaptation in promiscuous enzymes, we performed the laboratory evolution of an arylsulfatase to improve its initially weak phenylphosphonate hydrolase activity. The evolutionary trajectory led to a 100,000-fold enhancement of phenylphosphonate hydrolysis, while the native sulfate and promiscuous phosphate mono- and diester hydrolyses were only marginally affected (≤50-fold). Structural, kinetic, and in silico characterizations of the evolutionary intermediates revealed that two key mutations, T50A and M72V, locally reshaped the active site, improving access to the catalytic machinery for the phosphonate. Measured transition state (TS) charge changes along the trajectory suggest the creation of a new Michaelis complex (E•S, enzyme-substrate), with enhanced leaving group stabilization in the TS for the promiscuous phosphonate ( from -1.08 to -0.42). Rather than altering the catalytic machinery, evolutionary repurposing was achieved by fine-tuning the molecular recognition of the phosphonate in the Michaelis complex, and by extension, also in the TS. This molecular scenario constitutes a mechanistic alternative to adaptation solely based on enzyme flexibility and conformational selection. Instead, rapid functional transitions between distinct chemical reactions rely on the high reactivity of permissive active-site architectures that allow multiple substrate binding modes.

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Transition State Interactions in a Promiscuous Enzyme: Sulfate and Phosphate Monoester Hydrolysis byPseudomonas aeruginosaArylsulfatase

Cited by 2 publications

References 91 publications

New functional identity of the essential inner membrane protein YejM: the cardiolipin translocator is also a metalloenzyme

New functional identity of the essential inner membrane protein YejM: the cardiolipin translocator is also a metalloenzyme

Evolutionary repurposing of a sulfatase: A new Michaelis complex leads to efficient transition state charge offset

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