Enzymes in the Gcn5-related N-acetyltransferase (GNAT) superfamily are widespread and critically involved in multiple cellular processes ranging from antibiotic resistance to histone modification. While acetyl transfer is the most widely catalyzed reaction, recent studies have revealed that these enzymes are also capable of performing succinylation, condensation, decarboxylation, and methylcarbamoylation reactions. The canonical chemical mechanism attributed to GNATs is a general acid/base mechanism; however, mounting evidence has cast doubt on the applicability of this mechanism to all GNATs. This study shows that the Pseudomonas aeruginosa PA3944 enzyme uses a nucleophilic serine residue and a hybrid ping-pong mechanism for catalysis instead of a general acid/base mechanism. To simplify this enzyme’s kinetic characterization, we synthesized a polymyxin B substrate analog and performed molecular docking experiments. We performed site-directed mutagenesis of key active site residues (S148 and E102) and determined the structure of the E102A mutant. We found that the serine residue is essential for catalysis toward the synthetic substrate analog and polymyxin B, but the glutamate residue is more likely important for substrate recognition or stabilization. Our results challenge the current paradigm of GNAT mechanisms and show that this common enzyme scaffold utilizes different active site residues to accomplish a diversity of catalytic reactions.
Human ornithine aminotransferase
(hOAT) is a pyridoxal 5′-phosphate
(PLP)-dependent enzyme that contains a similar active site to that
of γ-aminobutyric acid aminotransferase (GABA-AT). Recently,
pharmacological inhibition of hOAT was recognized as a potential therapeutic
approach for hepatocellular carcinoma. In this work, we first studied
the inactivation mechanisms of hOAT by two well-known GABA-AT inactivators
(CPP-115 and OV329). Inspired by the inactivation
mechanistic difference between these two aminotransferases, a series
of analogues were designed and synthesized, leading to the discovery
of analogue 10b as a highly selective and potent hOAT
inhibitor. Intact protein mass spectrometry, protein crystallography,
and dialysis experiments indicated that 10b was converted
to an irreversible tight-binding adduct (34) in the active
site of hOAT, as was the unsaturated analogue (11). The
comparison of kinetic studies between 10b and 11 suggested that the active intermediate (17b) was only
generated in hOAT and not in GABA-AT. Molecular docking studies and
pK
a computational calculations highlighted
the importance of chirality and the endocyclic double bond for inhibitory
activity. The turnover mechanism of 10b was supported
by mass spectrometric analysis of dissociable products and fluoride
ion release experiments. Notably, the stopped-flow experiments were
highly consistent with the proposed mechanism, suggesting a relatively
slow hydrolysis rate for hOAT. The novel second-deprotonation mechanism
of 10b contributes to its high potency and significantly
enhanced selectivity for hOAT inhibition.
We report the atomic-resolution (1.3
Å) X-ray crystal structure
of an open conformation of the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase
(DapE, EC 3.5.1.18) from Neisseria meningitidis.
This structure [Protein Data Bank (PDB) entry 5UEJ] contains two bound
sulfate ions in the active site that mimic the binding of the terminal
carboxylates of the N-succinyl-l,l-diaminopimelic acid (l,l-SDAP) substrate. We demonstrated
inhibition of DapE by sulfate (IC50 = 13.8 ± 2.8 mM).
Comparison with other DapE structures in the PDB demonstrates the
flexibility of the interdomain connections of this protein. This high-resolution
structure was then utilized as the starting point for targeted molecular
dynamics experiments revealing the conformational change from the
open form to the closed form that occurs when DapE binds l,l-SDAP and cleaves the amide bond. These simulations demonstrated
closure from the open to the closed conformation, the change in RMS
throughout the closure, and the independence in the movement of the
two DapE subunits. This conformational change occurred in two phases
with the catalytic domains moving toward the dimerization domains
first, followed by a rotation of catalytic domains relative to the
dimerization domains. Although there were no targeting forces, the
substrate moved closer to the active site and bound more tightly during
the closure event.
An α-amido
cyclobutanone possessing a C10 hydrocarbon tail
was designed as a potential transition-state mimetic for the quorum-quenching
metallo-γ-lactonase autoinducer inactivator A (AiiA) with the
support of in-house modeling techniques and found to be a competitive
inhibitor of dicobalt(II) AiiA with an inhibition constant of K
i
= 0.007 ± 0.002 mM.
The catalytic mechanism of AiiA was further explored using our product-based
transition-state modeling (PBTSM) computational approach, providing
substrate-intermediate models arising during enzyme turnover and further
insight into substrate–enzyme interactions governing native
substrate catalysis. These interactions were targeted in the docking
of cyclobutanone hydrates into the active site of AiiA. The X-ray
crystal structure of dicobalt(II) AiiA cocrystallized with this cyclobutanone
inhibitor unexpectedly revealed an N-(2-oxocyclobutyl)decanamide
ring-opened acyclic product bound to the enzyme active site (PDB 7L5F). The C10 alkyl
chain and its interaction with the hydrophobic phenylalanine clamp
region of AiiA adjacent to the active site enabled atomic placement
of the ligand atoms, including the C10 alkyl chain. A mechanistic
hypothesis for the ring opening is proposed involving a radical-mediated
process.
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