Steroid Hydroxylation by Basidiomycete Peroxygenases: a Combined Experimental and Computational Study

ABSTRACTThe goal of this study is the selective oxyfunctionalization of steroids under mild and environmentally friendly conditions using fungal enzymes. With this purpose, peroxygenases from three basidiomycete species were tested for the hydroxylation of a variety of steroidal compounds, using H2O2as the only cosubstrate. Two of them are wild-type enzymes fromAgrocybe aegeritaandMarasmius rotula, and the third one is a recombinant enzyme fromCoprinopsis cinerea. The enzymatic reactions on free and esterified sterols, steroid hydrocarbons, and ketones were monitored by gas chromatography, and the products were identified by mass spectrometry. Hydroxylation at the side chain over the steroidal rings was preferred, with the 25-hydroxyderivatives predominating. Interestingly, antiviral and other biological activities of 25-hydroxycholesterol have been reported recently (M. Blanc et al., Immunity 38:106–118, 2013,http://dx.doi.org/10.1016/j.immuni.2012.11.004). However, hydroxylation in the ring moiety and terminal hydroxylation at the side chain also was observed in some steroids, the former favored by the absence of oxygenated groups at C-3 and by the presence of conjugated double bonds in the rings. To understand the yield and selectivity differences between the different steroids, a computational study was performed using Protein Energy Landscape Exploration (PELE) software for dynamic ligand diffusion. These simulations showed that the active-site geometry and hydrophobicity favors the entrance of the steroid side chain, while the entrance of the ring is energetically penalized. Also, a direct correlation between the conversion rate and the side chain entrance ratio could be established that explains the various reaction yields observed.

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Coordinated Biosynthesis of the Purine Nucleoside Antibiotics Aristeromycin and Coformycin in Actinomycetes

Applied and Environmental Microbiology ◽

10.1128/aem.01860-18 ◽

2018 ◽

Vol 84 (22) ◽

Cited By ~ 5

Author(s):

Gudan Xu ◽

Liyuan Kong ◽

Rong Gong ◽

Liudong Xu ◽

Yaojie Gao ◽

...

Keyword(s):

Biological Activities ◽

Genome Mining ◽

Combinatorial Biosynthesis ◽

Purine Nucleoside ◽

Enzymatic Reactions ◽

Single Strain ◽

Content Type ◽

Precursor Pool ◽

Irreversible Reaction ◽

Chemical Structures

ABSTRACT Purine nucleoside antibiotic pairs, concomitantly produced by a single strain, are an important group of microbial natural products. Here, we report a target-directed genome mining approach to elucidate the biosynthesis of the purine nucleoside antibiotic pair aristeromycin (ARM) and coformycin (COF) in Micromonospora haikouensis DSM 45626 (a new producer for ARM and COF) and Streptomyces citricolor NBRC 13005 (a new COF producer). We also provide biochemical data that MacI and MacT function as unusual phosphorylases, catalyzing an irreversible reaction for the tailoring assembly of neplanocin A (NEP-A) and ARM. Moreover, we demonstrate that MacQ is shown to be an adenosine-specific deaminase, likely relieving the potential “excess adenosine” for producing cells. Finally, we report that MacR, an annotated IMP dehydrogenase, is actually an NADPH-dependent GMP reductase, which potentially plays a salvage role for the efficient supply of the precursor pool. Hence, these findings illustrate a fine-tuned pathway for the biosynthesis of ARM and also open the way for the rational search for purine antibiotic pairs. IMPORTANCE ARM and COF are well known for their prominent biological activities and unusual chemical structures; however, the logic of their biosynthesis has long been poorly understood. Actually, the new insights into the ARM and COF pathway will not only enrich the biochemical repertoire for interesting enzymatic reactions but may also lay a solid foundation for the combinatorial biosynthesis of this group of antibiotics via a target-directed genome mining strategy.

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Functional Characterization of Three Specific Acyl-Coenzyme A Synthetases Involved in Anaerobic Cholesterol Degradation inSterolibacterium denitrificansChol1S

Applied and Environmental Microbiology ◽

10.1128/aem.02721-17 ◽

2018 ◽

Vol 84 (7) ◽

Cited By ~ 7

Author(s):

Markus Warnke ◽

Tobias Jung ◽

Christian Jacoby ◽

Michael Agne ◽

Franziska Maria Feller ◽

...

Keyword(s):

Coenzyme A ◽

Functional Characterization ◽

Enzymatic Reaction ◽

Side Chain ◽

Enzymatic Reactions ◽

Biological Degradation ◽

Content Type ◽

Aldehyde Dehydrogenases ◽

Degrading Bacteria ◽

Acyl Coenzyme A

ABSTRACTThe denitrifying betaproteobacteriumSterolibacterium denitrificansChol1S catabolizes steroids such as cholesterol via an oxygen-independent pathway. It involves enzyme reaction sequences described for aerobic cholesterol and bile acid degradation as well as enzymes uniquely found in anaerobic steroid-degrading bacteria. Recent studies provided evidence that inS. denitrificans, the cholest-4-en-3-one intermediate is oxygen-independently oxidized to Δ4-dafachronic acid (C26-oic acid), which is subsequently activated by a substrate-specific acyl-coenzyme A (acyl-CoA) synthetase (ACS). Further degradation was suggested to proceed via unconventional β-oxidation, where aldolases, aldehyde dehydrogenases, and additional ACSs substitute for classical β-hydroxyacyl-CoA dehydrogenases and thiolases. Here, we heterologously expressed three cholesterol-induced genes that putatively code for AMP-forming ACSs and characterized two of the products as specific 3β-hydroxy-Δ5-cholenoyl-CoA (C24-oic acid)- and pregn-4-en-3-one-22-oyl-CoA (C22-oic acid)-forming ACSs, respectively. A third heterologously produced ATP-dependent ACS was inactive with C26-, C24-, or C22-oic-acids but activated 3aα-H-4α-(3′propanoate)-7aβ-methylhexahydro-1,5-indanedione (HIP) to HIP-CoA, a rather late intermediate of aerobic cholesterol degradation that still contains the CD rings of the sterane skeleton. This work provides experimental evidence that anaerobic steroid degradation proceeds via numerous alternate CoA-ester-dependent or -independent enzymatic reaction sequences as a result of aldolytic side chain and hydrolytic sterane ring C—C bond cleavages. The aldolytic side chain degradation pathway comprising highly exergonic ACSs and aldehyde dehydrogenases is considered to be essential for driving the unfavorable oxygen-independent C26hydroxylation forward.IMPORTANCEThe biological degradation of ubiquitously abundant steroids is hampered by their low solubility and the presence of two quaternary carbon atoms. The degradation of cholesterol by aerobicActinobacteriahas been studied in detail for more than 30 years and involves a number of oxygenase-dependent reactions. In contrast, much less is known about the oxygen-independent degradation of steroids in denitrifying bacteria. In the cholesterol-degrading anaerobic model organismSterolibacterium denitrificansChol1S, initial evidence has been obtained that steroid degradation proceeds via numerous alternate coenzyme A (CoA)-ester-dependent/independent reaction sequences. Here, we describe the heterologous expression of three highly specific and characteristic acyl-CoA synthetases, two of which play key roles in the degradation of the side chain, whereas a third one is specifically involved in the B ring degradation. The results obtained shed light into oxygen-independent steroid degradation comprising more than 40 enzymatic reactions.

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Structural and kinetic mapping of side-chain exposure onto the protein energy landscape

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1103629108 ◽

2011 ◽

Vol 108 (26) ◽

pp. 10532-10537 ◽

Cited By ~ 14

Author(s):

R. Bernstein ◽

K. L. Schmidt ◽

P. B. Harbury ◽

S. Marqusee

Keyword(s):

Energy Landscape ◽

Side Chain ◽

Protein Energy ◽

Protein Energy Landscape

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Analogues of the cholecystokinin octapeptide modified in the central part of the molecule

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19911963 ◽

1991 ◽

Vol 56 (9) ◽

pp. 1963-1970 ◽

Cited By ~ 3

Author(s):

Jan Hlaváček ◽

Václav Čeřovský ◽

Jana Pírková ◽

Pavel Majer ◽

Lenka Maletínská ◽

...

Keyword(s):

Amino Acid ◽

Biological Activities ◽

Point Of View ◽

Biologically Active ◽

Side Chain ◽

Amino Acid Residues ◽

Cholecystokinin Octapeptide ◽

Biological Tests ◽

Biological Potency ◽

Biologically Active Conformation

In a series of analogues of the cholecystokinin octapeptide (CCK-8) the amino acid residues were gradually modified by substituting Gly by Pro in position 4, Trp by His in position 5, Met by Cle in position 6, or the Gly residue was inserted between Tyr and Met in positions 2 and 3 of the peptide chain, and in the case of the cholecystokinin heptapeptide (CCK-7) the Met residues were substituted by Nle or Aib. These peptides were investigated from the point of view of their biological potency in the peripheral and central region. From the results of the biological tests it follows that the modifications carried out in these analogues and in their Nα-Boc derivatives mean a suppression of the investigated biological activities by 2-3 orders of magnitude (at a maximum dose of the tested substance of 2 . 10-2 mg per animal).This means that a disturbance of the assumed biologically active conformation of CCK-8, connected with a considerable decrease of the biological potency of the molecule, takes place not only after introduction of the side chain into its centre (substitution of Gly4), but also after the modification of the side chains of the amino acids or by extension of the backbone in further positions around this central amino acid.

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Endo-fucoidan hydrolases from glycoside hydrolase family 107 (GH107) display structural and mechanistic similarities to α-l-fucosidases from GH29

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.005134 ◽

2018 ◽

Vol 293 (47) ◽

pp. 18296-18308 ◽

Cited By ~ 10

Author(s):

Chelsea Vickers ◽

Feng Liu ◽

Kento Abe ◽

Orly Salama-Alber ◽

Meredith Jenkins ◽

...

Keyword(s):

Glycoside Hydrolase ◽

Biological Activities ◽

Bacterial Species ◽

Structural Data ◽

Side Chain ◽

Glycoside Hydrolase Family ◽

X Ray Crystallography ◽

Catalytic Mechanisms ◽

Thickening Agents ◽

Hydrolase Family

Fucoidans are chemically complex and highly heterogeneous sulfated marine fucans from brown macro algae. Possessing a variety of physicochemical and biological activities, fucoidans are used as gelling and thickening agents in the food industry and have anticoagulant, antiviral, antitumor, antibacterial, and immune activities. Although fucoidan-depolymerizing enzymes have been identified, the molecular basis of their activity on these chemically complex polysaccharides remains largely uninvestigated. In this study, we focused on three glycoside hydrolase family 107 (GH107) enzymes: MfFcnA and two newly identified members, P5AFcnA and P19DFcnA, from a bacterial species of the genus Psychromonas. Using carbohydrate-PAGE, we show that P5AFcnA and P19DFcnA are active on fucoidans that differ from those depolymerized by MfFcnA, revealing differential substrate specificity within the GH107 family. Using a combination of X-ray crystallography and NMR analyses, we further show that GH107 family enzymes share features of their structures and catalytic mechanisms with GH29 α-l-fucosidases. However, we found that GH107 enzymes have the distinction of utilizing a histidine side chain as the proposed acid/base catalyst in its retaining mechanism. Further interpretation of the structural data indicated that the active-site architectures within this family are highly variable, likely reflecting the specificity of GH107 enzymes for different fucoidan substructures. Together, these findings begin to illuminate the molecular details underpinning the biological processing of fucoidans.

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“Newton’s cradle” proton relay with amide–imidic acid tautomerization in inverting cellulase visualized by neutron crystallography

Science Advances ◽

10.1126/sciadv.1500263 ◽

2015 ◽

Vol 1 (7) ◽

pp. e1500263 ◽

Cited By ~ 53

Author(s):

Akihiko Nakamura ◽

Takuya Ishida ◽

Katsuhiro Kusaka ◽

Taro Yamada ◽

Shinya Fushinobu ◽

...

Keyword(s):

Glycoside Hydrolases ◽

Side Chain ◽

Enzymatic Reactions ◽

X Ray Diffraction ◽

Peptide Bonds ◽

Proton Relay ◽

Newton’S Cradle ◽

Dynamics Simulations ◽

Hydrolysis Of

Hydrolysis of carbohydrates is a major bioreaction in nature, catalyzed by glycoside hydrolases (GHs). We used neutron diffraction and high-resolution x-ray diffraction analyses to investigate the hydrogen bond network in inverting cellulase PcCel45A, which is an endoglucanase belonging to subfamily C of GH family 45, isolated from the basidiomycete Phanerochaete chrysosporium. Examination of the enzyme and enzyme-ligand structures indicates a key role of multiple tautomerizations of asparagine residues and peptide bonds, which are finally connected to the other catalytic residue via typical side-chain hydrogen bonds, in forming the “Newton’s cradle”–like proton relay pathway of the catalytic cycle. Amide–imidic acid tautomerization of asparagine has not been taken into account in recent molecular dynamics simulations of not only cellulases but also general enzyme catalysis, and it may be necessary to reconsider our interpretation of many enzymatic reactions.

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Squalene-Hopene Cyclases

Applied and Environmental Microbiology ◽

10.1128/aem.00300-11 ◽

2011 ◽

Vol 77 (12) ◽

pp. 3905-3915 ◽

Cited By ~ 78

Author(s):

Gabriele Siedenburg ◽

Dieter Jendrossek

Keyword(s):

Common Ancestor ◽

Amino Acid Sequences ◽

Enzymatic Reactions ◽

Covalent Bonds ◽

Content Type ◽

Oxidosqualene Cyclase ◽

Ring Structures ◽

One Step ◽

Alicyclobacillus Acidocaldarius ◽

Eukaryotic Organisms

ABSTRACTHopanoids and sterols are members of a large group of cyclic triterpenoic compounds that have important functions in many prokaryotic and eukaryotic organisms. They are biochemically synthesized from linear precursors (squalene, 2,3-oxidosqualene) in only one enzymatic step that is catalyzed by squalene-hopene cyclase (SHC) or oxidosqualene cyclase (OSC). SHCs and OSCs are related in amino acid sequences and probably are derived from a common ancestor. The SHC reaction requires the formation of five ring structures, 13 covalent bonds, and nine stereo centers and therefore is one of the most complex one-step enzymatic reactions. We summarize the knowledge of the properties of triterpene cyclases and details of the reaction mechanism ofAlicyclobacillus acidocaldariusSHC. Properties of other SHCs are included.

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Mechanistic Basis for Ubiquitin Modulation of a Protein Energy Landscape

Biophysical Journal ◽

10.1016/j.bpj.2020.11.974 ◽

2021 ◽

Vol 120 (3) ◽

pp. 126a

Author(s):

Emma Carroll ◽

Naomi R. Latorraca ◽

Johanna Lindner ◽

Jeff Pelton ◽

Brendan Maguire ◽

...

Keyword(s):

Energy Landscape ◽

Protein Energy ◽

Mechanistic Basis ◽

Protein Energy Landscape

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Three-dimensional computational study in a corrugated pipe inserted system filled with phase change material

International Journal of Numerical Methods for Heat &amp Fluid Flow ◽

10.1108/hff-05-2021-0336 ◽

2021 ◽

Vol ahead-of-print (ahead-of-print) ◽

Author(s):

Ömer Akbal ◽

Hakan F. Öztop ◽

Nidal H. Abu-Hamdeh

Keyword(s):

Phase Change ◽

Phase Change Material ◽

Computational Analysis ◽

Computational Study ◽

Three Dimensional ◽

Simple Algorithm ◽

Melting Time ◽

Geometrical Parameters ◽

Content Type ◽

Change Material

Purpose The purpose of this paper is to make a three-dimensional computational analysis of melting in corrugated pipe inserted system filled with phase change material (PCM). The system was heated from the inner pipe, and temperature of the outer pipe was lower than that of inner pipe. Different geometrical ratio cases and two different temperature differences were tested for their effect on melting time. Design/methodology/approach A computational analysis through a pipe with corrugated pipe filled with PCM is analyzed. Finite volume method was applied with the SIMPLE algorithm method to solve the governing equations. Findings The results indicate that the geometrical parameters can be used to control the melting time inside the heat exchanger which, in turn, affect the energy efficiency. The fastest melting time is seen in Case 4 at the same temperature difference which is the major observation of the current work. Originality/value Originality of this work is to perform a three-dimensional analysis of melting of PCM in a corrugated pipe inserted pipe.

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