14 research outputs found
Mutation of Archaeal Isopentenyl Phosphate Kinase Highlights Mechanism and Guides Phosphorylation of Additional Isoprenoid Monophosphates
The biosynthesis of isopentenyl diphosphate (IPP) from either the mevalonate (MVA) or the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway provides the key metabolite for primary and secondary isoprenoid biosynthesis. Isoprenoid metabolism plays crucial roles in membrane stability, steroid biosynthesis, vitamin production, protein localization, defense and communication, photoprotection, sugar transport, and glycoprotein biosynthesis. Recently, an alternative branch of the MVA pathway was discovered in the archaeon Methanocaldococcus jannaschii involving a small molecule kinase, isopentenyl phosphate kinase (IPK). IPK belongs to the amino acid kinase (AAK) superfamily. <i>In vitro</i>, IPK phosphorylates isopentenyl monophosphate (IP) in an ATP and Mg<sup>2+</sup>-dependent reaction producing IPP. Here, we describe crystal structures of IPK from M. jannaschii refined to nominal resolutions of 2.0−2.8 Å. Notably, an active site histidine residue (His60) forms a hydrogen bond with the terminal phosphate of both substrate and product. This His residue serves as a marker for a subset of the AAK family that catalyzes phosphorylation of phosphate or phosphonate functional groups; the larger family includes carboxyl-directed kinases, which lack this active site residue. Using steady-state kinetic analysis of H60A, H60N, and H60Q mutants, the protonated form of the Nε<sub>2</sub> nitrogen of His60 was shown to be essential for catalysis, most likely through hydrogen bond stabilization of the transition state accompanying transphosphorylation. Moreover, the structures served as the starting point for the engineering of IPK mutants capable of the chemoenzymatic synthesis of longer chain isoprenoid diphosphates from monophosphate precursors
Stereochemical Basis for Engineered Pyrrolysyl-tRNA Synthetase and the Efficient <i>in Vivo</i> Incorporation of Structurally Divergent Non-native Amino Acids
Unnatural amino acids (Uaas) can be translationally incorporated into proteins <i>in vivo</i> using evolved tRNA/aminoacyl-tRNA synthetase (RS) pairs, affording chemistries inaccessible when restricted to the 20 natural amino acids. To date, most evolved RSs aminoacylate Uaas chemically similar to the native substrate of the wild-type RS; these conservative changes limit the scope of Uaa applications. Here, we adapt <i>Methanosarcina mazei</i> PylRS to charge a noticeably disparate Uaa, <i>O</i>-methyl-l-tyrosine (Ome). In addition, the 1.75 Ã… X-ray crystal structure of the evolved PylRS complexed with Ome and a non-hydrolyzable ATP analogue reveals the stereochemical determinants for substrate selection. Catalytically synergistic active site mutations remodel the substrate-binding cavity, providing a shortened but wider active site. In particular, mutation of Asn346, a residue critical for specific selection and turnover of the Pyl chemical core, accommodates different side chains while the central role of Asn346 in aminoacylation is rescued through compensatory hydrogen bonding provided by A302T. This multifaceted analysis provides a new starting point for engineering PylRS to aminoacylate a significantly more diverse selection of Uaas than previously anticipated
Mechanism-Based Post-Translational Modification and Inactivation in Terpene Synthases
Terpenes are ubiquitous natural chemicals
with diverse biological
functions spanning all three domains of life. In specialized metabolism,
the active sites of terpene synthases (TPSs) evolve in shape and reactivity
to direct the biosynthesis of a myriad of chemotypes for organismal
fitness. As most terpene biosynthesis mechanistically involves highly
reactive carbocationic intermediates, the protein surfaces catalyzing
these cascade reactions possess reactive regions possibly prone to
premature carbocation capture and potentially enzyme inactivation.
Here, we show using proteomic and X-ray crystallographic analyses
that cationic intermediates undergo capture by conserved active site
residues leading to inhibitory self-alkylation. Moreover, the level
of cation-mediated inactivation increases with mutation of the active
site, upon changes in the size and structure of isoprenoid diphosphate
substrates, and alongside increases in reaction temperatures. TPSs
that individually synthesize multiple products are less prone to self-alkylation
then TPSs possessing relatively high product specificity. In total,
the results presented suggest that mechanism-based alkylation represents
an overlooked mechanistic pressure during the evolution of cation-derived
terpene biosynthesis
Structure, Biochemistry, and Inhibition of Essential 4′-Phosphopantetheinyl Transferases from Two Species of <i>Mycobacteria</i>
4′-Phosphopantetheinyl
transferases (PPTase) post-translationally
modify carrier proteins with a phosphopantetheine moiety, an essential
reaction in all three domains of life. In the bacterial genus <i>Mycobacteria</i>, the Sfp-type PPTase activates pathways necessary
for the biosynthesis of cell wall components and small molecule virulence
factors. We solved the X-ray crystal structures and biochemically
characterized the Sfp-type PPTases from two of the most prevalent
Mycobacterial pathogens, PptT of <i>M. tuberculosis</i> and
MuPPT of <i>M. ulcerans</i>. Structural analyses reveal
significant differences in cofactor binding and active site composition
when compared to previously characterized Sfp-type PPTases. Functional
analyses including the efficacy of Sfp-type PPTase-specific inhibitors
also suggest that the Mycobacterial Sfp-type PPTases can serve as
therapeutic targets against Mycobacterial infections
Dynamic Conformational States Dictate Selectivity toward the Native Substrate in a Substrate-Permissive Acyltransferase
Hydroxycinnamoyl-CoA:shikimate
hydroxycinnamoyltransferase (HCT)
is an essential acyltransferase that mediates flux through plant phenylpropanoid
metabolism by catalyzing a reaction between <i>p</i>-coumaroyl-CoA
and shikimate, yet it also exhibits broad substrate permissiveness <i>in vitro</i>. How do enzymes like HCT avoid functional derailment
by cellular metabolites that qualify as non-native substrates? Here,
we combine X-ray crystallography and molecular dynamics to reveal
distinct dynamic modes of HCT under native and non-native catalysis.
We find that essential electrostatic and hydrogen-bonding interactions
between the ligand and active site residues, permitted by active site
plasticity, are elicited more effectively by shikimate than by other
non-native substrates. This work provides a structural basis for how
dynamic conformational states of HCT favor native over non-native
catalysis by reducing the number of futile encounters between the
enzyme and shikimate
Dynamic Conformational States Dictate Selectivity toward the Native Substrate in a Substrate-Permissive Acyltransferase
Hydroxycinnamoyl-CoA:shikimate
hydroxycinnamoyltransferase (HCT)
is an essential acyltransferase that mediates flux through plant phenylpropanoid
metabolism by catalyzing a reaction between <i>p</i>-coumaroyl-CoA
and shikimate, yet it also exhibits broad substrate permissiveness <i>in vitro</i>. How do enzymes like HCT avoid functional derailment
by cellular metabolites that qualify as non-native substrates? Here,
we combine X-ray crystallography and molecular dynamics to reveal
distinct dynamic modes of HCT under native and non-native catalysis.
We find that essential electrostatic and hydrogen-bonding interactions
between the ligand and active site residues, permitted by active site
plasticity, are elicited more effectively by shikimate than by other
non-native substrates. This work provides a structural basis for how
dynamic conformational states of HCT favor native over non-native
catalysis by reducing the number of futile encounters between the
enzyme and shikimate
Dynamic Conformational States Dictate Selectivity toward the Native Substrate in a Substrate-Permissive Acyltransferase
Hydroxycinnamoyl-CoA:shikimate
hydroxycinnamoyltransferase (HCT)
is an essential acyltransferase that mediates flux through plant phenylpropanoid
metabolism by catalyzing a reaction between <i>p</i>-coumaroyl-CoA
and shikimate, yet it also exhibits broad substrate permissiveness <i>in vitro</i>. How do enzymes like HCT avoid functional derailment
by cellular metabolites that qualify as non-native substrates? Here,
we combine X-ray crystallography and molecular dynamics to reveal
distinct dynamic modes of HCT under native and non-native catalysis.
We find that essential electrostatic and hydrogen-bonding interactions
between the ligand and active site residues, permitted by active site
plasticity, are elicited more effectively by shikimate than by other
non-native substrates. This work provides a structural basis for how
dynamic conformational states of HCT favor native over non-native
catalysis by reducing the number of futile encounters between the
enzyme and shikimate
Coevolutionary Information Captures Catalytic Functions and Reveals Divergent Roles of Terpene Synthase Interdomain Connections
Inferring the historical
and biophysical causes of diversity within
protein families is a complex puzzle. A key to unraveling this problem
is characterizing the rugged topography of sequence–function
adaptive landscapes. Using biochemical data from a 29 =
512 combinatorial library of tobacco 5-epi-aristolochene
synthase (TEAS) mutants engineered to make the native major product
of Egyptian henbane premnaspirodiene synthase (HPS) and a complementary
512 mutant HPS library, we address the question of how product specificity
is controlled. These data sets reveal that HPS is far more robust
and resistant to mutations than TEAS, where most mutants are promiscuous.
We also combine experimental data with a sequence Potts Hamiltonian
model and direct coupling analysis to quantify mutant fitness. Our
results demonstrate that the Hamiltonian captures variation in product
outputs across both libraries, clusters native family members based
on their substrate specificities, and exposes the divergent catalytic
roles of couplings between the catalytic and noncatalytic domains
of TEAS versus HPS. Specifically, we found that the role of the interdomain
connectivities in specifying product output is more important in TEAS
than connectivities within the catalytic domain. Despite being 75%
identical, this property is not shared by HPS, where connectivities
within the catalytic domain are more important for specificity. By
solving the X-ray crystal structure of HPS, we assessed structural
bases for their interdomain network differences. Last, we calculate
the product profile Shannon entropies of the two libraries, which
showcases that site–site connectivities also play divergent
roles in catalytic accuracy
Coevolutionary Information Captures Catalytic Functions and Reveals Divergent Roles of Terpene Synthase Interdomain Connections
Inferring the historical
and biophysical causes of diversity within
protein families is a complex puzzle. A key to unraveling this problem
is characterizing the rugged topography of sequence–function
adaptive landscapes. Using biochemical data from a 29 =
512 combinatorial library of tobacco 5-epi-aristolochene
synthase (TEAS) mutants engineered to make the native major product
of Egyptian henbane premnaspirodiene synthase (HPS) and a complementary
512 mutant HPS library, we address the question of how product specificity
is controlled. These data sets reveal that HPS is far more robust
and resistant to mutations than TEAS, where most mutants are promiscuous.
We also combine experimental data with a sequence Potts Hamiltonian
model and direct coupling analysis to quantify mutant fitness. Our
results demonstrate that the Hamiltonian captures variation in product
outputs across both libraries, clusters native family members based
on their substrate specificities, and exposes the divergent catalytic
roles of couplings between the catalytic and noncatalytic domains
of TEAS versus HPS. Specifically, we found that the role of the interdomain
connectivities in specifying product output is more important in TEAS
than connectivities within the catalytic domain. Despite being 75%
identical, this property is not shared by HPS, where connectivities
within the catalytic domain are more important for specificity. By
solving the X-ray crystal structure of HPS, we assessed structural
bases for their interdomain network differences. Last, we calculate
the product profile Shannon entropies of the two libraries, which
showcases that site–site connectivities also play divergent
roles in catalytic accuracy
Coevolutionary Information Captures Catalytic Functions and Reveals Divergent Roles of Terpene Synthase Interdomain Connections
Inferring the historical
and biophysical causes of diversity within
protein families is a complex puzzle. A key to unraveling this problem
is characterizing the rugged topography of sequence–function
adaptive landscapes. Using biochemical data from a 29 =
512 combinatorial library of tobacco 5-epi-aristolochene
synthase (TEAS) mutants engineered to make the native major product
of Egyptian henbane premnaspirodiene synthase (HPS) and a complementary
512 mutant HPS library, we address the question of how product specificity
is controlled. These data sets reveal that HPS is far more robust
and resistant to mutations than TEAS, where most mutants are promiscuous.
We also combine experimental data with a sequence Potts Hamiltonian
model and direct coupling analysis to quantify mutant fitness. Our
results demonstrate that the Hamiltonian captures variation in product
outputs across both libraries, clusters native family members based
on their substrate specificities, and exposes the divergent catalytic
roles of couplings between the catalytic and noncatalytic domains
of TEAS versus HPS. Specifically, we found that the role of the interdomain
connectivities in specifying product output is more important in TEAS
than connectivities within the catalytic domain. Despite being 75%
identical, this property is not shared by HPS, where connectivities
within the catalytic domain are more important for specificity. By
solving the X-ray crystal structure of HPS, we assessed structural
bases for their interdomain network differences. Last, we calculate
the product profile Shannon entropies of the two libraries, which
showcases that site–site connectivities also play divergent
roles in catalytic accuracy