Lid Domain Research Articles

A Lipase Gene of Thermomyces lanuginosus: Sequence Analysis and High-Efficiency Expression in Pichia pastoris.

Lipase, a type of enzyme that decomposes and synthesizes triglycerides, plays an important role in lipid processing. In this study, a heat-resisting lipase gene (lip4) from Thermomyces lanuginosus was subcloned into the pPICZαA vector and then transformed into Pichia pastoris X33. The recombinant yeast cell concentration reached the maximum (119.5 g/L) at 144 h, and the lipase (Lip4) activity reached the maximum (3900 U/mL) at 168 h in 10 L bioreactor. Through bioinformatics analysis, S168, as the key site of Lip4, participated in the formation of the catalytic triads S168-D223-H280 and G166-H167-S168-L169-G170. Furthermore, S168 and seven conserved amino acids of G104/288, S105, A195, P196, V225 and I287 constitute the active center of Lip4. Specifically, the structure modeling showed two α-helices of the lid domain, outside the active pocket domain, controlling the entry of the substrate on Lip4. The potential glycosylation of Asn-33 may be involved in exhibiting the high stable temperature for lipase activity. Therefore, the eukaryotic system was constructed to express Lip4 efficiently, and the amino acid sites related to the catalytic efficiency of Lip4 were clarified, providing a new way for its subsequent property research and industrial application.

Vitis vinifera Lipoxygenase LoxA is an Allosteric Dimer Activated by Lipidic Surfaces

Lipoxygenases catalyze the peroxidation of poly-unsaturated fatty acid chains either free or esterified in membrane lipids. Vitis vinifera LoxA is transcriptionally induced at ripening onset and localizes at the inner chloroplastic membrane where it is responsible for galactolipid regiospecific mono- and di-peroxidation. Here we present a kinetic and structural characterization of LoxA. Our X-ray structures reveal a constitutive dimer with detergent induced conformational changes affecting substrate binding and catalysis. In a closed conformation, a LID domain prevents substrate access to the catalytic site by steric hindrance. Detergent addition above the CMC destabilizes the LID and opens the dimer with both catalytic sites accessible from the same surface framed by the PLAT domains. As a consequence, detergent molecules occupy allosteric sites in the PLAT/catalytic domain interfaces. These structural changes are mirrored by increased enzymatic activity and positive cooperativity when the substrate is provided in micelles. The ability to interact with micelles is lost upon dimer destabilization by site-directed mutagenesis as assessed by tryptophan fluorescence. Our data allow to propose a model for protein activation at the membrane, classifying LoxA as an interfacial enzyme acting on fatty acid chains directly from the membrane similar to mammalian 15-LOX and 5-LOX.

Characterization of two bacterial tyrosinases from the halophilic bacterium Hahella sp. CCB MM4 relevant for phenolic compounds oxidation in wetlands.

Tyrosinases (TYRs) are type-3 copper proteins that are widely distributed in nature. They can hydroxylate and oxidize phenolic molecules and are mostly known for producing melanins that confer protection against photo induced damage. TYRs are also thought to play an important role in the 'latch mechanism', where high concentrations of phenolic compounds inhibit oxidative decomposition of organic biomass and subsequent CO2 release, especially relevant in wetland environments. In the present study, we describe two TYRs, HcTyr1 and HcTyr2, from halophilic bacterium Hahella sp. CCB MM4 previously isolated at Matang mangrove forest in Perak, Malaysia. The structure of HcTyr1 was determined by X-ray crystallography at a resolution of 1.9 Å and represents an uncharacterized group of prokaryotic TYRs as demonstrated by a sequence similarity network analysis. The genes encoding the enzymes were cloned, expressed, purified and thoroughly characterized by biochemical methods. HcTyr1 was able to self-cleave its lid-domain (LID) in a protease independent manner, whereas the LID of HcTyr2 was essential for activity and stability. Both enzymes showed variable activity in the presence of different metals, surfactants and NaCl, and were able to oxidize lignin constituents. The high salinity tolerance of HcTyr1 indicates that the enzyme can be an efficient catalyst in the habitat of the host.

Characterization of the Three DHFRs and K65P Variant: Enhanced Substrate Affinity and Molecular Dynamics Analysis.

Dihydrofolate reductase (DHFR) is ubiquitously present in all living organisms and plays a crucial role in the growth of the fungal pathogen R.solani. Sequence alignment confirmed the evolutionary conservation of the essential lid domain, with the amino acid 'P' within the PEKN lid domain appearing with a frequency of 89.5% in higher organisms and 11.8% in lower organisms. Consequently, a K65P variant was introduced into R.solani DHFR (rDHFR). Subsequent enzymatic kinetics assays were conducted for human DHFR (hDHFR), rDHFR, E. coli DHFR (eDHFR), and the K65P variant. hDHFR exhibited the highest kcat of 0.95s-1, followed by rDHFR with 0.14s-1, while eDHFR displayed the lowest kcat of 0.09s-1. Remarkably, the K65P variant induced a significant reduction in Km, resulting in a 1.8-fold enhancement in catalytic efficiency (kcat/Km) relative to the wild type. Differential scanning fluorimetry and binding free energy calculations confirmed the enhanced substrate affinity for both folate and NADPH in the K65P variant. These results suggest that the K65P mutation enhances substrate affinity and catalytic efficiency in DHFR, highlighting the evolutionary and functional importance of the K65 residue.

Rescue of Familial Lecithin:Cholesterol Acyltranferase Deficiency Mutations with an Allosteric Activator.

Lecithin:cholesterol acyltransferase (LCAT) deficiencies represent severe disorders characterized by aberrant cholesterol esterification in plasma, leading to life-threatening conditions. This study investigates the efficacy of Compound 2, a piperidinyl pyrazolopyridine allosteric activator that binds the membrane-binding domain of LCAT, in rescuing the activity of LCAT variants associated with disease. The variants K218N, N228K, and G230R, all located in the cap and lid domains of LCAT, demonstrated notable activity restoration in response to Compound 2. Molecular dynamics simulations and structural modeling indicate that these mutations disrupt the lid and membrane binding domain, with Compound 2 potentially dampening these structural alterations. Conversely, variants such as M252K and F382V in the cap and α/β-hydrolase domain, respectively, exhibited limited or no rescue by Compound 2. Future research should prioritize in vivo investigations that would validate the therapeutic potential of Compound 2 and related activators in familial LCAT deficiency patients with mutations in the cap and lid of the enzyme. SIGNIFICANCE STATEMENT: Lecithin:cholesterol acyltranferase (LCAT) catalyzes the first step of reverse cholesterol transport, namely the esterification of cholesterol in high density lipoprotein particles. Somatic mutations in LCAT lead to excess cholesterol in blood plasma and, in severe cases, kidney failure. In this study, we show that recently discovered small molecule activators can rescue function in LCAT-deficient variants when the mutations occur in the lid and cap domains of the enzyme.

Hydrophobic Residues Promote Interfacial Activation of Candida rugosa Lipase: A Study of Rotational Dynamics.

Microbial lipases constitute a class of biocatalysts with the ability to cleave ester linkages of long-chain triglycerides. This property makes them particularly attractive for industrial applications ranging from food processing to pharmaceutical preparation. Among such enzymes, Candida rugosa lipase (CRL) is one of the most frequently used in biotransformation. A notable feature of CRL, among many lipases, is its propensity for interfacial activation: these enzymes exhibit elevated catalytic rates when acting at the interface between aqueous and hydrophobic phases. Notably, this phenomenon can be attributed to the presence of a mobile lid domain, which in its closed state occludes the enzyme active site. To advance our understanding of interfacial activation, we explore the dynamics of CRL rotation at the octane-water interface in this work. To do so, we employ molecular dynamics and umbrella sampling to evaluate the free energy of rotation of the enzyme at the interface. We identify a global minimum in the rotational landscape that coincides with lid opening at the interface. Additionally, we investigate the role of surface residues outside the lid domain as they serve to instigate rotation of the lid toward the aqueous phase. In doing so, we identify a patch of leucine residues which when mutated to glycine impose a barrier to rotation that maintains the enzyme in the inactive (closed lid) state on the order of 1 μs. Importantly, this study presents a novel quantification of the rotational free energy corresponding to CRL lid opening at the octane-water interface. The accompanying mutagenesis study likewise clarifies the role of hydrophobic surface residues in the transition. As such, this work provides valuable insight into the phenomenon of interfacial activation that might open up new avenues for manipulating the microenvironment of industrially relevant lipases, affording enhanced control over the enzyme state.

Structures of BlEst2 from Bacillus licheniformis in its propeptide and mature forms reveal autoinhibitory effects of the C-terminal domain.

Carboxylesterases comprise a major class of α/β-fold hydrolases responsible for the cleavage and formation of ester bonds. Found ubiquitously in nature, these enzymes are crucial for the metabolism of both endogenous and exogenous carboxyl esters in animals, plants and microorganisms. Beyond their essential physiological roles, carboxylesterases stand out as one of the important classes of biocatalysts for biotechnology. BlEst2, an enzyme previously classified as Bacillus licheniformis esterase, remains largely uncharacterized. In the present study, we elucidate the structural biology, molecular dynamics and biochemical features of BlEst2. Our findings reveal a canonical α/β-hydrolase fold similar to the ESTHER block L of lipases, further augmented by two additional accessory C-terminal domains. Notably, the catalytic domain demonstrates two insertions, which occupy conserved locations in α/β-hydrolase proteins and commonly form the lid domain in lipase structures. Intriguingly, our in vitro cleavage of C-terminal domains revealed the structure of the active form of BlEst2. Upon activation, BlEst2 showed a markedly elevated hydrolytic activity. This observation implies that the intramolecular C-terminal domain serves as a regulatory intramolecular inhibitor. Interestingly, despite exhibiting esterase-like activity, BlEst2 structural characteristics align more closely with lipases. This suggests that BlEst2 could potentially represent a previously unrecognized subgroup within the realm of carboxyl ester hydrolases.

Structure of epoxide hydrolase 2 from Mangifera indica throws light on the substrate specificity determinants of plant epoxide hydrolases

Epoxide hydrolases (EHs) are a group of ubiquitous enzymes that catalyze hydrolysis of chemically reactive epoxides to yield corresponding dihydrodiols. Despite extensive studies on EHs from different clades, generic rules governing their substrate specificity determinants have remained elusive. Here, we present structural, biochemical and molecular dynamics simulation studies on MiEH2, a plant epoxide hydrolase from Mangifera indica. Comparative structure-function analysis of nine homologs of MiEH2, which include a few AlphaFold structural models, show that the two conserved tyrosines (MiEH2Y152 and MiEH2Y232) from the lid domain dissect substrate binding tunnel into two halves, forming substrate-binding-pocket one (BP1) and two (BP2). This compartmentalization offers diverse binding modes to their substrates, as exemplified by the binding of smaller aromatic substrates, such as styrene oxide (SO). Docking and molecular dynamics simulations reveal that the linear epoxy fatty acid substrates predominantly occupy BP1, while the aromatic substrates can bind to either BP1 or BP2. Furthermore, SO preferentially binds to BP2, by stacking against catalytically important histidine (MiEH2H297) with the conserved lid tyrosines engaging its epoxide oxygen. Residue (MiEH2L263) next to the catalytic aspartate (MiEH2D262) modulates substrate binding modes. Thus, the divergent binding modes correlate with the differential affinities of the EHs for their substrates. Furthermore, long-range dynamical coupling between the lid and core domains critically influences substrate enantioselectivity in plant EHs.

The Biochemical Impact of Extracting an Embedded Adenylate Kinase Domain Using Circular Permutation.

Adenylate kinases (AKs) have evolved AMP-binding and lid domains that are encoded as continuous polypeptides embedded at different locations within the discontinuous polypeptide encoding the core domain. A prior study showed that AK homologues of different stabilities consistently retain cellular activity following circular permutation that splits a region with high energetic frustration within the AMP-binding domain into discontinuous fragments. Herein, we show that mesophilic and thermophilic AKs having this topological restructuring retain activity and substrate-binding characteristics of the parental AK. While permutation decreased the activity of both AK homologues at physiological temperatures, the catalytic activity of the thermophilic AK increased upon permutation when assayed >30 °C below the melting temperature of the native AK. The thermostabilities of the permuted AKs were uniformly lower than those of native AKs, and they exhibited multiphasic unfolding transitions, unlike the native AKs, which presented cooperative thermal unfolding. In addition, proteolytic digestion revealed that permutation destabilized each AK in differing manners, and mass spectrometry suggested that the new termini within the AMP-binding domain were responsible for the increased proteolysis sensitivity. These findings illustrate how changes in contact order can be used to tune enzyme activity and alter folding dynamics in multidomain enzymes.

Inhibition of Pancreatic Lipase by Flavonoid Derivatives: In Vitro and In Silico Investigations.

Obesity, characterized by excessive adipose tissue accumulation, has emerged as a crucial determinant for a wide range of chronic medical conditions. The identification of effective interventions for obesity is of utmost importance. Widely researched antiobesity agents focus on pancreatic lipase, a significant therapeutic target. This study presented the evaluation of ten flavonoid compounds in terms of their inhibitory activities against pancreatic lipase, utilizing both in vitro and in silico approaches. The results indicated that all tested compounds demonstrated modest and weaker inhibitory activities compared to the reference compound, orlistat. Among the compounds investigated, F01 exhibited the highest potency, with an IC50 value of 17.68 ± 1.43 µM. The enzymatic inhibition kinetic analysis revealed that F01 operated through a competitive inhibition mechanism with a determined Ki of 7.16 μM. This value suggested a moderate binding affinity for the pancreatic lipase enzyme. Furthermore, the associated Vmax value was quantified at 0.03272 ΔA·min-1. In silico studies revealed that F01 displayed a binding mode similar to that of orlistat, despite lacking an active functional group capable of forming a covalent bond with Ser152 of the catalytic triad. However, F01 formed a hydrogen bond with this crucial amino acid. Furthermore, F01 interacted with other significant residues at the enzyme's active site, particularly those within the lid domain. Based on these findings, F01 demonstrates substantial potential as a candidate for further investigations.

Molecular dynamics simulations suggest novel allosteric modes in the Hsp70 chaperone protein

The Hsp70 chaperone protein system is an essential component of the protein folding and homeostasis machinery in E.Coli. Hsp70 is a three domain, 70 kDa protein which functions as an allosteric system cycling between an ADP-bound state where the three domains are loosely coupled via a flexible interdomain linker and an ATP-bound state where they are tightly coupled into a single entity. The structure-function model of this protein proposes an allosteric connection between the 45 kDa Nucleotide Binding Domain (NBD) and the 25 kDa Substrate Binding Domain (SBD) and Lid Domain which operates through the inter NBD-SBD linker. X-Ray crystallography and NMR spectroscopy have provided structures of the end states of the functional cycle of this protein, bound to ADP and ATP. We have used MD simulations to study the transitions between these end states and allosteric communication in this system. Our results largely validate the experimentally derived allosteric model of function, but shed additional light on the flow of allosteric information in the SBD + Lid. Specifically, we find that the Lid domain has a double-hinged structure with the potential for greater conformational flexibility than was hitherto expected.Communicated by Ramaswamy H. Sarma

Discovery of a non-canonical prototype long-chain monoacylglycerol lipase through a structure-based endogenous reaction intermediate complex

The identification and characterization of enzyme function is largely lacking behind the rapidly increasing availability of large numbers of sequences and associated high-resolution structures. This is often hampered by lack of knowledge on in vivo relevant substrates. Here, we present a case study of a high-resolution structure of an unusual orphan lipase in complex with an endogenous C18 monoacylglycerol ester reaction intermediate from the expression host, which is insoluble under aqueous conditions and thus not accessible for studies in solution. The data allowed its functional characterization as a prototypic long-chain monoacylglycerol lipase, which uses a minimal lid domain to position the substrate through a hydrophobic tunnel directly to the enzyme’s active site. Knowledge about the molecular details of the substrate binding site allowed us to modulate the enzymatic activity by adjusting protein/substrate interactions, demonstrating the potential of our findings for future biotechnology applications.

Improved catalytic performance and molecular insight for lipoxygenase from Enterovibrio norvegicus via directed evolution.

Lipoxygenase (LOX) holds significant promise for food and pharmaceutical industries. However, albeit its application has been hampered by low catalytic activity and suboptimal thermostability. To address the drawbacks, a directed evolution strategy was explored to enhance the catalytic activity and thermostability of LOX from Enterovibrio norvegicus (EnLOX) for the first time. After two rounds of error-prone polymerase chain reaction (error-prone PCR) and one generations of sequential DNA shuffling, all of four different mutants showed a significant increase in the specific activity of EnLOX, ranging from 132.07 ± 9.34 to 330.17 ± 18.54U/mg. Among these mutants, D95E/T99A/A121H/S142N/N444W/S613G (EAHNWG) exhibited the highest specific activity, which was 8.25-fold higher than the wild-type enzyme (WT). Meanwhile, the catalytic efficiency (K cat /K m) of EAHNWG was also improved, which was 13.61 ± 1.67s-1μM-1, in comparison to that of WT (4.83 ± 0.38s-1μM-1). In addition, mutant EAHNWG had a satisfied thermostability with the t 1/2,50°C value of 6.44 ± 0.24h, which was 0.4h longer than that of the WT. Furthermore, the molecular dynamics simulation and structural analysis demonstrated that the reduction of hydrogen bonds number, the enhancement of hydrophobic interactions in the catalytic pocket, and the improvement of flexibility of the lid domain facilitated structural stability and the strength of substrate binding capacity for improved thermal stability and catalytic efficiency of mutant LOX after directed evolution. Overall, these results could provide the guidance for further enzymatic modification of LOX with high catalytic performance for industrial application.

Dynamic lid domain of Chloroflexus aurantiacus Malonyl-CoA reductase controls the reaction

Malonyl-Coenzyme A Reductase (MCR) in Chloroflexus aurantiacus, a characteristic enzyme of the 3-hydroxypropionate (3-HP) cycle, catalyses the reduction of malonyl-CoA to 3-HP. MCR is a bi-functional enzyme; in the first step, malonyl-CoA is reduced to the free intermediate malonate semialdehyde by the C-terminal region of MCR, and this is further reduced to 3-HP by the N-terminal region of MCR. Here we present the crystal structures of both N-terminal and C-terminal regions of the MCR from C. aurantiacus. A catalytic mechanism is suggested by ligand and substrate bound structures, and structural and kinetic studies of MCR variants. Both MCR structures reveal one catalytic, and one non-catalytic SDR (short chain dehydrogenase/reductase) domain. C-terminal MCR has a lid domain which undergoes a conformational change and controls the reaction. In the proposed mechanism of the C-terminal MCR, the conversion of malonyl-CoA to malonate semialdehyde is based on the reduction of malonyl-CoA by NADPH, followed by the decomposition of the hemithioacetal to produce malonate semialdehyde and coenzyme A. Conserved arginines, Arg734 and Arg773 are proposed to play key roles in the mechanism and conserved Ser719, and Tyr737 are other essential residues forming an oxyanion hole for the substrate intermediates.

Sequence identification and in silico characterization of novel thermophilic lipases from Geobacillus species.

Microbial lipases are utilized in various biotechnological areas, including pharmaceuticals, food, biodiesel, and detergents. In this study, we cloned and sequenced Lip21 and Lip33 genes from Geobacillus sp. GS21 and Geobacillus sp. GS33, then we in silico and experimentally analyzed the encoded lipases. For this purpose, Lip21 and Lip33 were cloned, sequenced, and their amino acid sequences were investigated for determination of biophysicochemical characteristics, evolutionary relationships, and sequence similarities. 3D models were built and computationally affirmed by various bioinformatics tools, and enzyme-ligand interactions were investigated by docking analysis using six ligands. Biophysicochemical property of Lip21 and Lip33 was also determined experimentally and the results demonstrated that they had similar isoelectric point (pI) (6.21) and Tm (75.5°C) values as Tm was revealed by denatured protein analysis of the circular dichroism spectrum and pI was obtained by isoelectric focusing. Phylogeny analysis indicated that Lip21 and Lip33 were the closest to lipases from Geobacillus sp. SBS-4S and Geobacillus thermoleovorans, respectively. Alignment analysis demonstrated that S144-D348-H389 was catalytic triad residues in Lip21 and Lip33, and enzymes possessed a conserved Gly-X-Ser-X-Gly motif containing catalytic serine. 3D structure analysis indicated that Lip21 and Lip33 highly resembled each other and they were α/β hydrolase-fold enzymes with large lid domains. BANΔIT analysis results showed that Lip21 and Lip33 had higher thermal stability, compared to other thermostable Geobacillus lipases. Docking results revealed that Lip21- and Lip33-docked complexes possessed common residues (H112, K115, Q162, E163, and S141) that interacted with the substrates, except paranitrophenyl (pNP)-C10 and pNP-C12, indicating that these residues might have a significant action on medium and short-chain fatty acid esters. Thus, Lip21 and Lip33 can be potential candidates for different industrial applications.

Enhancing the Hydrolytic Activity of a Lipase towards Larger Triglycerides through Lid Domain Engineering.

Lipases have valuable potential for industrial use, particularly those mostly active against water-insoluble substrates, such as triglycerides composed of long-carbon chain fatty acids. However, in most cases, engineered variants often need to be constructed to achieve optimal performance for such substrates. Protein engineering techniques have been reported as strategies for improving lipase characteristics by introducing specific mutations in the cap domain of esterases or in the lid domain of lipases or through lid domain swapping. Here, we improved the lipase activity of a lipase (WP_075743487.1, or LipMRD) retrieved from the Marine Metagenomics MarRef Database and assigned to the Actinoalloteichus genus. The improvement was achieved through site-directed mutagenesis and by substituting its lid domain (FRGTEITQIKDWLTDA) with that of Rhizopus delemar lipase (previously R. oryzae; UniProt accession number, I1BGQ3) (FRGTNSFRSAITDIVF). The results demonstrated that the redesigned mutants gain activity against bulkier triglycerides, such as glyceryl tridecanoate and tridodecanoate, olive oil, coconut oil, and palm oil. Residue W89 (LipMRD numbering) appears to be key to the increase in lipase activity, an increase that was also achieved with lid swapping. This study reinforces the importance of the lid domains and their amino acid compositions in determining the substrate specificity of lipases, but the generalization of the lid domain swapping between lipases or the introduction of specific mutations in the lid domain to improve lipase activity may require further investigation.

Structural insights into a bacterial β-glucosidase capable of degrading sesaminol triglucoside to produce sesaminol: toward the understanding of the aglycone recognition mechanism by the C-terminal lid domain.

The sesaminol triglucoside (STG)-hydrolyzing β-glucosidase from Paenibacillus sp. (PSTG1), which belongs to glycoside hydrolase family 3 (GH3), is a promising catalyst for the industrial production of sesaminol. We determined the X-ray crystal structure of PSTG1 with bound glycerol molecule in the putative active site. PSTG1 monomer contained typical three domains of GH3 with the active site in domain 1 (TIM barrel). In addition, PSTG1 contained an additional domain (domain 4) at the C-terminus that interacts with the active site of the other protomer as a lid in the dimer unit. Interestingly, the interface of domain 4 and the active site forms a hydrophobic cavity probably for recognizing the hydrophobic aglycone moiety of substrate. The short flexible loop region of TIM barrel was found to be approaching the interface of domain 4 and the active site. We found that n-heptyl-β-D-thioglucopyranoside detergent acts as an inhibitor for PSTG1. Thus, we propose that the recognition of hydrophobic aglycone moiety is important for PSTG1-catalyzed reactions. Domain 4 might be a potential target for elucidating the aglycone recognition mechanism of PSTG1 as well as for engineering PSTG1 to create a further excellent enzyme to degrade STG more efficiently to produce sesaminol.

HSPA8 regulates anti-bacterial autophagy through liquid-liquid phase separation

ABSTRACT HSPA8 (heat shock protein family A (Hsp70) member 8) plays a significant role in the autophagic degradation of proteins, however, its effect on protein stabilization and anti-bacterial autophagy remains unknown. Here, it is discovered that HSPA8, as a binding partner of RHOB and BECN1, induce autophagy for intracellular bacteria clearance. Using its NBD and LID domains, HSPA8 physically binds to RHOB residues 1–42 and 89–118 as well as to BECN1 ECD domain, preventing RHOB and BECN1 degradation. Intriguingly, HSPA8 contains predicted intrinsically disordered regions (IDRs), and drives liquid-liquid phase separation (LLPS) to concentrate RHOB and BECN1 into HSPA8-formed liquid-phase droplets, resulting in improved RHOB and BECN1 interactions. Our study reveals a novel role and mechanism of HSPA8 in modulating anti-bacterial autophagy, and highlights the effect of LLPS-related HSPA8-RHOB-BECN1 complex on enhancing protein interaction and stabilization, which improves the understanding of autophagy-mediated defense against bacteria.

Allosteric communication between ligand binding domains modulates substrate inhibition in adenylate kinase

Enzymes play a vital role in life processes; they control chemical reactions and allow functional cycles to be synchronized. Many enzymes harness large-scale motions of their domains to achieve tremendous catalytic prowess and high selectivity for specific substrates. One outstanding example is provided by the three-domain enzyme adenylate kinase (AK), which catalyzes phosphotransfer between ATP to AMP. Here we study the phenomenon of substrate inhibition by AMP and its correlation with domain motions. Using single-molecule FRET spectroscopy, we show that AMP does not block access to the ATP binding site, neither by competitive binding to the ATP cognate site nor by directly closing the LID domain. Instead, inhibitory concentrations of AMP lead to a faster and more cooperative domain closure by ATP, leading in turn to an increased population of the closed state. The effect of AMP binding can be modulated through mutations throughout the structure of the enzyme, as shown by the screening of an extensive AK mutant library. The mutation of multiple conserved residues reduces substrate inhibition, suggesting that substrate inhibition is an evolutionary well conserved feature in AK. Combining these insights, we developed a model that explains the complex activity of AK, particularly substrate inhibition, based on the experimentally observed opening and closing rates. Notably, the model indicates that the catalytic power is affected by the microsecond balance between the open and closed states of the enzyme. Our findings highlight the crucial role of protein motions in enzymatic activity.

Structure of YdjH from Acinetobacter baumannii revealed an active site of YdjH family sugar kinase

Bacterial sugar kinase is a central enzyme for proper sugar degradation in bacteria, essential for survival and growth. Therefore, this enzyme family is a primary target for antibacterial drug development, with YdjH most preferring to phosphorylate higher-order monosaccharides with a carboxylate terminus. Sugar kinases express diverse specificity and functions, making specificity determination of this family a prominent issue. This study examines the YdjH crystal structure from Acinetobacter baumannii (abYdjH), which has an exceptionally high antibiotic resistance and is considered a superbug. Our structural and biochemical study revealed that abYdjH has a widely open lid domain and is a solution dimer. In addition, the putative active site of abYdjH was determined based on structural analysis, sequence comparison, and in silico docking. Finally, we proposed the active site-forming residues that determine various sugar specificities from abYdjH. This study contributes towards a deeper understanding of the phosphorylation process and bacterial sugar metabolism of YdjH family to design the next-generation antibiotics for targeting A. baumannii.