Enzym-Substratkomplexe der Quinat-Dehydrogenase aus Corynebacterium glutamicum. Strukturen, Katalyse und Spezifitäten

In der vorliegenden Arbeit wurde die Quinat-Dehydrogenase aus Corynebacterium glutamicum (CglQDH) enzymkinetisch charakterisiert und ihre Struktur in verschiedenen funktionellen Zuständen röntgenkristallographisch untersucht. Die CglQDH gehört zu den NAD(H)-abhängigen Oxidoreduktasen. Sie ist im katabolen Quinat-Stoffwechsel lokalisiert und spielt eine wichtige Rolle bei der xenobiotischen Detoxifikation sowie beim Ligninabbau. Die Quinat-Dehydrogenase katalysiert die NAD(P)-abhängige Oxidation von Quinat zu 3-Dehydroquinat über einen Hydridionen-Transfer, sowie die entsprechende Rückreaktion. Durch die Ergebnisse der enzymkinetischen Untersuchungen konnte gezeigt werden, dass die Quinat-Dehydrogenase sowohl unter optimalen pH-Bedingungen, als auch unter physiologischem pH eine deutlich höhere Affinität und katalytische Effizienz gegenüber Quinat verglichen mit Shikimat besitzt. Des Weiteren ist sie strikt NAD-abhängig, NADP kann nicht umgesetzt werden. Die CglQDH konnte darüber hinaus im binären Komplex mit NAD und in zwei verschiedenen ternären Komplexen (Quinat und NADH bzw. Shikimat und NADH) kristallisiert und mit einer hohen Auflösung aufgeklärt werden (1,0 A bzw. 1,16 A). Diese Strukturinformationen ermöglichen erstmalig Aussagen zur Substrat- und Cofaktor-Bindung bzw. -Diskriminierung sowie dem möglichen Katalysemechanismus bei einer Quinat-Dehydrogenase. Im Vergleich zur Apostruktur der CglQDH (2nlo, Dissertation Jan Schoepe) zeigen die Domänen der Quinat-Dehydrogenase nach der Substrat- und Cofaktor-Bindung eine offene Konformation. Durch den Vergleich der Aminosäurezusammensetzung und der Architektur des aktiven Zentrums der CglQDH mit eng verwandten Enzymen kann sowohl die strikte NAD-Abhängigkeit, als auch der schlechtere Shikimat-Umsatz dieses Enzyms erklärt werden. Alle Ergebnisse der vorliegenden Arbeit bekräftigen den für den Hydridtransfer postulierten Reaktionsmechanismus der katalytischen Dyade, bei der ein Lysin als Protonenakzeptor agiert, während ein benachbartes Aspartat diesen Protonenabzug vom Substrat und den anschließenden Hydridionen-Transfer auf den Cofaktor erleichtert

Structural elements regulating the photochromicity in a cyanobacteriochrome

The three-dimensional (3D) crystal structures of the GAF3 domain of cyanobacteriochrome Slr1393 (Synechocystis PCC6803) carrying a phycocyanobilin chromophore could be solved in both 15-Z dark-adapted state, Pr, λmax = 649 nm, and 15-E photoproduct, Pg, λmax = 536 nm (resolution, 1.6 and 1.86 Å, respectively). The structural data allowed identifying the large spectral shift of the Pr-to-Pg conversion as resulting from an out-of-plane rotation of the chromophore’s peripheral rings and an outward movement of a short helix formed from a formerly unstructured loop. In addition, a third structure (2.1-Å resolution) starting from the photoproduct crystals allowed identification of elements that regulate the absorption maxima. In this peculiar form, generated during X-ray exposition, protein and chromophore conformation still resemble the photoproduct state, except for the D-ring already in 15-Z configuration and tilted out of plane akin the dark state. Due to its formation from the photoproduct, it might be considered an early conformational change initiating the parental state-recovering photocycle. The high quality and the distinct features of the three forms allowed for applying quantum-chemical calculations in the framework of multiscale modeling to rationalize the absorption maxima changes. A systematic analysis of the PCB chromophore in the presence and absence of the protein environment showed that the direct electrostatic effect is negligible on the spectral tuning. However, the protein forces the outer pyrrole rings of the chromophore to deviate from coplanarity, which is identified as the dominating factor for the color regulation

Structural elements regulating the photochromicity in a cyanobacteriochrome

Phytochromes and related photoreceptors distinguish themselves for their long-wavelength absorption and large spectral shift between parental state and photoproduct. Both features are not well understood, partly due to lack of high-resolution structural data and insufficient support from quantum-chemical calculations. The red–green switching cyanobacteriochrome Slr1393g3 shows an absorption shift larger than 110 nm. Both parental state and photoproduct could be crystallized with high resolution, together with a “hybrid” form carrying the chromophore in parental state geometry, whereas the protein remained in the photoproduct conformation. The crystal structures reveal how chromophore and protein mutually regulate their conformational changes, yielding the observed spectral shift. Quantum-chemical calculations, based on these structures, provide a deeper understanding of the spectral tuning mechanisms

Biochemical properties of ectoine hydroxylases from extremophiles and their wider taxonomic distribution among microorganisms.

Ectoine and hydroxyectoine are well-recognized members of the compatible solutes and are widely employed by microorganisms as osmostress protectants. The EctABC enzymes catalyze the synthesis of ectoine from the precursor L-aspartate-β-semialdehyde. A subgroup of the ectoine producers can convert ectoine into 5-hydroxyectoine through a region-selective and stereospecific hydroxylation reaction. This compatible solute possesses stress-protective and function-preserving properties different from those of ectoine. Hydroxylation of ectoine is carried out by the EctD protein, a member of the non-heme-containing iron (II) and 2-oxoglutarate-dependent dioxygenase superfamily. We used the signature enzymes for ectoine (EctC) and hydroxyectoine (EctD) synthesis in database searches to assess the taxonomic distribution of potential ectoine and hydroxyectoine producers. Among 6428 microbial genomes inspected, 440 species are predicted to produce ectoine and of these, 272 are predicted to synthesize hydroxyectoine as well. Ectoine and hydroxyectoine genes are found almost exclusively in Bacteria. The genome context of the ect genes was explored to identify proteins that are functionally associated with the synthesis of ectoines; the specialized aspartokinase Ask_Ect and the regulatory protein EctR. This comprehensive in silico analysis was coupled with the biochemical characterization of ectoine hydroxylases from microorganisms that can colonize habitats with extremes in salinity (Halomonas elongata), pH (Alkalilimnicola ehrlichii, Acidiphilium cryptum), or temperature (Sphingopyxis alaskensis, Paenibacillus lautus) or that produce hydroxyectoine very efficiently over ectoine (Pseudomonas stutzeri). These six ectoine hydroxylases all possess similar kinetic parameters for their substrates but exhibit different temperature stabilities and differ in their tolerance to salts. We also report the crystal structure of the Virgibacillus salexigens EctD protein in its apo-form, thereby revealing that the iron-free structure exists already in a pre-set configuration to incorporate the iron catalyst. Collectively, our work defines the taxonomic distribution and salient biochemical properties of the ectoine hydroxylase protein family and contributes to the understanding of its structure

Crystal structure of the sugar acid‐binding protein CxaP from a TRAP transporter in Advenella mimigardefordensis strain DPN7 T

Recently, CxaP, a sugar acid substrate binding protein (SBP) fromAdvenella mimigardefordensis strain DPN7 T, was identified as part of anovel sugar uptake strategy. In the present study, the protein was success-fully crystallized. Although several SBP structures of tripartite ATP-inde-pendent periplasmic transporters have already been solved, this is the firststructure of an SBP accepting multiple sugar acid ligands. Protein crystalswere obtained with bound D -xylonic acid, D-fuconic acid D -galactonic andD-gluconic acid, respectively. The protein shows the typical structure of anSBP of a tripartite ATP-independent periplasmic transporter consisting oftwo domains linked by a hinge and spanned by a long α-helix. By analysisof the structure, the substrate binding site of the protein was identified.The carboxylic group of the sugar acids interacts with Arg175, whereas thecoordination of the hydroxylic groups at positions C2 and C3 is mostprobably realized by Arg154 and Asn151. Furthermore, it was observedthat 2-keto-3-deoxy- D-gluconic acid is bound in protein crystals that werecrystallized without the addition of any ligand, indicating that this mole-cule is prebound to the protein and is displaced by the other ligands if theyare available

Structures and enzymatic mechanisms of phycobiliprotein lyases CpcE/F and PecE/F

The light-harvesting phycobilisome in cyanobacteria and red algae requires the lyase-catalyzed chromophorylation of phycobiliproteins. There are three functionally distinct lyase families known. The heterodimeric E/F type is specific for attaching bilins covalently to α-subunits of phycocyanins and phycoerythrins. Unlike other lyases, the lyase also has chromophore-detaching activity. A subclass of the E/F-type lyases is, furthermore, capable of chemically modifying the chromophore. Although these enzymes were characterized >25 y ago, their structures remained unknown. We determined the crystal structure of the heterodimer of CpcE/F from Nostoc sp. PCC7120 at 1.89-Å resolution. Both subunits are twisted, crescent-shaped α-solenoid structures. CpcE has 15 and CpcF 10 helices. The inner (concave) layer of CpcE (helices h2, 4, 6, 8, 10, 12, and 14) and the outer (convex) layer of CpcF (h16, 18, 20, 22, and 24) form a cavity into which the phycocyanobilin chromophore can be modeled. This location of the chromophore is supported by mutations at the interface between the subunits and within the cavity. The structure of a structurally related, isomerizing lyase, PecE/F, that converts phycocyanobilin into phycoviolobilin, was modeled using the CpcE/F structure as template. A H$_{87}$C$_{88}$ motif critical for the isomerase activity of PecE/F is located at the loop between h20 and h21, supporting the proposal that the nucleophilic addition of Cys-88 to C10 of phycocyanobilin induces the isomerization of phycocyanobilin into phycoviolobilin. Also, the structure of NblB, involved in phycobilisome degradation could be modeled using CpcE as template. Combined with CpcF, NblB shows a low chromophore-detaching activity

Role of the Extremolytes Ectoine and Hydroxyectoine as Stress Protectants and Nutrients: Genetics, Phylogenomics, Biochemistry, and Structural Analysis

Fluctuations in environmental osmolarity are ubiquitous stress factors in many natural habitats of microorganisms, as they inevitably trigger osmotically instigated fluxes of water across the semi-permeable cytoplasmic membrane. Under hyperosmotic conditions, many microorganisms fend off the detrimental effects of water efflux and the ensuing dehydration of the cytoplasm and drop in turgor through the accumulation of a restricted class of organic osmolytes, the compatible solutes. Ectoine and its derivative 5-hydroxyectoine are prominent members of these compounds and are synthesized widely by members of the Bacteria and a few Archaea and Eukarya in response to high salinity/osmolarity and/or growth temperature extremes. Ectoines have excellent function-preserving properties, attributes that have led to their description as chemical chaperones and fostered the development of an industrial-scale biotechnological production process for their exploitation in biotechnology, skin care, and medicine. We review, here, the current knowledge on the biochemistry of the ectoine/hydroxyectoine biosynthetic enzymes and the available crystal structures of some of them, explore the genetics of the underlying biosynthetic genes and their transcriptional regulation, and present an extensive phylogenomic analysis of the ectoine/hydroxyectoine biosynthetic genes. In addition, we address the biochemistry, phylogenomics, and genetic regulation for the alternative use of ectoines as nutrients

Structural features of antiviral DNA cytidine deaminases

The APOBEC3 (A3) family of cytidine deaminases plays a vital role for innate defense against retroviruses. Lentiviruses such as HIV-1 evolved the Vif protein that triggers A3 protein degradation. There are seven A3 proteins, A3A-A3H, found in humans. All A3 proteins can deaminate cytidines to uridines in single-stranded DNA (ssDNA), generated during viral reverse transcription. A3 proteins have either one or two cytidine deaminase domains (CD). The CDs coordinate a zinc ion, and their amino acid specificity classifies the A3s into A3Z1, A3Z2, and A3Z3. A3 proteins occur as monomers, dimers, and large oligomeric complexes. Studies on the nature of A3 oligomerization, as well as the mode of interaction of A3s with RNA and ssDNA are partially controversial. High-resolution structures of the catalytic CD2 of A3G and A3F as well as of the single CD proteins A3A and A3C have been published recently. The NMR and X-ray crystal structures show globular proteins with six α-helices and five β sheets arranged in a characteristic motif (α1-β1-β2/2'-α2-β3-α3-β4-α4-β5-α5-α6). However, the detailed arrangement and extension of individual structure elements and their relevance for A3 complex formation and activity remains a matter of debate and will be highlighted in this review

Crystal structures of a novel family IV esterase in free and substrate‐bound form

Bacterial lipolytic enzymes of family IV are homologs of the mammalian hormone-sensitive lipases (HSL) and have been successfully used for various biotechnological applications. The broad substrate specificity and ability for enantio-, regio-, and stereoselective hydrolysis are remarkable features of enzymes from this class. Many crystal structures are available for esterases and lipases, but structures of enzyme–substrate or enzyme–inhibitor complexes are less frequent although important to understand the molecular basis of enzyme–substrate interaction and to rationalize biochemical enzyme characteristics. Here, we report on the structures of a novel family IV esterase isolated from a metagenomic screen, which shows a broad substrate specificity. We solved the crystal structures in the apo form and with a bound substrate analogue at 1.35 and 1.81 Å resolution, respectively. This enzyme named PtEst1 hydrolyzed more than 60 out 96 structurally different ester substrates thus being substrate promiscuous. Its broad substrate specificity is in accord with a large active site cavity, which is covered by an α-helical cap domain. The substrate analogue methyl 4-methylumbelliferyl hexylphosphonate was rapidly hydrolyzed by the enzyme leading to a complete inactivation caused by covalent binding of phosphinic acid to the catalytic serine. Interestingly, the alcohol leaving group 4-methylumbelliferone was found remaining in the active site cavity, and additionally, a complete inhibitor molecule was found at the cap domain next to the entrance of the substrate tunnel. This unique situation allowed gaining valuable insights into the role of the cap domain for enzyme–substrate interaction of esterases belonging to family IV