Structural and mechanistic insights into a lysosomal membrane enzyme HGSNAT involved in Sanfilippo syndrome.

Heparan sulfate (HS) is degraded in lysosome by a series of glycosidases. Before the glycosidases can act, the terminal glucosamine of HS must be acetylated by the integral lysosomal membrane enzyme heparan-α-glucosaminide N-acetyltransferase (HGSNAT). Mutations of HGSNAT cause HS accumulation and consequently mucopolysaccharidosis IIIC, a devastating lysosomal storage disease characterized by progressive neurological deterioration and early death where no treatment is available. HGSNAT catalyzes a unique transmembrane acetylation reaction where the acetyl group of cytosolic acetyl-CoA is transported across the lysosomal membrane and attached to HS in one reaction. However, the reaction mechanism remains elusive. Here we report six cryo-EM structures of HGSNAT along the reaction pathway. These structures reveal a dimer arrangement and a unique structural fold, which enables the elucidation of the reaction mechanism. We find that a central pore within each monomer traverses the membrane and controls access of cytosolic acetyl-CoA to the active site at its luminal mouth where glucosamine binds. A histidine-aspartic acid catalytic dyad catalyzes the transfer reaction via a ternary complex mechanism. Furthermore, the structures allow the mapping of disease-causing variants and reveal their potential impact on the function, thus creating a framework to guide structure-based drug discovery efforts.

Cryo-EM structures of the human Elongator complex at work.

tRNA modifications affect ribosomal elongation speed and co-translational folding dynamics. The Elongator complex is responsible for introducing 5-carboxymethyl at wobble uridine bases (cm5U34) in eukaryotic tRNAs. However, the structure and function of human Elongator remain poorly understood. In this study, we present a series of cryo-EM structures of human ELP123 in complex with tRNA and cofactors at four different stages of the reaction. The structures at resolutions of up to 2.9 Å together with complementary functional analyses reveal the molecular mechanism of the modification reaction. Our results show that tRNA binding exposes a universally conserved uridine at position 33 (U33), which triggers acetyl-CoA hydrolysis. We identify a series of conserved residues that are crucial for the radical-based acetylation of U34 and profile the molecular effects of patient-derived mutations. Together, we provide the high-resolution view of human Elongator and reveal its detailed mechanism of action.

Chemoproteomics Yields a Selective Molecular Host for Acetyl-CoA.

Chemoproteomic profiling is a powerful approach to define the selectivity of small molecules and endogenous metabolites with the human proteome. In addition to mechanistic studies, proteome specificity profiling also has the potential to identify new scaffolds for biomolecular sensing. Here, we report a chemoproteomics-inspired strategy for selective sensing of acetyl-CoA. First, we use chemoproteomic capture experiments to validate the N-terminal acetyltransferase NAA50 as a protein capable of differentiating acetyl-CoA and CoA. A Nanoluc-NAA50 fusion protein retains this specificity and can be used to generate a bioluminescence resonance energy transfer (BRET) signal in the presence of a CoA-linked fluorophore. This enables the development of a ligand displacement assay in which CoA metabolites are detected via their ability to bind the Nanoluc-NAA50 protein "host" and compete binding of the CoA-linked fluorophore "guest". We demonstrate that the specificity of ligand displacement reflects the molecular recognition of the NAA50 host, while the window of dynamic sensing can be controlled by tuning the binding affinity of the CoA-linked fluorophore guest. Finally, we show that the method's specificity for acetyl-CoA can be harnessed for gain-of-signal optical detection of enzyme activity and quantification of acetyl-CoA from cellular samples. Overall, our studies demonstrate the potential of harnessing insights from chemoproteomics for molecular sensing and provide a foundation for future applications in target engagement and selective metabolite detection.

Characterization of Methyl- and Acetyl-Ni Intermediates in Acetyl CoA Synthase Formed during Anaerobic CO2 and CO Fixation.

The Wood-Ljungdahl Pathway is a unique biological mechanism of carbon dioxide and carbon monoxide fixation proposed to operate through nickel-based organometallic intermediates. The most unusual steps in this metabolic cycle involve a complex of two distinct nickel-iron-sulfur proteins: CO dehydrogenase and acetyl-CoA synthase (CODH/ACS). Here, we describe the nickel-methyl and nickel-acetyl intermediates in ACS completing the characterization of all its proposed organometallic intermediates. A single nickel site (Nip) within the A cluster of ACS undergoes major geometric and redox changes as it transits the planar Nip, tetrahedral Nip-CO and planar Nip-Me and Nip-Ac intermediates. We propose that the Nip intermediates equilibrate among different redox states, driven by an electrochemical-chemical (EC) coupling process, and that geometric changes in the A-cluster linked to large protein conformational changes control entry of CO and the methyl group.

How to Build a Metalloenzyme: Lessons from a Protein-Based Model of Acetyl Coenzyme A Synthase.

"What I cannot create, I do not understand"─Richard Feynman. This sentiment motivates the entire field of artificial metalloenzymes. Naturally occurring enzymes catalyze reactions with efficiencies, rates, and selectivity that generally cannot be achieved in synthetic systems. Many of these processes represent vital building blocks for a sustainable society, including CO2 conversion, nitrogen fixation, water oxidation, and liquid fuel synthesis. Our inability as chemists to fully reproduce the functionality of naturally occurring enzymes implicates yet-unknown contributors to reactivity. To identify these properties, it is necessary to consider all of the components of naturally occurring metalloenzymes, from the active site metal(s) to large-scale dynamics. In this Account, we describe the holistic development of a metalloprotein-based model that functionally reproduces the acetyl coenzyme A synthase (ACS) enzyme.ACS catalyzes the synthesis of a thioester, acetyl coenzyme A, from gaseous carbon monoxide, a methyl group donated by a cobalt corrinoid protein, and coenzyme A. The active site of ACS contains a bimetallic nickel site coupled to a [4Fe-4S] cluster. This reaction mimics Monsanto's acetic acid synthesis and represents an ancient process for incorporating inorganic carbon into cellular biomass through the primordial Wood-Ljungdahl metabolic pathway. From a sustainability standpoint, the reversible conversion of C1 substrates into an acetyl group and selective downstream transfer to a thiolate nucleophile offer opportunities to expand this reactivity to the anthropogenic synthesis of liquid fuels. However, substantial gaps in our understanding of the ACS catalytic mechanism coupled with the enzyme's oxygen sensitivity and general instability have limited these applications. It is our hope that development of an artificial metalloenzyme that carries out ACS-like reactions will advance our mechanistic understanding and enable synthesis of robust compounds with the capacity for similar reactivity.To construct this model, we first focused on the catalytic proximal nickel (NiP) site, which has a single metal center bound by three bridging cysteine residues in a "Y"-shaped arrangement. With an initial emphasis on reproducing the general structure of a low-coordinate metal binding site, the type I cupredoxin, azurin, was selected as the protein scaffold, and a nickel center was incorporated into the mononuclear site. Using numerous spectroscopic and computational techniques, including electron paramagnetic resonance (EPR) spectroscopy, nickel-substituted azurin was shown to have similar electronic and geometric structures to the NiP center in ACS. A substrate access channel was installed, and both carbon monoxide and a methyl group were shown to bind individually to the reduced NiI center. The elusive EPR-active S = 1/2 Ni-CH3 species, which has never been detected in native ACS, was observed in the azurin-based model, establishing the capacity of a biological NiI species to support two-electron organometallic reactions. Pulsed EPR studies on the S = 1/2 Ni-CH3 species in azurin suggested a noncanonical electronic structure with an inverted ligand field, which was proposed to prevent irreversible site degradation. This model azurin protein was ultimately shown to perform carbon-carbon and carbon-sulfur bond formation using sequential, ordered substrate addition for selective, stoichiometric thioester synthesis. X-ray spectroscopic methods were used to provide characterization of the remaining catalytic intermediates, resolving some debate over key mechanistic details.The overall approach and strategies that we employed for the successful construction of a functional protein-based model of ACS are described in this Account. We anticipate that these principles can be adapted across diverse metalloenzyme classes, providing essential mechanistic details and guiding the development of next-generation, functional artificial metalloenzymes.

Installing a Green Engine To Drive an Enzyme Cascade: A Light-Powered In Vitro Biosystem for Poly(3-hydroxybutyrate) Synthesis.

Many existing in vitro biosystems harness power from the chemical energy contained in substrates and co-substrates, and light or electric energy provided from abiotic parts, leading to a compromise in atom economy, incompatibility between biological and abiotic parts, and most importantly, incapability to spatiotemporally co-regenerate ATP and NADPH. In this study, we developed a light-powered in vitro biosystem for poly(3-hydroxybutyrate) (PHB) synthesis using natural thylakoid membranes (TMs) to regenerate ATP and NADPH for a five-enzyme cascade. Through effective coupling of cofactor regeneration and mass conversion, 20 mM PHB was yielded from 50 mM sodium acetate with a molar conversion efficiency of carbon of 80.0 % and a light-energy conversion efficiency of 3.04 %, which are much higher than the efficiencies of similar in vitro PHB synthesis biosystems. This suggests the promise of installing TMs as a green engine to drive more enzyme cascades.

Engineering potassium activation into biosynthetic thiolase.

Activation of enzymes by monovalent cations (M+) is a widespread phenomenon in biology. Despite this, there are few structure-based studies describing the underlying molecular details. Thiolases are a ubiquitous and highly conserved family of enzymes containing both K+-activated and K+-independent members. Guided by structures of naturally occurring K+-activated thiolases, we have used a structure-based approach to engineer K+-activation into a K+-independent thiolase. To our knowledge, this is the first demonstration of engineering K+-activation into an enzyme, showing the malleability of proteins to accommodate M+ ions as allosteric regulators. We show that a few protein structural features encode K+-activation in this class of enzyme. Specifically, two residues near the substrate-binding site are sufficient for K+-activation: A tyrosine residue is required to complete the K+ coordination sphere, and a glutamate residue provides a compensating charge for the bound K+ ion. Further to these, a distal residue is important for positioning a K+-coordinating water molecule that forms a direct hydrogen bond to the substrate. The stability of a cation-π interaction between a positively charged residue and the substrate is determined by the conformation of the loop surrounding the substrate-binding site. Our results suggest that this cation-π interaction effectively overrides K+-activation, and is, therefore, destabilised in K+-activated thiolases. Evolutionary conservation of these amino acids provides a promising signature sequence for predicting K+-activation in thiolases. Together, our structural, biochemical and bioinformatic work provide important mechanistic insights into how enzymes can be allosterically activated by M+ ions.

The role of the phosphate groups of trinitrophenyl adenosine 5'-triphosphate (TNP-ATP) in allosteric activation of pyruvate carboxylase and the inhibition of acetyl CoA-dependent activation.

A previous study showed that 2'-3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) was a weak allosteric activator of Rhizobium etli pyruvate carboxylase (RePC) in the absence of acetyl-CoA. On the other hand, TNP-ATP inhibited the allosteric activation of RePC by acetyl-CoA. Here, we aimed to study the role of triphosphate group of TNP-ATP on its allosteric activation of the enzyme and inhibition of acetyl-CoA-dependent activation of RePC using TNP-ATP and its derivatives, including TNP-ADP, TNP-AMP and TNP-adenosine. The pyruvate carboxylation activity was assayed to determine the effect of reducing the number of phosphate groups in TNP-ATP derivatives on allosteric activation and inhibition of acetyl-CoA activation of RePC and chicken liver pyruvate carboxylase (CLPC). Reducing the number of phosphate groups in TNP-ATP derivatives decreased the activation efficacy for both RePC and CLPC compared to TNP-ATP. The apparent binding affinity and inhibition of activation of the enzymes by acetyl-CoA were also diminished when the number of phosphate groups in the TNP-ATP derivatives was reduced. Whilst TNP-AMP activated RePC, it did not activate CLPC, but it did inhibit acetyl-CoA activation of both RePC and CLPC. Similarly, TNP-adenosine did not activate RePC; however, it did inhibit acetyl-CoA activation using a different mechanism compared to phosphorylated TNP-derivatives. These findings indicate that mechanisms of PC activation and inhibition of acetyl-CoA activation by TNP-ATP and its derivatives are different. This study provides the basis for possible drug development for treatment of metabolic diseases and cancers with aberrant expression of PC.

Structure-guided selection of puromycin N-acetyltransferase mutants with enhanced selection stringency for deriving mammalian cell lines expressing recombinant proteins.

Puromycin and the Streptomyces alboniger-derived puromycin N-acetyltransferase (PAC) enzyme form a commonly used system for selecting stably transfected cultured cells. The crystal structure of PAC has been solved using X-ray crystallography, revealing it to be a member of the GCN5-related N-acetyltransferase (GNAT) family of acetyltransferases. Based on structures in complex with acetyl-CoA or the reaction products CoA and acetylated puromycin, four classes of mutations in and around the catalytic site were designed and tested for activity. Single-residue mutations were identified that displayed a range of enzymatic activities, from complete ablation to enhanced activity relative to wild-type (WT) PAC. Cell pools of stably transfected HEK293 cells derived using two PAC mutants with attenuated activity, Y30F and A142D, were found to secrete up to three-fold higher levels of a soluble, recombinant target protein than corresponding pools derived with the WT enzyme. A third mutant, Y171F, appeared to stabilise the intracellular turnover of PAC, resulting in an apparent loss of selection stringency. Our results indicate that the structure-guided manipulation of PAC function can be utilised to enhance selection stringency for the derivation of mammalian cell lines secreting elevated levels of recombinant proteins.

Structural and Molecular Dynamics Analysis of Plant Serotonin N-Acetyltransferase Reveal an Acid/Base-Assisted Catalysis in Melatonin Biosynthesis.

Serotonin N-acetyltransferase (SNAT) is the key rate-limiting enzyme in melatonin biosynthesis. It mediates melatonin biosynthesis in plants by using serotonin and 5-methoxytryptamine (5-MT), but little is known of its underlying mechanisms. Herein, we present a detailed reaction mechanism of a SNAT from Oryza sativa through combined structural and molecular dynamics (MD) analysis. We report the crystal structures of plant SNAT in the apo and binary/ternary complex forms with acetyl-CoA (AcCoA), serotonin, and 5-MT. OsSNAT exhibits a unique enzymatically active dimeric fold not found in the known structures of arylalkylamine N-acetyltransferase (AANAT) family. The key residues W188, D189, D226, N220, and Y233 located around the active pocket are important in catalysis, confirmed by site-directed mutagenesis. Combined with MD simulations, we hypothesize a novel plausible catalytic mechanism in which D226 and Y233 function as catalytic base and acid during the acetyl-transfer reaction.

Characterization of two enzymes from Psychrobacter cryohalolentis that are required for the biosynthesis of an unusual diacetamido-d-sugar.

Psychrobacter cryohalolentis strain K5T is a Gram-negative organism first isolated in 2006. It has a complex O-antigen that contains, in addition to l-rhamnose and d-galactose, two diacetamido- and a triacetamido-sugar. The biochemical pathways for the production of these unusual sugars are presently unknown. Utilizing the published genome sequence of the organism, we hypothesized that the genes 0612, 0638, and 0637 encode for a 4,6-dehydratase, an aminotransferase, and an N-acetyltransferase, respectively, which would be required for the biosynthesis of one of the diacetamido-sugars, 2,4-diacetamido-2,4,6-trideoxy-d-glucose, starting from UDP-N-acetylglucosamine. Here we present functional and structural data on the proteins encoded by the 0638 and 0637 genes. The kinetic properties of these enzymes were investigated by a discontinuous HPLC assay. An X-ray crystallographic structure of 0638, determined in its external aldimine form to 1.3 Å resolution, demonstrated the manner in which the UDP ligand is positioned into the active site. It is strikingly different from that previously observed for PglE from Campylobacter jejuni, which functions on the same substrate. Four X-ray crystallographic structures were also determined for 0637 in various complexed states at resolutions between 1.3 and 1.55 Å. Remarkably, a tetrahedral intermediate mimicking the presumed transition state was trapped in one of the complexes. The data presented herein confirm the hypothesized functions of these enzymes and provide new insight into an unusual sugar biosynthetic pathway in Gram-negative bacteria. We also describe an efficient method for acetyl-CoA synthesis that allowed us to overcome its prohibitive cost for this analysis.

Structural and functional characterization of the N-terminal acetyltransferase Naa50.

The majority of eukaryotic proteins is modified by N-terminal acetylation, which plays a fundamental role in protein homeostasis, localization, and complex formation. N-terminal acetyltransferases (NATs) mainly act co-translationally on newly synthesized proteins at the ribosomal tunnel exit. NatA is the major NAT consisting of Naa10 catalytic and Naa15 auxiliary subunits, and with Naa50 forms the NatE complex. Naa50 has recently been identified in Arabidopsis thaliana and is important for plant development and stress response regulation. Here, we determined high-resolution X-ray crystal structures of AtNaa50 in complex with AcCoA and a bisubstrate analog. We characterized its substrate specificity, determined its enzymatic parameters, and identified functionally important residues. Even though Naa50 is conserved among species, we highlight differences between Arabidopsis and yeast, where Naa50 is catalytically inactive but binds CoA conjugates. Our study provides insights into Naa50 conservation, species-specific adaptations, and serves as a basis for further studies of NATs in plants.

Ligand Field Inversion as a Mechanism to Gate Bioorganometallic Reactivity: Investigating a Biochemical Model of Acetyl CoA Synthase Using Spectroscopy and Computation.

The biological global carbon cycle is largely regulated through microbial nickel enzymes, including carbon monoxide dehydrogenase (CODH), acetyl coenzyme A synthase (ACS), and methyl coenzyme M reductase (MCR). These systems are suggested to utilize organometallic intermediates during catalysis, though characterization of these species has remained challenging. We have established a mutant of nickel-substituted azurin as a scaffold upon which to develop protein-based models of enzymatic intermediates, including the organometallic states of ACS. In this work, we report the comprehensive investigation of the S = 1/2 Ni-CO and Ni-CH3 states using pulsed EPR spectroscopy and computational techniques. While the Ni-CO state shows conventional metal-ligand interactions and a classical ligand field, the Ni-CH3 hyperfine interactions between the methyl protons and the nickel indicate a closer distance than would be expected for an anionic methyl ligand. Structural analysis instead suggests a near-planar methyl ligand that can be best described as cationic. Consistent with this conclusion, the frontier molecular orbitals of the Ni-CH3 species indicate a ligand-centered LUMO, with a d9 population on the metal center, rather than the d7 population expected for a typical metal-alkyl species generated by oxidative addition. Collectively, these data support the presence of an inverted ligand field configuration for the Ni-CH3 Az species, in which the lowest unoccupied orbital is centered on the ligands rather than the more electropositive metal. These analyses provide the first evidence for an inverted ligand field within a biological system. The functional relevance of the electronic structures of both the Ni-CO and Ni-CH3 species are discussed in the context of native ACS, and an inverted ligand field is proposed as a mechanism by which to gate reactivity both within ACS and in other thiolate-containing metalloenzymes.

Steric-Free Bioorthogonal Labeling of Acetylation Substrates Based on a Fluorine-Thiol Displacement Reaction.

We have developed a novel bioorthogonal reaction that can selectively displace fluorine substitutions alpha to amide bonds. This fluorine-thiol displacement reaction (FTDR) allows for fluorinated cofactors or precursors to be utilized as chemical reporters, hijacking acetyltransferase-mediated acetylation both in vitro and in live cells, which cannot be achieved with azide- or alkyne-based chemical reporters. The fluoroacetamide labels can be further converted to biotin or fluorophore tags using FTDR, enabling the general detection and imaging of acetyl substrates. This strategy may lead to a steric-free labeling platform for substrate proteins, expanding our chemical toolbox for functional annotation of post-translational modifications in a systematic manner.

Involvement of subdomain II in the recognition of acetyl-CoA revealed by the crystal structure of homocitrate synthase from Sulfolobus acidocaldarius.

Homocitrate synthase (HCS) catalyzes the aldol condensation of α-ketoglutarate and acetyl coenzyme A to form homocitrate, which is the first committed step of lysine biosynthesis through the α-aminoadipate pathway in yeast, fungi, and some prokaryotes. We determined the crystal structure of a truncated form of HCS from a hyperthermophilic acidophilic archaeon, Sulfolobus acidocaldarius, which lacks the RAM (Regulation of amino acid metabolism) domain at the C terminus serving as the regulatory domain for the feedback inhibition by lysine, in complex with α-ketoglutarate, Mg2+ , and CoA. This structure coupled with mutational analysis revealed that a subdomain, subdomain II, connecting the N-terminal catalytic domain and C-terminal RAM domain is involved in the recognition of acetyl-CoA. This is the first structural evidence of the function of subdomain II in the related enzyme family, which will lead to a better understanding of the catalytic mechanism of HCS. DATABASES: Structural data are available in the RCSB PDB database under the accession number 6KTQ.

Gas channel rerouting in a primordial enzyme: Structural insights of the carbon-monoxide dehydrogenase/acetyl-CoA synthase complex from the acetogen Clostridium autoethanogenum.

Clostridium autoethanogenum, the bacterial model for biological conversion of waste gases into biofuels, grows under extreme carbon-monoxide (CO) concentrations. The strictly anaerobic bacterium derives its entire cellular energy and carbon from this poisonous gas, therefore requiring efficient molecular machineries for CO-conversion. Here, we structurally and biochemically characterized the key enzyme of the CO-converting metabolism: the CO-dehydrogenase/Acetyl-CoA synthase (CODH/ACS). We obtained crystal structures of natively isolated complexes from fructose-grown and CO-grown C. autoethanogenum cultures. Both contain the same isoforms and if the overall structure adopts the classic α2ß2 architecture, comparable to the model enzyme from Moorella thermoacetica, the ACS binds a different position on the CODH core. The structural characterization of a proteolyzed complex and the conservation of the binding interface in close homologs rejected the possibility of a crystallization artefact. Therefore, the internal CO-channeling system, critical to transfer CO generated at the C-cluster to the ACS active site, drastically differs in the complex from C. autoethanogenum. The 1.9-Å structure of the CODH alone provides an accurate picture of the new CO-routes, leading to the ACS core and reaching the surface. Increased gas accessibility would allow the simultaneous CO-oxidation and acetyl-CoA production. Biochemical experiments showed higher flexibility of the ACS subunit from C. autoethanogenum compared to M. thermoacetica, albeit monitoring similar CO-oxidation and formation rates. These results show a reshuffling of internal CO-tunnels during evolution of these Firmicutes, putatively leading to a bidirectional complex that ensure a high flux of CO-conversion toward energy conservation, acting as the main cellular powerplant.

Ustethylin Biosynthesis Implies Phenethyl Derivative Formation in Aspergillus ustus.

A highly oxygenated phenethyl derivative ustethylin A was isolated from Aspergillus ustus. Gene deletion, isotope labeling, and heterologous expression proved that the phenethyl core structure is assembled from malonyl-CoA by a polyketide synthase harboring a methyltransferase domain. Propionate was converted via acetyl-CoA to malonyl-CoA and incorporated into the molecule. Modifications on the core structure by three different oxidoreductases and one O-methyltransferase lead to the final product, ustethylin A.

13C Electron Nuclear Double Resonance Spectroscopy Shows Acetyl-CoA Synthase Binds Two Substrate CO in Multiple Binding Modes and Reveals the Importance of a CO-Binding "Alcove".

EPR and Electron Nuclear Double Resonance spectroscopies here characterize CO binding to the active-site A cluster of wild-type (WT) Acetyl-CoA Synthase (ACS) and two variants, F229W and F229A. The A-cluster binds CO to a proximal Ni (Nip) that bridges a [4Fe-4S] cluster and a distal Nid. An alcove seen in the ACS crystal structure near the A-cluster, defined by hydrophobic residues including F229, forms a cage surrounding a Xe mimic of CO. Previously, we only knew WT ACS bound a single CO to form the Ared-CO intermediate, containing Nip(I)-CO with CO located on the axis of the dz2 odd-electron orbital (g⊥ > g|| ∼ 2). Here, the two-dimensional field-frequency pattern of 2K-35 GHz 13C-ENDOR spectra collected across the Ared-CO EPR envelope reveals a second CO bound in the dz2 orbital's equatorial plane. This WT A-cluster conformer dominates the nearly conservative F229W variant, but 13C-ENDOR reveals a minority "A" conformation with (g|| > g⊥ ∼ 2) characteristic of a "cloverleaf" (e.g., dx2-y2) odd-electron orbital, with Nip binding two, apparently "in-plane" CO. Disruption of the alcove through introduction of the smaller alanine residue in the F229A variant diminishes conversion to Ni(I) ∼ 10-fold and introduces extensive cluster flexibility. 13C-ENDOR shows the F229A cluster is mostly (60%) in the "A" conformation but with ∼20% each of the WT conformer and an "O" state in which dz2 Nip(I) (g⊥ > g|| ∼ 2) surprisingly lacks CO. This paper thus demonstrates the importance of an intact alcove in forming and stabilizing the Ni(I)-CO intermediate in the Wood-Ljungdahl pathway of anaerobic CO and CO2 fixation.

Structural analysis of a novel substrate-free form of the aminoglycoside 6'-N-acetyltransferase from Enterococcus faecium.

Aminoglycoside acetyltransferases (AACs) catalyze the transfer of an acetyl group between acetyl-CoA and an aminoglycoside, producing CoA and an acetylated aminoglycoside. AAC(6')-Ii enzymes target the amino group linked to the 6' C atom in an aminoglycoside. Several structures of the AAC(6')-Ii from Enterococcus faecium [Ef-AAC(6')-Ii] have been reported to date. However, the detailed mechanism of its enzymatic function remains elusive. In this study, the crystal structure of Ef-AAC(6')-Ii was determined in a novel substrate-free form. Based on structural analysis, it is proposed that Ef-AAC(6')-Ii sequentially undergoes conformational selection and induced fit for substrate binding. These results therefore provide a novel viewpoint on the mechanism of action of Ef-AAC(6')-Ii.

Evidence of a preferred kinetic pathway in the carnitine acetyltransferase reaction.

Mammalian carnitine acetyltransferase (CrAT) is a mitochondrial enzyme that catalyzes the reversible transfer of an acetyl group from acetyl-CoA to carnitine. CrAT knockout studies have shown that this enzyme is critical to sustain metabolic flexibility, or the ability to switch between different fuel types, an underlying theme of the metabolic syndrome. These recent physiological findings imply that CrAT dysfunction, or its catalytic impairment, may lead to disease. To gain insight into the CrAT kinetic mechanism, we conducted stopped-flow experiments in various enzyme substrate/product conditions and analyzed full progress curves by global fitting. Simultaneous mixing of both substrates with CrAT produced relatively fast kinetics that follows an ordered bi bi mechanism. A great preference for ordered binding is supported by stopped-flow double mixing experiments such that premixed CrAT with acetyl-CoA or CoA demonstrated a biphasic decrease in initial rate that produces about a 100-fold attenuation in catalysis. Double mixing experiments also revealed that the CrAT initial rate is inhibited by 50% in approximately 8 s by either acetyl-CoA or CoA premixing. Analysis of available CrAT structures support a substrate conformational change between acetyl-CoA/CoA binary versus ternary complexes. Additional viscosity-based kinetic experiments yielded strong evidence that product release is the rate limiting step in the CrAT-catalyzed reaction.