Substrate Carboxylate Research Articles

The Reaction Mechanism of Loganic Acid Methyltransferase: A Molecular Dynamics Simulation and Quantum Mechanics Study.

In this work, the catalytic mechanism of loganic acid methyltransferase was characterized at the molecular level. This enzyme is responsible for the biosynthesis of loganin, which is a precursor for a wide range of biologically active compounds. Due to the lack of detailed knowledge about this process, the aim of this study was the analysis of the structure and activity of loganic acid methyltransferase. Using molecular dynamics (MD) simulations, the native structure of the complex was reconstructed, and the key interactions between the substrate and loganic acid methyltransferase were investigated. Subsequently, the structures obtained from the simulations were used for quantum chemical (QM) calculations. The QM calculations allowed for the exploration of the energetic aspects of the reaction and the characterization of its mechanism. The results obtained in this study suggest the existence of two patterns of interactions between loganic acid methyltransferase and the substrate. The role of residue Q38 in the binding and orientation of the substrate's carboxyl group was also demonstrated. By employing a combined MD and QM approach, the experimental reaction barrier was reproduced, and detailed insights into the enzymatic activity mechanism of loganic acid methyltransferase were revealed.

Biochemical and structural basis of polyamine, lysine and ornithine acetylation catalyzed by spermine/spermidine N-acetyl transferase in moss and maize.

Polyamines such as spermidine and spermine are essential regulators of cell growth, differentiation, maintenance of ion balance and abiotic stress tolerance. Their levels are controlled by the spermidine/spermine N1 -acetyltransferase (SSAT) via acetylation to promote either their degradation or export outside the cell as shown in mammals. Plant genomes contain at least one gene coding for SSAT (also named NATA for N-AcetylTransferase Activity). Combining kinetics, HPLC-MS and crystallography, we show that three plant SSATs, one from the lower plant moss Physcomitrium patens and two from the higher plant Zea mays, acetylate various aliphatic polyamines and two amino acids lysine (Lys) and ornithine (Orn). Thus, plant SSATs exhibit a broad substrate specificity, unlike more specific human SSATs (hSSATs) as hSSAT1 targets polyamines, whereas hSSAT2 acetylates Lys and thiaLys. The crystal structures of two PpSSAT ternary complexes, one with Lys and CoA, the other with acetyl-CoA and polyethylene glycol (mimicking spermine), reveal a different binding mode for polyamine versus amino acid substrates accompanied by structural rearrangements of both the coenzyme and the enzyme. Two arginine residues, unique among plant SSATs, hold the carboxyl group of amino acid substrates. The most abundant acetylated compound accumulated in moss was N6 -acetyl-Lys, whereas N5 -acetyl-Orn, known to be toxic for aphids, was found in maize. Both plant species contain very low levels of acetylated polyamines. The present study provides a detailed biochemical and structural basis of plant SSAT enzymes that can acetylate a wide range of substrates and likely play various roles in planta.

Characterization of Unexpected Self-Acylation Activity of Acyl Carrier Proteins in a Modular Type I Apicomplexan Polyketide Synthase.

Natural products play critical roles as antibiotics, anticancer therapeutics, and biofuels. Polyketides are a distinct natural product class of structurally diverse secondary metabolites that are synthesized by polyketide synthases (PKSs). The biosynthetic gene clusters that encode PKSs have been found across nearly all realms of life, but those from eukaryotic organisms are relatively understudied. A type I PKS from the eukaryotic apicomplexan parasite Toxoplasma gondii,TgPKS2, was recently discovered through genome mining, and the functional acyltransferase (AT) domains were found to be selective for malonyl-CoA substrates. To further characterize TgPKS2, we resolved assembly gaps within the gene cluster, which confirmed that the encoded protein consists of three distinct modules. We subsequently isolated and biochemically characterized the four acyl carrier protein (ACP) domains within this megaenzyme. We observed self-acylation─or substrate acylation without an AT domain─for three of the four TgPKS2 ACP domains with CoA substrates. Furthermore, CoA substrate specificity and kinetic parameters were determined for all four unique ACPs. TgACP2-4 were active with a wide scope of CoA substrates, while TgACP1 from the loading module was found to be inactive for self-acylation. Previously, self-acylation has only been observed in type II systems, which are enzymes that act in-trans with one another, and this represents the first report of this activity in a modular type I PKS whose domains function in-cis. Site-directed mutagenesis of specific TgPKS2 ACP3 acidic residues near the phosphopantetheinyl arm demonstrated that they influence self-acylation activity and substrate specificity, possibly by influencing substrate coordination or phosphopantetheinyl arm activation. Further, the lack of TgPKS2 ACP self-acylation with acetoacetyl-CoA, which is utilized by previously characterized type II PKS systems, suggests that the substrate carboxyl group may be critical for TgPKS2 ACP self-acylation. The unexpected properties observed from T. gondii PKS ACP domains highlight their distinction from well-characterized microbial and fungal systems. This work expands our understanding of ACP self-acylation beyond type II systems and helps pave the way for future studies on biosynthetic enzymes from eukaryotes.

Emerging roles for thiol dioxygenases as oxygen sensors.

Cysteine dioxygenases, 3-mercaptopropionate dioxygenases and mercaptosuccinate dioxygenases are all thiol dioxygenases (TDOs) that catalyse oxidation of thiol molecules to sulphinates. They are Fe(II)-dependent dioxygenases with a cupin fold that supports a 3xHis metal-coordinating triad at the active site. They also have other, broadly common features including arginine residues involved in substrate carboxylate binding and a conserved trio of residues at the active site featuring a tyrosine important in substrate binding catalysis. Recently, N-terminal cysteinyl dioxygenase enzymes (NCOs) have been identified in plants (plant cysteine oxidases, PCOs), while human 2-aminoethanethiol dioxygenase (ADO) has been shown to act as both an NCO and a small molecule TDO. Although the cupin fold and 3xHis Fe(II)-binding triad seen in the small molecule TDOs are conserved in NCOs, other active site features and aspects of the overall protein architecture are quite different. Furthermore, the PCOs and ADO appear to act as biological O2 sensors, as shown by kinetic analyses and hypoxic regulation of the stability of their biological targets (N-terminal cysteine oxidation triggers protein degradation via the N-degron pathway). Here, we discuss the emergence of these two subclasses of TDO including structural features that could dictate their ability to bind small molecule or polypeptide substrates. These structural features may also underpin the O2 -sensing capability of the NCOs. Understanding how these enzymes interact with their substrates, including O2 , could reveal strategies to manipulate their activity, relevant to hypoxic disease states and plant adaptive responses to flooding.

Mechanism of a Standalone β-Lactone Synthetase: New Continuous Assay for a Widespread ANL Superfamily Enzyme.

Enzyme-catalyzed β-lactone formation from β-hydroxy acids is a crucial step in bacterial biosynthesis of β-lactone natural products and membrane hydrocarbons. We developed a novel, continuous assay for β-lactone synthetase activity using synthetic β-hydroxy acid substrates with alkene or alkyne moieties. β-Lactone formation is followed by rapid decarboxylation to form a conjugated triene chromophore for real-time evaluation by UV/Vis spectroscopy. The assay was used to determine steady-state kinetics of a long-chain β-lactone synthetase, OleC, from the plant pathogen Xanthomonas campestris. Site-directed mutagenesis was used to test the involvement of conserved active site residues in Mg2+ and ATP binding. A previous report suggested OleC adenylated the substrate hydroxy group. Here we present several lines of evidence, including hydroxylamine trapping of the AMP intermediate, to demonstrate the substrate carboxyl group is adenylated prior to making the β-lactone final product. A panel of nine substrate analogues were used to investigate the substrate specificity of X. campestris OleC by HPLC and GC-MS. Stereoisomers of 2-hexyl-3hydroxyoctanoic acid were synthesized and OleC preferred the (2R,3S) diastereomer consistent with the stereo-preference of upstream and downstream pathway enzymes. This biochemical knowledge was used to guide phylogenetic analysis of the β-lactone synthetases to map their functional diversity within the acyl-CoA synthetase, NRPS adenylation domain, and luciferase superfamily.

Different Behaviors of a Substrate in P450 Decarboxylase and Hydroxylase Reveal Reactivity-Enabling Actors

Biological routes to the production of fuels from renewable feedstocks hold significant promise in our efforts towards a sustainable future. The fatty acid decarboxylase enzyme (OleTJE) is a cytochrome P450 enzyme that converts long and medium chain fatty acids to terminal alkenes and shares significant similarities in terms of structure, substrate scope and mechanism with the hydroxylase cytochrome P450 (P450BSβ). Recent reports have demonstrated that catalytic pathways in these enzymes bifurcate when the heme is in its iron-hydroxo (compound II) state. In spite of significant similarities, the fundamental underpinnings of their different characteristic wild-type reactivities remain ambiguous. Here, we develop point charges, modified parameters and report molecular simulations of this crucial intermediate step. Water occupancies and substrate mobility at the active site are observed to be vital differentiating aspects between the two enzymes in the compound II state and corroborate recent experimental hypotheses. Apart from increased substrate mobility in the hydroxylase, which could have implications for enabling the rebound mechanism for hydroxylation, OleTJE is characterized by much stronger binding of the substrate carboxylate group to the active site arginine, implicating it as an important enabling actor for decarboxylation.

Glucuronoxylan recognition by GH 30 xylanases: A study with enzyme and substrate variants

XynA from Erwinia chrysanthemi (EcXyn30A), belonging to glycoside hydrolase family 30 subfamily 8, is specialized for hydrolysis of 4-O-methylglucuronoxylan (GX). Carboxyl group of 4-O-methylglucuronic acid serves as a substrate recognition element interacting ionically with positively charged Arg293 of the enzyme. We determined kinetic parameters of EcXyn30A on GX, its methyl ester (GXE) and 4-O-methylglucoxylan (GXR) and compared them with behavior of the enzyme variant in which Arg293 was replaced by Ala. The modifications of the substrate carboxyl groups resulted in several thousand-fold decrease in catalytic efficiency of EcXyn30A. In contrast, the R293A replacement reduced catalytic efficiency on GX only 18-times. The main difference was in catalytic rate (kcat) which was much lower for EcXyn30A acting on the modified substrates than for the variant which exhibited similar kcat values on all three polymers. The R293A variant cleaved GX, GXE and GXR on the second glycosidic bond from branch towards the reducing end, similarly to EcXyn30A. The R293A replacement caused 15–times decrease in specific activity on MeGlcA3Xyl4, but it did not influence low activity on linear xylooligosaccharides. Docking experiments showed that MeGlcA3Xyl4 and its esterified and reduced forms were bound to both enzymes in analogous way but with different binding energies.

Structure and function of the cytochrome P450 peroxygenase enzymes

The cytochromes P450 (P450s or CYPs) constitute a large heme enzyme superfamily, members of which catalyze the oxidative transformation of a wide range of organic substrates, and whose functions are crucial to xenobiotic metabolism and steroid transformation in humans and other organisms. The P450 peroxygenases are a subgroup of the P450s that have evolved in microbes to catalyze the oxidative metabolism of fatty acids, using hydrogen peroxide as an oxidant rather than NAD(P)H-driven redox partner systems typical of the vast majority of other characterized P450 enzymes. Early members of the peroxygenase (CYP152) family were shown to catalyze hydroxylation at the α and β carbons of medium-to-long-chain fatty acids. However, more recent studies on other CYP152 family P450s revealed the ability to oxidatively decarboxylate fatty acids, generating terminal alkenes with potential applications as drop-in biofuels. Other research has revealed their capacity to decarboxylate and to desaturate hydroxylated fatty acids to form novel products. Structural data have revealed a common active site motif for the binding of the substrate carboxylate group in the peroxygenases, and mechanistic and transient kinetic analyses have demonstrated the formation of reactive iron-oxo species (compounds I and II) that are ultimately responsible for hydroxylation and decarboxylation of fatty acids, respectively. This short review will focus on the biochemical properties of the P450 peroxygenases and on their biotechnological applications with respect to production of volatile alkenes as biofuels, as well as other fine chemicals.

Product specificity of fungal 8R- and 9S-dioxygenases of the peroxidase-cyclooxygenase superfamily with amino acid derivatized polyenoic fatty acids

Pathogenic fungi express fatty acid dioxygenases (DOX) fused to cytochromes P450 with diol or allene oxide synthase activities. The orientation of the fatty acids in the active sites of DOX was investigated with amino acid conjugates of 18:3n-3 and 18:2n-6. 9S-DOX-allene oxide synthase (AOS) oxidized the Gly, Ile, and Trp derivatives at C-9, which suggests that these conjugates enter the substrate recognition site with the omega end in analogy with fatty acids bound to cyclooxygenases and coral 8R-lipoxygenase (8R-LOX). In contrast, 7,8-diol synthases (7,8-LDS), 5,8-LDS, and 8R-DOX-AOS oxidized the Gly conjugates in most case only to small amounts of metabolites, but with retention of hydrogen abstraction at C-8 and relatively minor hydrogen abstraction at C-11. The Ile and Trp conjugates were not oxidized at C-8, and often insignificantly at C-9/C-13. The 8-DOX domains of these enzymes likely position the carboxyl group of substrates at the end of the active site in analogy with plant α-DOX and 9-LOX. Tyr radicals of the 9S-DOX and 8R-DOX domains catalyze antarafacial hydrogen abstraction and oxygen insertion in 18:3n-3. This occurs by abstraction of the proR and proS hydrogens at C-11 and C-8, respectively, in agreement with different “head to tail” orientation in the active site.

Catalytic Determinants of Alkene Production by the Cytochrome P450 Peroxygenase OleTJE

The Jeotgalicoccus sp. peroxygenase cytochrome P450 OleTJE (CYP152L1) is a hydrogen peroxide-driven oxidase that catalyzes oxidative decarboxylation of fatty acids, producing terminal alkenes with applications as fine chemicals and biofuels. Understanding mechanisms that favor decarboxylation over fatty acid hydroxylation in OleTJE could enable protein engineering to improve catalysis or to introduce decarboxylation activity into P450s with different substrate preferences. In this manuscript, we have focused on OleTJE active site residues Phe79, His85, and Arg245 to interrogate their roles in substrate binding and catalytic activity. His85 is a potential proton donor to reactive iron-oxo species during substrate decarboxylation. The H85Q mutant substitutes a glutamine found in several peroxygenases that favor fatty acid hydroxylation. H85Q OleTJE still favors alkene production, suggesting alternative protonation mechanisms. However, the mutant undergoes only minor substrate binding-induced heme iron spin state shift toward high spin by comparison with WT OleTJE, indicating the key role of His85 in this process. Phe79 interacts with His85, and Phe79 mutants showed diminished affinity for shorter chain (C10–C16) fatty acids and weak substrate-induced high spin conversion. F79A OleTJE is least affected in substrate oxidation, whereas the F79W/Y mutants exhibit lower stability and cysteine thiolate protonation on reduction. Finally, Arg245 is crucial for binding the substrate carboxylate, and R245E/L mutations severely compromise activity and heme content, although alkene products are formed from some substrates, including stearic acid (C18:0). The results identify crucial roles for the active site amino acid trio in determining OleTJE catalytic efficiency in alkene production and in regulating protein stability, heme iron coordination, and spin state.

Facile modulation of enantioselectivity of thermophilic Geobacillus zalihae lipase by regulating hydrophobicity of its Q114 oxyanion.

Site-directed mutagenesis of the oxyanion-containing amino acid Q114 in the recombinant thermophilic T1 lipase previously isolated from Geobacillus zalihae was performed to elucidate its role in the enzyme's enantioselectivity and reactivity. Substitution of Q114 with a hydrophobic methionine to yield mutant Q114M increased enantioselectivity (3.2-fold) and marginally improved reactivity (1.4-fold) of the lipase in catalysing esterification of ibuprofen with oleyl alcohol. The improved catalytic efficiency of Q114L was concomitant with reduced flexibility in the active site while the decreased enantioselectivity of Q114L could be directly attributed to diminished electrostatic repulsion of the substrate carboxylate ion that rendered partial loss in steric hindrance and thus enantioselectivity. The highest E-values for both Q114L (E-value 14.6) and Q114M (E-value 48.5) mutant lipases were attained at 50°C, after 12-16h, with a molar ratio of oleyl alcohol to ibuprofen of 1.5:1 and at 2.0% (w/v) enzyme load without addition of molecular sieves. Pertinently, site-directed mutagenesis on the Q114 oxyanion of T1 resulted in improved enantioselectivity and such approach may be applicable to other lipases of the same family. We demonstrated that electrostatic repulsion phenomena could affect flexibility/rigidity of the enzyme-substrate complex, aspects vital for enzyme activity and enantioselectivity of T1.

Mechanism and Catalytic Potential of a Cytochrome P450 (OleT) that Catalyzes C‐C Bond Scission

OleT (CYP152L1) is a recently discovered cytochrome P450 that is structurally similar to peroxide dependent P450s that catalyze fatty acid hydroxylations. OleT has been shown to catalyze the cleavage of n chain length fatty acids to form n‐1 alkenes through C‐C bond scission of the terminal carboxylate. Using headspace GC‐MS and FTIR with isotopically labeled substrates, we have confirmed that OleT metabolizes eicosanoic acid to 1‐nonadecene, forming carbon dioxide as a co‐product of the reaction. No oxygen deriving from peroxide is observed in either product. In order to probe the mechanistic deviation of OleT from P450 peroxygenases, we have employed a “decoy” approach where a short chain length fatty acid provides the acidic group necessary for peroxide heterolysis, and also allows access of small molecule probe substrates. Using this approach, we confirm that OleT also catalyzes epoxidation and hydroxylation reactions. Products derived from the metabolism of radical clock substrates directly show that OleT is capable of efficient oxygen rebound with radical lifetimes that are similar to other P450 monooxygenases. A site‐directed mutant, in which the acidic group is instead provided by the protein framework, circumvents the need for a substrate carboxylate, and shows efficient metabolism of a wide range of hydrocarbon substrates.Support or Funding InformationThis work is supported by startup funds from the University of South Carolina, and funding from the U.S.C. Office of Research through the Aspire program to T.M.M.

Solution-State (17)O Quadrupole Central-Transition NMR Spectroscopy in the Active Site of Tryptophan Synthase.

Oxygen is an essential participant in the acid-base chemistry that takes place within many enzyme active sites, yet has remained virtually silent as a probe in NMR spectroscopy. Here, we demonstrate the first use of solution-state (17)O quadrupole central-transition NMR spectroscopy to characterize enzymatic intermediates under conditions of active catalysis. In the 143 kDa pyridoxal-5'-phosphate-dependent enzyme tryptophan synthase, reactions of the α-aminoacrylate intermediate with the nucleophiles indoline and 2-aminophenol correlate with an upfield shift of the substrate carboxylate oxygen resonances. First principles calculations suggest that the increased shieldings for these quinonoid intermediates result from the net increase in the charge density of the substrate-cofactor π-bonding network, particularly at the adjacent α-carbon site.

Solution‐State 17O Quadrupole Central‐Transition NMR Spectroscopy in the Active Site of Tryptophan Synthase

AbstractOxygen is an essential participant in the acid–base chemistry that takes place within many enzyme active sites, yet has remained virtually silent as a probe in NMR spectroscopy. Here, we demonstrate the first use of solution‐state 17O quadrupole central‐transition NMR spectroscopy to characterize enzymatic intermediates under conditions of active catalysis. In the 143 kDa pyridoxal‐5′‐phosphate‐dependent enzyme tryptophan synthase, reactions of the α‐aminoacrylate intermediate with the nucleophiles indoline and 2‐aminophenol correlate with an upfield shift of the substrate carboxylate oxygen resonances. First principles calculations suggest that the increased shieldings for these quinonoid intermediates result from the net increase in the charge density of the substrate–cofactor π‐bonding network, particularly at the adjacent α‐carbon site.

Speeding up the product release: a second‐sphere contribution from Tyr191 to the reactivity of l‐lactate oxidase revealed in crystallographic and kinetic studies of site‐directed variants

Among α-hydroxy acid-oxidizing flavoenzymes l-lactate oxidase (LOX) is unique in featuring a second-sphere tyrosine (Tyr191 in Aerococcus viridans LOX; avLOX) at the binding site for the substrate's carboxylate group. Y191F, Y191L and Y191A variants of avLOX were constructed to affect a hydrogen-bond network connecting Tyr191 to the carboxylate of the bound ligand via the conserved Tyr40 and to examine consequent effects on enzymatic reactivity. Kinetic studies at 20 °C and pH 6.5 revealed that release of pyruvate product was decreased 4.7-fold (Y191F), 19-fold (Y191L) and 28-fold (Y191A) compared with wild-type enzyme (~ 141 s(-1)) and thus became mainly rate limiting for l-lactate oxidation by the variants at a steady-state under air-saturated conditions. In the Y191L and the Y191A variants, but not in the Y191F variant, l-lactate binding was also affected strongly by the site-directed substitution. Reduction of the flavin cofactor by l-lactate and its reoxidation by molecular oxygen were, however, comparatively weakly affected by the replacements of Tyr191. Unlike the related lactate monooxygenase, which prevents the fast dissociation of pyruvate to promote its oxidative decarboxylation by H2 O2 into acetate, CO2 and water as final reaction products, all avLOX variants retained their native oxidase activity where catalytic turnover results in the equivalent formation of H2O2. The 1.9 Å crystal structure of the Y191F variant bound with FMN and pyruvate revealed a strictly locally disruptive effect of the site-directed substitution. Product off-rates appear to be dictated by partitioning of residues including Tyr191 from an active-site lid loop into bulk solvent and modulation of the hydrogen bond strength that links Tyr40 with the pyruvate's carboxylate group. Overall, this study emphasizes the possibly high importance of contributions from second-sphere substrate-binding residues to the fine-tuning of reactivity in α-hydroxy acid-oxidizing flavoenzymes, requiring that the catalytic steps of flavin reduction and oxidation are properly timed with the physical step of α-keto acid product release.

Structural studies of a thermophilic esterase from a new Planctomycetes species, Thermogutta terrifontis

Thermogutta terrifontis esterase (TtEst), a carboxyl esterase identified in the novel thermophilic bacterium T. terrifontis from the phylum Planctomycetes, has been cloned and over-expressed in Escherichia coli. The enzyme has been characterized biochemically and shown to have activity towards small p-nitrophenyl (pNP) carboxylic esters, with optimal activity for pNP-propionate. The enzyme retained 95% activity after incubation for 1 h at 80 °C. The crystal structures of the native TtEst and its complexes with the substrate analogue D-malate and the product acetate have been determined to high resolution. The bound ligands have allowed the identification of the carboxyl and alcohol binding pockets in the enzyme active site. Comparison of TtEst with structurally related enzymes provides insight into how differences in their catalytic activity can be rationalized based upon the properties of the amino acid residues in their active site pockets. The mutant enzymes L37A and L251A have been constructed to extend the substrate range of TtEst towards the larger butyrate and valerate pNP-esters. These mutant enzymes have also shown a significant increase in activity towards acetate and propionate pNP esters. A crystal structure of the L37A mutant was determined with the butyrate product bound in the carboxyl pocket of the active site. The mutant structure shows an expansion of the pocket that binds the substrate carboxyl group, which is consistent with the observed increase in activity towards pNP-butyrate.

Second-Sphere Interactions between the C93–Y157 Cross-Link and the Substrate-Bound Fe Site Influence the O2 Coupling Efficiency in Mouse Cysteine Dioxygenase

Cysteine dioxygenase (CDO) is a non-heme iron enzyme that catalyzes the O₂-dependent oxidation of l-cysteine (l-Cys) to produce cysteinesulfinic acid (CSA). Adjacent to the Fe site of CDO is a covalently cross-linked cysteine-tyrosine pair (C93-Y157). While several theories have been proposed for the function of the C93-Y157 pair, the role of this post-translational modification remains unclear. In this work, the steady-state kinetics and O₂/CSA coupling efficiency were measured for wild-type CDO and selected active site variants (Y157F, C93A, and H155A) to probe the influence of second-sphere enzyme-substrate interactions on catalysis. In these experiments, it was observed that both kcat and the O₂/CSA coupling efficiency were highly sensitive to the presence of the C93-Y157 cross-link and its proximity to the substrate carboxylate group. Complementary electron paramagnetic resonance (EPR) experiments were performed to obtain a more detailed understanding of the second-sphere interactions identified in O₂/CSA coupling experiments. Samples of the catalytically inactive substrate-bound Fe(III)-CDO species were treated with cyanide, resulting in a low-spin (S = ¹/₂) ternary complex. Remarkably, both the presence of the C93-Y157 pair and interactions with the Cys carboxylate group could be readily identified by perturbations to the rhombic EPR signal. Spectroscopically validated active site quantum mechanics/molecular mechanics and density functional theory computational models are provided to suggest a potential role for Y157 in the positioning of the substrate Cys in the active site and to verify the orientation of the g-tensor relative to the CDO Fe site molecular axis.

Mechanism of Acyl–Enzyme Complex Formation from the Henry–Michaelis Complex of Class C β-Lactamases with β-Lactam Antibiotics

Bacteria that cause most of the hospital-acquired infections make use of class C β-lactamase (CBL) among other enzymes to resist a wide spectrum of modern antibiotics and pose a major public health concern. Other than the general features, details of the defensive mechanism by CBL, leading to the hydrolysis of drug molecules, remain a matter of debate, in particular the identification of the general base and role of the active site residues and substrate. In an attempt to unravel the detailed molecular mechanism, we carried out extensive hybrid quantum mechanical/molecular mechanical Car-Parrinello molecular dynamics simulation of the reaction with the aid of the metadynamics technique. On this basis, we report here the mechanism of the formation of the acyl-enzyme complex from the Henry-Michaelis complex formed by β-lactam antibiotics and CBL. We considered two β-lactam antibiotics, namely, cephalothin and aztreonam, belonging to two different subfamilies. A general mechanism for the formation of a β-lactam antibiotic-CBL acyl-enzyme complex is elicited, and the individual roles of the active site residues and substrate are probed. The general base in the acylation step has been identified as Lys67, while Tyr150 aids the protonation of the β-lactam nitrogen through either the substrate carboxylate group or a water molecule.

Structural Basis for the Divergence of Substrate Specificity and Biological Function within HAD Phosphatases in Lipopolysaccharide and Sialic Acid Biosynthesis

The haloacid dehalogenase enzyme superfamily (HADSF) is largely composed of phosphatases that have been particularly successful at adaptating to novel biological functions relative to members of other phosphatase families. Herein, we examine the structural basis for the divergence of function in two bacterial homologues: 2-keto-3-deoxy-d-manno-octulosonate 8-phosphate phosphohydrolase (KDO8P phosphatase, KDO8PP) and 2-keto-3-deoxy-9-O-phosphonononic acid phosphohydrolase (KDN9P phosphatase, KDN9PP). KDO8PP and KDN9PP catalyze the final step in KDO and KDN synthesis, respectively, prior to transfer to CMP to form the activated sugar nucleotide. KDO8PP and KDN9PP orthologs derived from an evolutionarily diverse collection of bacterial species were subjected to steady-state kinetic analysis to determine their specificities toward catalyzed KDO8P and KDN9P hydrolysis. Although each enzyme was more active with its biological substrate, the degree of selectivity (as defined by the ratio of kcat/Km for KDO8P vs KDN9P) varied significantly. High-resolution X-ray structure determination of Haemophilus influenzae KDO8PP bound to KDO/VO3(-) and Bacteriodes thetaiotaomicron KDN9PP bound to KDN/VO3(-) revealed the substrate-binding residues. The structures of the KDO8PP and KDN9PP orthologs were also determined to reveal the differences in their active-site structures that underlie the variation in substrate preference. Bioinformatic analysis was carried out to define the sequence divergence among KDN9PP and KDO8PP orthologs. The KDN9PP orthologs were found to exist as single-domain proteins or fused with the pathway nucleotidyl transferases; the fusion of KDO8PP with the transferase is rare. The KDO8PP and KDN9PP orthologs share a stringently conserved Arg residue that forms a salt bridge with the substrate carboxylate group. The split of the KDN9PP lineage from the KDO8PP orthologs is easily tracked by the acquisition of a Glu/Lys pair that supports KDN9P binding. Moreover, independently evolved lineages of KDO8PP orthologs exist, and are separated by diffuse active-site sequence boundaries. We infer a high tolerance of the KDO8PP catalytic platform to amino acid replacements that in turn influence substrate specificity changes and thereby facilitate the divergence in biological function.

Structural basis for dynamic mechanism of proton-coupled symport by the peptide transporter POT.

Proton-dependent oligopeptide transporters (POTs) are major facilitator superfamily (MFS) proteins that mediate the uptake of peptides and peptide-like molecules, using the inwardly directed H(+) gradient across the membrane. The human POT family transporter peptide transporter 1 is present in the brush border membrane of the small intestine and is involved in the uptake of nutrient peptides and drug molecules such as β-lactam antibiotics. Although previous studies have provided insight into the overall structure of the POT family transporters, the question of how transport is coupled to both peptide and H(+) binding remains unanswered. Here we report the high-resolution crystal structures of a bacterial POT family transporter, including its complex with a dipeptide analog, alafosfalin. These structures revealed the key mechanistic and functional roles for a conserved glutamate residue (Glu310) in the peptide binding site. Integrated structural, biochemical, and computational analyses suggested a mechanism for H(+)-coupled peptide symport in which protonated Glu310 first binds the carboxyl group of the peptide substrate. The deprotonation of Glu310 in the inward open state triggers the release of the bound peptide toward the intracellular space and salt bridge formation between Glu310 and Arg43 to induce the state transition to the occluded conformation.