Biochemical and structural characteristics of S-adenosyl-L-homocysteine hydrolases from selected microorganisms

Pomiary dyfrakcyjne z wykorzystaniem promieniowania rentgenowskiego ze źródła synchrotronowego wykonano w ośrodku HZB, BESSY w Berlinie na linii pomiarowej BL14.1 w ramach realizacji grantu pomiarowego pn. “Structural studies of S-adenosyl-L-homocysteine hydrolase from human pathogens”.Wykaz publikacji wchodzących w skład cyklu stanowiącego podstawę do ubiegania się o nadanie stopnia doktora. D1. Brzezinski, K., Czyrko, J., Sliwiak, J., Nalewajko-Sieliwoniuk, E., Jaskolski, M., Nocek, B., Dauter, Z. S-adenosyl-L-homocysteine hydrolase from a hyperthermophile (Thermotoga maritima) is expressed in Escherichia coli in inactive form - Biochemical and structural studies. (2017). International Journal of Biological Macromolecules 104, 584-596. D2. Czyrko, J., Sliwiak, J., Imiolczyk, B., Gdaniec, Z., Jaskolski, M., Brzezinski, K. Metal-cation regulation of enzyme dynamics is a key factor influencing the activity of S-adenosyl-L-homocysteine hydrolase from Pseudomonas aeruginosa. (2018). Scientific Reports 8, 11334. D3. Czyrko, J., Jaskolski, M., Brzezinski, K. Crystal structure of S-adenosyl-Lhomocysteine hydrolase from Cytophaga hutchinsonii, a case of combination of crystallographic and non-crystallographic symmetry. (2018). Croatica Chemica Acta 91, 153-162.Wyniki badań przedstawionych w publikacji D1 były podstawą do opracowania dwóch zgłoszeń patentowych z dnia 8 września 2016. WP1. Sposób syntezy enzymatycznej S-adenozylo-L-homocysteiny. Numer zgłoszenia: P.418595 WP2. Sposób uzyskania rekombinowanego enzymu bakteryjnego z Thermotoga maritima, hydrolazy S-adenozylo-L-homocysteiny. Numer zgłoszenia: P.418596Przedstawione osiągnięcie naukowe stanowiące podstawę do nadania stopnia doktora dotyczy badań biochemicznych i strukturalnych bakteryjnych hydrolaz S-adenozylo-L-homocysteiny (SAHazy) pochodzących z Thermotogamaritima, Pseudomonasaeruginosa i Cytophagahutchinsonii. SAHazy regulują kluczowe dla metabolizmu komórki reakcje metylacji zależne od S-adenozylo-L-metioniny (SAM), poprzez kontrolę stężenia S-adenozylo-L-homocysteiny (SAH), silnego inhibitora szeregu procesów metylacyjnych.Enzymy te są najczęściej aktywne w formie homotetrameru, w którym każda z podjednostek: (i) ma budowę trójdomenową oraz (ii) wiąże po jednej cząsteczce substratu i kofaktoranikotynoamidowoadeninowego w formie utlenionej (NAD+). Obecność kofaktora jest konieczna do przeprowadzenia reakcji enzymatycznej. W trakcie cyklu katalitycznego, każda z podjednostek oscyluje pomiędzy dwiema konformacjami – otwartą i zamkniętą, co wynika z ruchu dwóch głównych domen w trakcie wiązania substratu i uwalniania produktu. Prezentowane przeze mnie badania miały na celu poszerzenie wiedzy dotyczącej tej grupy enzymów. Interdyscyplinarność badań opisanych w niniejszym osiągnięciu naukowym pozwoliła mi na rozwiązanie wielu problemów pojawiających się w trakcie realizacji poszczególnych etapów badawczych, a co za tym idzie, pozwoliło na osiągnięcie założonych celów. W trakcie prowadzenia badań nad hipertermofilnąSAHazą z T.maritima wykazałam, że rekombinowane białko uzyskane w temperaturze pokojowej nie wykazuje aktywności katalitycznej. Analiza modelu krystalograficznego wskazywała, że poszczególne podjednostki przyjmują dwie nietypowe konformacje, wykluczające aktywność enzymatyczną tak zwiniętego białka. Dodatkowo, tylko dwie z czterech podjednostek wiążą kofaktor, który ponadto występuje głównie w formie zredukowanej (NADH), co również uniemożliwia katalityczny rozkład SAH. W oparciu o opracowaną przeze mnie procedurę pomiaru aktywności enzymatycznej tej SAHazy wykazałam, że białko zyskuje pełną aktywność katalityczną w wysokiej temperaturze, jedynie w obecności utlenionej formy kofaktora. W oparciu o porównanie modeli krystalograficznych SAHazy z T. maritima w formie nieaktywnej i aktywnej wyjaśniłam podłoże molekularne procesu termoaktywacji tego białka, polegające na przestrzennej rearanżacji poprawnie ufałdowanych domen. W ramach badań związanych z SAHaząz P. aeruginosa zwróciłam uwagę na to, że aktywność katalityczna tego enzymu mocno zależy od typu jonu metalu alkalicznego obecnego w mieszaninie reakcyjnej. Wykazałam, że enzym osiągał najwyższą aktywność w obecności kationów K+ poprzez odpowiedni wpływ na dynamikę białka. Ponadto wykazałam, że wiązanie innych kationów wpływających na dynamikę tego białka może również prowadzić do inhibicji aktywności katalitycznej SAHazy. Zaobserwowałam to dla kationów Rb+ i Zn2+, które hamują aktywność enzymatyczną w sposób niekompetycyjny. W ramach badań scharakteryzowałam biochemicznie i krystalograficznie SAHazę z C. hutchinsonii. Badania strukturalne oparte o metody biokrystalografii dotyczyły enzymu w konformacji zamkniętej w kompleksie z kofaktorem w formie utlenionej (NAD+), adenozyną (produkt reakcji rozkładu SAH) oraz kationem Na+ związanym w pobliżu kieszeni wiążącej substrat. Dodatkowo, przeanalizowałam sposób upakowania cząsteczek białka w sieci krystalicznej, zwracając uwagę na interesujący przypadek generowania homotetramerów odpowiadających aktywnej formie enzymu w oparciu o kombinację elementów symetrii krystalograficznej oraz translacyjnej symetrii niekrystalograficznej. Podsumowując, uzyskane rezultaty stanowią nowe odkrycia i pozwalają na szersze zrozumienie aspektów biochemicznych i strukturalnych hydrolazy S-adenozylo-L-homocysteiny, dotyczących zwłaszcza nieznanych mechanizmów regulacji aktywności katalitycznej tej grupy enzymów.The scientific achievement, described therein, which constitutes the basis for being conferred a doctoral degree, concerns the biochemical and structural characterization of S-adenosyl-L-homocysteine hydrolases (SAHases) from various bacteria, such as Thermotogamaritima, Pseudomonas aeruginosa and Cytophagahutchinsonii. SAHase is an essential element of cell metabolism, involved in the regulation of methylation reactions that utilize S-adenosyl-L-methionine (SAM) as a methyl group donor. SAM-Dependent methylation generates equimolar amounts of S-adenosyl-L-homocysteine (SAH), a potentinhibitorof SAM-dependent methylation processes. Therefore, cellular concentration of SAH has to be strictly controlled, and this function is fulfilled by SAHase. The enzyme is usually active as a homotetramer with a subunit folded into three domains. Each subunit binds one substrate and one nicotinamide adenine dinucleotide cofactor in its oxidized state (NAD+). A presence of the cofactor is required for the enzyme activity. Two principal domains, involved in substrate and cofactor binding, are connected by a two-part hinge element and the enzyme oscillates between two conformational states: open (ligand-free) and closed (with ligand bound) during the catalytic cycle.The studies presented hereinwere performed to broaden abiochemical and structural knowledge about SAHases. The interdisciplinary nature of the research were of key importance for the success of the research. In the course of research onhyperthermophilicSAHase from T. maritima, I revealed that a recombinant protein expressed and purified at room temperature is not active. A closer inspection of a crystallographic model of the protein showed, that individual subunits adopt two distinct and atypical conformations, which do not permit a protein folded in such manner to be enzymatically active. In addition, only two of the four subunits bind the cofactor, however, mainly in its reduced form (NADH). This fact alsoprecludes the enzymatic degradation of SAH. I have developed a new assay that indicated that the protein could gain a full catalytic activity only at a high temperaturein the presence of the oxidized form of the cofactor (NAD+). Based on crystallographic models of both, active and inactive forms of SAHase from T. maritima, I elucidated a mechanism of thermoactivation of the enyzme that is based on a spatial rearrangement of properly folded domains. While conducting research related to SAHase from P. aeruginosa, I noticed that the catalytic activity of the enzyme varies considerably, depending on the alkali metal ion present in the reaction mixture. Among tested cations, the K+ ion stimulates the highest enzymatic activity. An explanation of this phenomenon is that K+, but not other alkali cations, enables unique dynamic properties of the enzyme to ensure its maximum catalytic activity.The enzymatic activity can be influenced via regulation of protein dynamics, which depends on the type of coordinated cation. This mechanism can also be exploited for noncompetitive inhibition of the enzyme, as I observed for reactions performed in a presence of Rb+ and Zn2+ cations. The interdisciplinary research was also aimed at biochemical and structural characterization of SAHase from C.hutchinsonii. Within the study, I presented the crystal structure of recombinant enzyme in a ternary complex with NAD+, a reaction product/substrate (adenosine). Additionally, a sodium cation was identified in close proximity of the active site. The crystal contains two translational NCS-related dimers in the asymmetric unit. Two complete tetrameric enzyme molecules are generated from these dimers within the crystal lattice through the operation of two separate crystallographic twofold axes. To summarize, the results obtained are new discoveries, which allow a deeper understanding of numerous biochemical and structural aspects of S-adenosyl-L-homocysteine hydrolase, especially these related to previously unknown regulation mechanisms of the enzyme activity.Prowadzone badania naukowe w ramach rozprawy doktorskiej zostały sfinansowane ze środków pochodzących z grantu OPUS 5 nr UMO-2013/09/B/NZ1/01880 (Narodowe Centrum Nauki) pn. „Enzymologia strukturalna hydrolazy S-adenozylo-L-homocysteiny: poszukiwania nowych celów strukturalnych dla selektywnych inhibitorów”.W trakcie badań naukowych wykorzystano sprzęt zakupiony w projekcie realizowanym zgodnie z umową nr POPW.01.03.00-20-034/09-00 w ramach Programu Operacyjnego Rozwój Polski Wschodniej 2007-2013, Osi priorytetowej Nowoczesna Gospodarka, Działanie I.3 Wspieranie Innowacji.Uniwersytet w Białymstoku. Wydział Chemi

The Highly Efficient Expression System of Recombinant Human Prolidase and the Effect of N-Terminal His-Tag on the Enzyme Activity

Prolidase is an enzyme hydrolyzing dipeptides containing proline or hydroxyprolineat the C-terminus and plays an important role in collagen turnover. Human prolidase is active as a dimer with the C-terminal domain containing two Mn2+ ions in its active site. The study aimed to develop a highly efficient expression system of recombinant human prolidase (rhPEPD) and to evaluate the effect of the N-terminal His-Tag on its enzymatic and biological activity. An optimized bacterial expression system and an optimized purification procedure for rhPEPD included the two-step rhPEPD purification procedure based on (i) affinity chromatography on an Ni2+ ion-bound chromatography column and (ii) gel filtration with the possibility of tag removal by selective digestion with protease Xa. As the study showed, a high concentration of IPTGand high temperature of induction led to a fast stimulation of gene expression, which as a result forced the host into an intensive and fast production of rhPEPD. The results demonstrated that a slow induction of gene expression (low concentration of inducing factor, temperature, and longer induction time) led to efficient protein production in the soluble fraction. Moreover, the study proved that the presence of His-Tag changed neither the expression pattern of EGFR-downstream signaling proteins nor the prolidase catalytic activity

Recombinant Human Prolidase (rhPEPD) Induces Wound Healing in Experimental Model of Inflammation through Activation of EGFR Signalling in Fibroblasts

The potential of recombinant human prolidase (rhPEPD) to induce wound healing in an experimental model of IL-1β-induced inflammation in human fibroblasts was studied. It was found that rhPEPD significantly increased cell proliferation and viability, as well as the expression of the epidermal growth factor receptor (EGFR) and downstream signaling proteins, such as phosphorylated PI3K, AKT, and mTOR, in the studied model. Moreover, rhPEPD upregulated the expression of the β1 integrin receptor and its downstream signaling proteins, such as p-FAK, Grb2 and p-ERK 1/2. The inhibition of EGFR signaling by gefitinib abolished rhPEPD-dependent functions in an experimental model of inflammation. Subsequent studies showed that rhPEPD augmented collagen biosynthesis in IL-1β-treated fibroblasts as well as in a wound healing model (wound closure/scratch test). Although IL-1β treatment of fibroblasts increased cell migration, rhPEPD significantly enhanced this process. This effect was accompanied by an increase in the activity of MMP-2 and MMP-9, suggesting extracellular matrix (ECM) remodeling during the inflammatory process. The data suggest that rhPEPD may play an important role in EGFR-dependent cell growth in an experimental model of inflammation in human fibroblasts, and this knowledge may be useful for further approaches to the treatment of abnormalities of wound healing and other skin diseases

Flexible loops of New Delhi metallo-β-lactamase modulate its activity towards different substrates

Two accessory loop regions that are present in numerous variants of New Delhi metallo-β-lactamases (NDM) are important for the enzymatic activity. The first one is a flexible loop L3 that is located near the active site and is thought to play an important role in the catalytic process. The second region, Ω loop is located close to a structural element that coordinates two essential zinc ions. Both loops are not involved in any specific interactions with a substrate. Herein, we investigated how the length and hydrophobicity of loop L3 influence the enzymatic activity of NDMs, by analyzing mutants of NDM-1 with various deletions/point mutations within the L3 loop. We also investigated NDM variants with sequence variations/artificial deletions within the Ω loop. For all these variants we determined kinetic parameters for the hydrolysis of ampicillin, imipenem, and a chromogenic cephalosporin (CENTA). None of the mutations in the L3 loop completely abolished the enzymatic activity of NDM-1. Our results suggest that various elements of the loop play different roles in the hydrolysis of different substrates and the flexibility of the loop seems necessary to fulfill the requirements imposed by various substrates. Deletions within the Ω loop usually enhanced the enzymatic activity, particularly for the hydrolysis of ampicillin and imipenem. However, the exact role of the Ω loop in the catalytic reaction remains unclear. In our kinetic tests, the NDM enzymes were inhibited in the β-lactamase reaction by the CENTA substrate. We also present the X-ray crystal structures of the NDM-1, NDM-9 and NDM-12 proteins

Biochemical and structural insights into an unusual, alkali-metal-independent S -adenosyl- L -homocysteine hydrolase from Synechocystis sp. PCC 6803

The mesophilic cyanobacterium Synechocystis sp. PCC 6803 encodes an S-adenosyl-L-homocysteine hydrolase (SAHase) of archaeal origin in its genome. SAHases are essential enzymes involved in the regulation of cellular S-adenosyl-L-methionine (SAM)-dependent methylation reactions. They are usually active as homotetramers or, less commonly, as homodimers. A SAHase subunit is composed of two major domains: a cofactor (NAD+)-binding domain and a substrate (S-adenosyl-L-homocysteine)-binding domain. These are connected by a hinge element that is also a coordination site for an alkali-metal cation that influences domain movement during the catalytic cycle. Typically, the highest activity and strongest substrate binding of bacterial SAHases are observed in the presence of K+ ions. The SAHase from Synechocystis (SynSAHase) is an exception in this respect. Enzymatic and isothermal titration calorimetry studies demonstrated that in contrast to K+-dependent SAHases, the activity and ligand binding of SynSAHase are not affected by the presence of any particular alkali ion. Moreover, in contrast to other SAHases, the cyanobacterial enzyme is in an equilibrium of two distinct oligomeric states corresponding to its dimeric and tetrameric forms in solution. To explain these phenomena, crystal structures of SynSAHase were determined for the enzyme crystallized in the presence of adenosine (a reaction byproduct or substrate) and sodium or rubidium cations. The structural data confirm that while SynSAHase shares common structural features with other SAHases, no alkali metal is coordinated by the cyanobacterial enzyme as a result of a different organization of the macromolecular environment of the site that is normally supposed to coordinate the metal cation. This inspired the generation of SynSAHase mutants that bind alkali-metal cations analogously to K+-dependent SAHases, as confirmed by crystallographic studies. Structural comparisons of the crystal structure of SynSAHase with other experimental models of SAHases suggest a possible explanation for the occurrence of the cyanobacterial enzyme in the tetrameric state. On the other hand, the reason for the existence of SynSAHase in the dimeric state in solution remains elusive