Biochemistry Biochemistry最新文献

This study reveals dual catalytic activities (β-lactamase and esterase) in a new penicillin-recognizing protein (IITRS), found in two closely related species, Enterococcus faecium and Enterococcus lactis. IITRS is distinct from other β-lactamase classes, showing only limited structural and functional similarity to class C β-lactamases. The conserved KTG motif, which helps in substrate recognition in class C, is not present in this enzyme. The enzyme is different from class C in terms of different conserved loops, such as R₂ and Ω loops, which are involved in the recognition, specificity, and hydrolysis of β-lactams. Nevertheless, the involvement of Ser64 and Tyr150 residues in β-lactam hydrolysis as found in class C enzymes has been demonstrated by site-directed mutagenesis. The study also highlights Tyr150 from the catalytic triad Tyr–Asp–Lys as being responsible for the esterase activity. This dual functionality confers catalytic promiscuity, enabling IITRS to function through two different mechanisms. The enzyme exhibits hydrolysis of p-NP esters (acetate, butyrate, hexanoate, decanoate, and laurate) displaying progressively higher activity with increasing alkyl chain lengths. Since Tyr150 has been found as a common ligand-binding residue for both of the activities, the β-lactamase inhibition by diisopropyl fluorophosphate (DFP), a reported inhibitor of bacterial esterase, also has been demonstrated. This promising albeit unexplored biocatalyst also might be used in the production of chiral compounds, investigating its enantioselective nature similar to other bacterial esterases. Overall, this research upholds a new promiscuous enzyme and proposes a distinct active site, narrower than that of a β-lactamase and wider than that of an esterase.

Copalyl diphosphate synthase from Penicillium verruculosum (PvCPS) is a bifunctional class II terpene synthase containing a prenyltransferase that produces geranylgeranyl diphosphate (GGPP) and a class II cyclase that utilizes GGPP as a substrate to generate the bicyclic diterpene copalyl diphosphate. Various stereoisomers of copalyl diphosphate establish the greater family of labdane natural products, many of which have environmental and medicinal impact. Understanding structure–function relationships in class II diterpene synthases is crucial for guiding protein engineering campaigns aimed at the generation of diverse bicyclic diterpene scaffolds. However, only a limited number of structures are available for class II cyclases from bacteria, plants, and humans, and no structures are available for a class II cyclase from a fungus. Further, bifunctional class II terpene synthases have not been investigated with regard to substrate channeling between the prenyltransferase and the cyclase. Here, we report the 2.9 Å-resolution cryo-EM structure of the 63-kD class II cyclase domain from PvCPS. Comparisons with bacterial and plant copalyl diphosphate synthases reveal conserved residues that likely guide the formation of the bicyclic labdane core but divergent catalytic dyads that mediate the final deprotonation step of catalysis. Substrate competition experiments reveal preferential GGPP transit from the PvCPS prenyltransferase to the cyclase, even when the enzymes are prepared as separate constructs. These results are consistent with a model in which transient prenyltransferase–cyclase association facilitates substrate channeling due to active-site proximity.

Histone trimethyllysine (Kme3) reader proteins are emerging therapeutic targets. However, development of selective inhibitors has proven challenging given the conserved nature of the aromatic cage which binds Kme3 as well as the myriad reader proteins which bind Kme3 at the same position on histone tails. These readers rely on both the presence of Kme3 as well as the appropriate surrounding histone tail sequence to bind, suggesting that binding is highly cooperative. We recently found that a small subset of Kme3 readers bind with equal or tighter affinity to histone tail peptides which replace Kme3 with its neutral isostere, tBuNle. This unexpected result offers promise for therapeutic design. Herein, we utilize histone 3 tail peptides containing Kme3 or the unnatural tBuNle to probe cooperativity in reader protein binding. Through three case studies, we quantitatively determine that the degree of cooperativity in a reader protein binding histone Kme3 influences the degree of its aromatic cage preference for cationic versus neutral ligands. Moreover, we find that the degree of cooperativity differs for each reader, suggesting that such differences in cooperativity could be utilized strategically for selective inhibitor design and that mutation to either histones or readers to alter cooperativity could significantly affect a reader protein’s selectivity for a specific post-translational modification.

Methyl-coenzyme M reductase (Mcr) catalyzes the terminal carbon reducing step in the methanogenesis cycle and has been closely scrutinized since its discovery nearly five decades ago. One critical gap in our knowledge is the structure of the protein complex necessary for the reductive activation of the nickel atom coordinated within the Mcr coenzyme F₄₃₀. Phylogenomic analysis previously identified 17 genes of unknown function that were found only in the genomes of sequenced methanogens and encode so-called “methanogenesis marker proteins” (Mmp1 through Mmp17). The functions of most Mmps remain largely unknown. Here we describe a complex formed from methanogenesis marker proteins 3, 5, 6, 7, 15, 17, AtwA, McrC, and two proteins with domains of unknown function (DUF2098 and DUF2111). Expression of the operon encoding these mmp genes from Methanosarcina acetivorans in Escherichia coli resulted in the formation of a large iron–sulfur cluster containing protein complex. Subsequent structural modeling revealed a putative complex comprised of a dimer of heterodecamers containing a total of ten [8Fe-9S–C] clusters, four Mg²⁺-ATPs, three [4Fe-4S] clusters, two Zn²⁺ ions, and two Mg²⁺-FAD ligands that interact with two Mcr holoenzymes. Systematic individual overexpression of the components of the complex in a native host, Methanococcus maripaludis, with affinity chromatography pull-downs and analysis by tandem mass spectrometry revealed a native protein complex formed in agreement with the predicted structure. These results provide a more complete molecular model of the activation complex catalyzing the ATP-dependent reductive activation of Mcr.

X-succinate synthases (XSSs) are a class of glycyl radical enzymes (GREs) that enable anaerobic hydrocarbon functionalization, granting anaerobes access to petroleum-derived substrates for metabolism. Owing to their ability to functionalize components of crude oil and catalyze selective olefin hydroalkylation, XSSs hold significant biotechnological promise. However, mechanistic understanding has been limited due to long-standing barriers to installing their essential glycyl radical in vitro, which have only recently been overcome. Unlike most GREs, XSSs contain accessory subunits that bind to the periphery of the catalytic subunit. The most well-studied XSS, benzylsuccinate synthase (BSS), includes two [4Fe–4S] cluster-binding accessory subunits, BSSγ and BSSβ. The full structure of BSSγ and the catalytic role of BSSβ have remained unclear. Here, we report the crystal structure of BSSγ with its [4Fe–4S] cluster intact, revealing a HiPP-like fold similar to that of BSSβ. Through biochemical and spectroscopic studies, we provide evidence that BSSβ promotes thiyl radical formation, even in the absence of a substrate. This finding contrasts with recent models, in which substrate binding is required to trigger thiyl radical formation. With this mechanistic insight, we optimized reaction conditions to achieve total turnover numbers of ∼17,000, representing an over 340-fold improvement compared to prior reports. We further show that in the absence of BSSβ, activated BSSαγ remains catalytically active for up to 11 days. Together, these results clarify the unique regulatory architecture of BSS and lay the groundwork for the use of XSSs in biocatalytic applications.

Phosphopantetheine adenylyltransferase (PPAT) catalyzes the transfer of an adenylyl group from adenosine triphosphate (ATP) to 4′-phosphopantetheine (PNS) to generate dephosphocoenzyme A (dPCoA) and pyrophosphate (PP_i). The dPCoA is required for the biosynthesis of coenzyme A (CoA), which is a vital cofactor in several essential biochemical reactions. PPAT enzyme from Enterobacter spp. (EbPPAT), cloned with a 30-residue-long N-terminal tag, was purified and crystallized. The structure determination of EbPPAT revealed the presence of six protein molecules, A, B, C, D, E, and F, in the asymmetric unit, which formed three homodimers designated as A–B, C–D and E–F. At the N-termini of molecules B and F, 17 additional residues belonging to the expression tag were observed. These 17-residue segments of molecules B and F were located deep inside the PNS-binding sites of the adjacent molecules. In addition to this, six citric acid (CIT) molecules were observed in the ATP-binding sites of all six EbPPAT molecules. Thus, the 17-mer peptide and CIT molecules filled the substrate-binding cleft of EbPPAT completely. In order to estimate the binding affinity, the 17-mer tag peptide was synthesized. The K_D value for the 17-mer peptide was found to be 1.7 × 10^–8 M. The K_D value for the CIT molecule was 2.13 × 10^-–5 M. These values indicated higher binding affinities of the 17-mer peptide and CIT molecule than those of the substrates, PNS and ATP, respectively. These results suggest that expression-tag fragments can be used to design the required peptide inhibitors of enzymes.

Nitrous oxide (N₂O) is a serious concern due to its role in global warming and ozone destruction. Agricultural practices account for ∼80% of all anthropogenic N₂O produced in the US, due in large part to the stimulation of microbial denitrification. Stable isotopes are uniquely suited to examine both microbial N₂O sources and the mechanism of N₂O biosynthesis through the use of Site Preference (δ¹⁵N^SP; the difference in δ¹⁵N between the central and outer N atoms in N₂O) and kinetic isotope effects (KIEs), respectively. Using trace gas isotope ratio mass spectrometry (TG-IRMS), we determined the δ¹⁵N, δ¹⁵N^α, δ¹⁵N^β, and δ¹⁸O of N₂O produced by a purified cytochrome c nitric oxide reductase (cNOR) from Paracoccus denitrificans. We also calculated δ¹⁵N^SP, the KIEs, and associated isotopic enrichment factors (ε) for N^bulk, N^α, and N^β. A normal isotope effect was observed for bulk ¹⁵N, with a KIE value of 1.0086 ± 0.0009 (ε = −8.6 ± 0.9‰). The isotope effects for both ¹⁵N^α and ¹⁵N^β were also normal, with position-specific KIEs of 1.0072 ± 0.0010 (ε = −7.2 ± 1.0‰) and 1.0100 ± 0.0010 (ε = −9.9 ± 1.0‰), respectively, and δ¹⁵N^SP values ranged from 0.5 to 8.7‰ with no significant trend as the reaction proceeded. Values of δ¹⁸O increased with N₂O production (slope of δ¹⁸O against [−f ln f/(1 – f)] = −19.9 ± 1.9‰). We present implications for the mechanism of N₂O production from cNOR based on our data.

Ca²⁺/CaM-dependent protein kinase kinase (CaMKK) phosphorylates and activates downstream kinases, including CaMKI, CaMKIV, PKB, and AMPK, regulating various cellular functions such as neuronal morphogenesis, metabolic control, and pathophysiological pathways, such as cancer progression. CaMKKα/1 is tightly regulated by an autoinhibitory mechanism. CaMKKβ/2 activity is highly Ca²⁺/CaM-independent (autonomous activity) in vitro and Ca²⁺/CaM-dependent in cultured cells. Whether these two activity states of CaMKKβ/2 exist in vivo and the detailed regulatory mechanisms for the transition of both activity states remain unclear due to the difficulty in distinguishing the two activity states. In this study, we detected Ca²⁺-dependent and autonomous CaMKK activity in HeLa cells and successfully separated both activity states of CaMKKβ/2 in mouse brain and testis extracts using a recently developed CaMKK inhibitor (TIM-063)-coupled sepharose, which binds to the catalytic domain in the active state but not in the autoinhibited state. Furthermore, lambda protein phosphatase treatment converted the Ca²⁺/CaM-dependent form to the autonomous form of CaMKKβ/2, which was not affected by Ala mutation of Ser128, Ser132, and Ser136. The two activity forms of CaMKKβ/2 had equivalent Ca²⁺/CaM-binding ability. The findings demonstrate the presence of autonomous and Ca²⁺/CaM-dependent forms of CaMKKβ/2 independently in mouse tissues and cultured cells. The transition of these states of CaMKKβ/2 may be dynamically regulated by the phosphorylation/dephosphorylation of serine residues in the N-terminal regulatory domain.

With an extraordinary rate enhancement of 10¹⁷ compared to the uncatalyzed reaction and no need for a cofactor, orotidine 5′-monophosphate decarboxylase (OMPDC) is considered one of the most efficient enzymes. Its mechanism has fascinated researchers for over 50 years. In this study, we used high-resolution X-ray crystallography to examine the molecular interactions between the active site of human OMPDC and various natural and synthetic ligands, including transition-state and product analogues, at the atomic level. Additionally, we evaluated their binding affinities with isothermal titration calorimetry (ITC). During protein expression and subsequent structure analysis, we identified nucleotides xanthosine-5′-monophosphate (XMP) and thymidine-5′-monophosphate (dTMP) bound to the active sites of OMPDC and its Thr321Asn variant, respectively, and confirmed their high binding affinities through ITC. Chemically, we investigated the role of the ribose 2′–OH group using 2′-deoxy OMP and 2′-SH UMP, focusing on validating key binding interactions within the nucleoside moiety. To further explore these interactions, we modified the heterocycles (e.g., GMP and CMP) and synthesized a new transition-state analogue, cyanuryl-5′-monophosphate (YMP). YMP exhibited strong affinity for OMPDC and formed an additional hydrogen bond with a nearby water molecule. However, this enthalpically favorable interaction resulted in an entropic penalty compared to the best-known OMPDC inhibitor, BMP, leading to similar affinities. To address this, we synthesized 5-methyl OMP to further improve ligand-enzyme interactions. This modification enhanced stabilization within the hydrophobic pocket through van der Waals forces, paving the way for designing more effective OMPDC inhibitors with specific substitutions aimed at optimizing binding affinity and enzyme inhibition.

The thiol group of the Cys side chain is known to participate in hydrogen bonds as an acceptor or donor. Similarly, the backbone nitrogens in proteins are involved in forming hydrogen bonds as donors that provide stability to protein secondary structures. In this study, we have identified more than 400 examples of self-contacting and inter-residue contacts from nearly 6000 high-resolution protein crystal structures in which the S–H group of the Cys side chain and the backbone nitrogen satisfy the geometric criteria to form hydrogen bonds. Very few studies have investigated the role of backbone nitrogen as a hydrogen-bond acceptor. Relative energy profiles calculated by varying the Cys χ¹ side chain dihedral angle of self-contacting Cys residues revealed that the energy difference between crystal structure and minimum energy conformations is between 0–3 kcal/mol. Quantum chemical calculations using DFT and MP2 theories indicated that the interaction energies of model systems with S–H···N self-contacts were only marginally favorable. However, the model systems representing S–H···N inter-residue contacts showed reasonably stable interaction. Natural bond orbital (NBO) analysis and NCIPLOT studies do not exhibit any hydrogen-bond interaction between the S–H donor and acceptor backbone nitrogen. The favorable interaction energies may be due to electrostatic and dispersion interactions. We found that the interactions due to S–H···N inter-residue contacts stabilize two secondary structural elements, and a large number of them occur between two β-strands. The structural role of S–H···N interactions can be further investigated by mutation studies of specific Cys residues involved in S–H···N contacts.