Crystal structure of bacterial cyclopropane-fatty-acyl-phospholipid synthase with phospholipid

2019 ◽  
Vol 166 (2) ◽  
pp. 139-147 ◽  
Author(s):  
Yulong Ma ◽  
Chunli Pan ◽  
Qihai Wang
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Cyclopropane Ring ◽  
Fatty Acyl ◽  
Structural Basis ◽  
Cyclopropane Fatty Acid ◽  
Adverse Conditions ◽  
General Base ◽  
Acyl Chains ◽  
Methylene Group

AbstractThe lipids containing cyclopropane-fatty-acid (CFA) protect bacteria from adverse conditions such as acidity, freeze-drying desiccation and exposure to pollutants. CFA is synthesized when cyclopropane-fatty-acyl-phospholipid synthase (CFA synthase, CFAS) transfers a methylene group from S-adenosylmethionine (SAM) across the cis double bonds of unsaturated fatty acyl chains. Here, we reported a 2.7-Å crystal structure of CFAS from Lactobacillus acidophilus. The enzyme is composed of N- and C-terminal domain, which belong to the sterol carrier protein and methyltransferase superfamily, respectively. A phospholipid in the substrate binding site and a bicarbonate ion (BCI) acting as a general base in the active site were discovered. To elucidate the mechanism, a docking experiment using CFAS from L. acidophilus and SAM was carried out. The analysis of this structure demonstrated that three groups, the carbons from the substrate, the BCI and the methyl of S(CHn)3 group, were close enough to form a cyclopropane ring with the help of amino acids in the active site. Therefore, the structure supports the hypothesis that CFAS from L. acidophilus catalyzes methyl transfer via a carbocation mechanism. These findings provide a structural basis to more deeply understand enzymatic cyclopropanation.

The crystal structure of human mitochondrial 3-ketoacyl-CoA thiolase (T1): insight into the reaction mechanism of its thiolase and thioesterase activities

2014 ◽  
Vol 70 (12) ◽  
pp. 3212-3225 ◽  
Author(s):  
Tiila-Riikka Kiema ◽  
Rajesh K. Harijan ◽  
Malgorzata Strozyk ◽  
Toshiyuki Fukao ◽  
Stefan E. H. Alexson ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Reaction Mechanism ◽  
Active Site ◽  
Quaternary Structure ◽  
Fatty Acyl ◽  
Physiological Importance ◽  
Loop Domain ◽  
Solution Studies ◽  
Hydrolysis Of ◽  
Insight Into

Crystal structures of human mitochondrial 3-ketoacyl-CoA thiolase (hT1) in the apo form and in complex with CoA have been determined at 2.0 Å resolution. The structures confirm the tetrameric quaternary structure of this degradative thiolase. The active site is surprisingly similar to the active site of theZoogloea ramigerabiosynthetic tetrameric thiolase (PDB entries 1dm3 and 1m1o) and different from the active site of the peroxisomal dimeric degradative thiolase (PDB entries 1afw and 2iik). A cavity analysis suggests a mode of binding for the fatty-acyl tail in a tunnel lined by the Nβ2–Nα2 loop of the adjacent subunit and the Lα1 helix of the loop domain. Soaking of the apo hT1 crystals with octanoyl-CoA resulted in a crystal structure in complex with CoA owing to the intrinsic acyl-CoA thioesterase activity of hT1. Solution studies confirm that hT1 has low acyl-CoA thioesterase activity for fatty acyl-CoA substrates. The fastest rate is observed for the hydrolysis of butyryl-CoA. It is also shown that T1 has significant biosynthetic thiolase activity, which is predicted to be of physiological importance.

scholarly journals Structural basis for UFM1 transfer from UBA5 to UFC1

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Manoj Kumar ◽  
Prasanth Padala ◽  
Jamal Fahoum ◽  
Fouad Hassouna ◽  
Tomer Tsaban ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Structural Basis ◽  
Adenylation Domain ◽  
Post Translational Modification ◽  
Target Proteins ◽  
Cellular Processes ◽  
Enzyme Cascade ◽  
Linear Sequence ◽  
C Terminus

AbstractUfmylation is a post-translational modification essential for regulating key cellular processes. A three-enzyme cascade involving E1, E2 and E3 is required for UFM1 attachment to target proteins. How UBA5 (E1) and UFC1 (E2) cooperatively activate and transfer UFM1 is still unclear. Here, we present the crystal structure of UFC1 bound to the C-terminus of UBA5, revealing how UBA5 interacts with UFC1 via a short linear sequence, not observed in other E1-E2 complexes. We find that UBA5 has a region outside the adenylation domain that is dispensable for UFC1 binding but critical for UFM1 transfer. This region moves next to UFC1’s active site Cys and compensates for a missing loop in UFC1, which exists in other E2s and is needed for the transfer. Overall, our findings advance the understanding of UFM1’s conjugation machinery and may serve as a basis for the development of ufmylation inhibitors.

scholarly journals High-Resolution Crystal Structure of the Subclass B3 Metallo-β-Lactamase BJP-1: Rational Basis for Substrate Specificity and Interaction with Sulfonamides

2010 ◽  
Vol 54 (10) ◽  
pp. 4343-4351 ◽  
Author(s):  
Jean-Denis Docquier ◽  
Manuela Benvenuti ◽  
Vito Calderone ◽  
Magdalena Stoczko ◽  
Nicola Menciassi ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Bradyrhizobium Japonicum ◽  
Active Sites ◽  
Structural Diversity ◽  
Binding Pocket ◽  
Structural Basis ◽  
Side Chain ◽  
Functional Heterogeneity ◽  
Hydrophobic Binding

ABSTRACT Metallo-β-lactamases (MBLs) are important enzymatic factors in resistance to β-lactam antibiotics that show important structural and functional heterogeneity. BJP-1 is a subclass B3 MBL determinant produced by Bradyrhizobium japonicum that exhibits interesting properties. BJP-1, like CAU-1 of Caulobacter vibrioides, overall poorly recognizes β-lactam substrates and shows an unusual substrate profile compared to other MBLs. In order to understand the structural basis of these properties, the crystal structure of BJP-1 was obtained at 1.4-Å resolution. This revealed significant differences in the conformation and locations of the active-site loops, determining a rather narrow active site and the presence of a unique N-terminal helix bearing Phe-31, whose side chain binds in the active site and represents an obstacle for β-lactam substrate binding. In order to probe the potential of sulfonamides (known to inhibit various zinc-dependent enzymes) to bind in the active sites of MBLs, the structure of BJP-1 in complex with 4-nitrobenzenesulfonamide was also obtained (at 1.33-Å resolution), thereby revealing the mode of interaction of these molecules in MBLs. Interestingly, sulfonamide binding resulted in the displacement of the side chain of Phe-31 from its hydrophobic binding pocket, where the benzene ring of the molecule is now found. These data further highlight the structural diversity shown by MBLs but also provide interesting insights in the structure-function relationships of these enzymes. More importantly, we provided the first structural observation of MBL interaction with sulfonamides, which might represent an interesting scaffold for the design of MBL inhibitors.

scholarly journals Structural basis for regiospecific midazolam oxidation by human cytochrome P450 3A4

2016 ◽  
Vol 114 (3) ◽  
pp. 486-491 ◽  
Author(s):  
Irina F. Sevrioukova ◽  
Thomas L. Poulos
Keyword(s):  
Crystal Structure ◽  
Cytochrome P450 ◽  
Active Site ◽  
Structural Information ◽  
Binding Mode ◽  
Structural Basis ◽  
Cytochrome P450 3A4 ◽  
Human Cytochrome ◽  
Cyp3a4 Substrate

Human cytochrome P450 3A4 (CYP3A4) is a major hepatic and intestinal enzyme that oxidizes more than 60% of administered therapeutics. Knowledge of how CYP3A4 adjusts and reshapes the active site to regioselectively oxidize chemically diverse compounds is critical for better understanding structure–function relations in this important enzyme, improving the outcomes for drug metabolism predictions, and developing pharmaceuticals that have a decreased ability to undergo metabolism and cause detrimental drug–drug interactions. However, there is very limited structural information on CYP3A4–substrate interactions available to date. Despite the vast variety of drugs undergoing metabolism, only the sedative midazolam (MDZ) serves as a marker substrate for the in vivo activity assessment because it is preferentially and regioselectively oxidized by CYP3A4. We solved the 2.7 Å crystal structure of the CYP3A4–MDZ complex, where the drug is well defined and oriented suitably for hydroxylation of the C1 atom, the major site of metabolism. This binding mode requires H-bonding to Ser119 and a dramatic conformational switch in the F–G fragment, which transmits to the adjacent D, E, H, and I helices, resulting in a collapse of the active site cavity and MDZ immobilization. In addition to providing insights on the substrate-triggered active site reshaping (an induced fit), the crystal structure explains the accumulated experimental results, identifies possible effector binding sites, and suggests why MDZ is predominantly metabolized by the CYP3A enzyme subfamily.

scholarly journals Structural basis for the function and regulation of the receptor protein tyrosine phosphatase CD45

2005 ◽  
Vol 201 (3) ◽  
pp. 441-452 ◽  
Author(s):  
Hyun-Joo Nam ◽  
Florence Poy ◽  
Haruo Saito ◽  
Christin A. Frederick
Keyword(s):  
Crystal Structure ◽  
Protein Tyrosine Phosphatase ◽  
Active Site ◽  
Active Sites ◽  
Hydrophobic Interactions ◽  
Tyrosine Phosphatase ◽  
Receptor Protein ◽  
Structural Basis ◽  
Salt Bridges ◽  
Protein Tyrosine

CD45 is the prototypic member of transmembrane receptor-like protein tyrosine phosphatases (RPTPs) and has essential roles in immune functions. The cytoplasmic region of CD45, like many other RPTPs, contains two homologous protein tyrosine phosphatase domains, active domain 1 (D1) and catalytically impaired domain 2 (D2). Here, we report crystal structure of the cytoplasmic D1D2 segment of human CD45 in native and phosphotyrosyl peptide-bound forms. The tertiary structures of D1 and D2 are very similar, but doubly phosphorylated CD3ζ immunoreceptor tyrosine-based activation motif peptide binds only the D1 active site. The D2 “active site” deviates from the other active sites significantly to the extent that excludes any possibility of catalytic activity. The relative orientation of D1 and D2 is very similar to that observed in leukocyte common antigen–related protein with both active sites in an open conformation and is restrained through an extensive network of hydrophobic interactions, hydrogen bonds, and salt bridges. This crystal structure is incompatible with the wedge model previously suggested for CD45 regulation.

scholarly journals Crystal Structure of Bacterial Cystathionine Γ-Lyase in The Cysteine Biosynthesis Pathway of Staphylococcus aureus

Crystals ◽  
2019 ◽  
Vol 9 (12) ◽  
pp. 656 ◽  
Author(s):  
Dukwon Lee ◽  
Soyeon Jeong ◽  
Jinsook Ahn ◽  
Nam-Chul Ha ◽  
Ae-Ran Kwon
Keyword(s):  
Crystal Structure ◽  
Staphylococcus Aureus ◽  
Active Site ◽  
Structural Changes ◽  
Enzymatic Reactions ◽  
Biosynthesis Pathway ◽  
Active Site Residues ◽  
Structural Comparison ◽  
General Base ◽  
Bond Network

Many enzymes require pyridoxal 5’-phosphate (PLP) as an essential cofactor and share active site residues in mediating diverse enzymatic reactions. Methionine can be converted into cysteine by cystathionine γ-lyases (CGLs) through a transsulfuration reaction dependent on PLP. In bacteria, MccB, also known as YhrB, exhibits CGL activity that cleaves the C–S bond of cystathionine at the γ position. In this study, we determined the crystal structure of MccB from Staphylococcus aureus in its apo- and PLP-bound forms. The structures of MccB exhibited similar molecular arrangements to those of MetC-mediating β-elimination with the same substrate and further illustrated PLP-induced structural changes in MccB. A structural comparison to MetC revealed a longer distance between the N-1 atom of the pyridine ring of PLP and the Oδ atom of the Asp residue, as well as a wider and more flexible active site environment in MccB. We also found a hydrogen bond network in Ser-water-Ser-Glu near the Schiff base nitrogen atom of the PLP molecule and propose the Ser-water-Ser-Glu motif as a general base for the γ-elimination process. Our study suggests the molecular mechanism for how homologous enzymes that use PLP as a cofactor catalyze different reactions with the same active site residues.

scholarly journals Mechanistic and structural basis for inhibition of thymidylate synthase ThyX

Open Biology ◽  
2012 ◽  
Vol 2 (10) ◽  
pp. 120120 ◽  
Author(s):  
Tamara Basta ◽  
Yap Boum ◽  
Julien Briffotaux ◽  
Hubert F. Becker ◽  
Isabelle Lamarre-Jouenne ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Thymidylate Synthase ◽  
Active Site ◽  
De Novo ◽  
Tight Binding ◽  
Binding Pocket ◽  
Structural Basis ◽  
Microbial Genomes ◽  
Thymidylate Synthases ◽  
Thymidylate Synthesis

Nature has established two mechanistically and structurally unrelated families of thymidylate synthases that produce de novo thymidylate or dTMP, an essential DNA precursor. Representatives of the alternative flavin-dependent thymidylate synthase family, ThyX, are found in a large number of microbial genomes, but are absent in humans. We have exploited the nucleotide binding pocket of ThyX proteins to identify non-substrate-based tight-binding ThyX inhibitors that inhibited growth of genetically modified Escherichia coli cells dependent on thyX in a manner mimicking a genetic knockout of thymidylate synthase. We also solved the crystal structure of a viral ThyX bound to 2-hydroxy-3-(4-methoxybenzyl)-1,4-naphthoquinone at a resolution of 2.6 Å. This inhibitor was found to bind within the conserved active site of the tetrameric ThyX enzyme, at the interface of two monomers, partially overlapping with the dUMP binding pocket. Our studies provide new chemical tools for investigating the ThyX reaction mechanism and establish a novel mechanistic and structural basis for inhibition of thymidylate synthesis. As essential ThyX proteins are found e.g. in Mycobacterium tuberculosis and Helicobacter pylori , our studies have also potential to pave the way towards the development of new anti-microbial compounds.

scholarly journals Crystal Structure of the Mycobacterium fortuitum Class A β-Lactamase: Structural Basis for Broad Substrate Specificity

2006 ◽  
Vol 50 (7) ◽  
pp. 2516-2521 ◽  
Author(s):  
Eric Sauvage ◽  
Eveline Fonzé ◽  
Birgit Quinting ◽  
Moreno Galleni ◽  
Jean-Marie Frère ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Broad Spectrum ◽  
Active Site ◽  
Bacterial Resistance ◽  
Specific Activity ◽  
Structural Basis ◽  
Mycobacterium Fortuitum ◽  
Bacterial Strains ◽  
Class A ◽  
Extended Spectrum

ABSTRACT β-Lactamases are the main cause of bacterial resistance to penicillins and cephalosporins. Class A β-lactamases, the largest group of β-lactamases, have been found in many bacterial strains, including mycobacteria, for which no β-lactamase structure has been previously reported. The crystal structure of the class A β-lactamase from Mycobacterium fortuitum (MFO) has been solved at 2.13-Å resolution. The enzyme is a chromosomally encoded broad-spectrum β-lactamase with low specific activity on cefotaxime. Specific features of the active site of the class A β-lactamase from M. fortuitum are consistent with its specificity profile. Arg278 and Ser237 favor cephalosporinase activity and could explain its broad substrate activity. The MFO active site presents similarities with the CTX-M type extended-spectrum β-lactamases but lacks a specific feature of these enzymes, the VNYN motif (residues 103 to 106), which confers on CTX-M-type extended-spectrum β-lactamases a more efficient cefotaximase activity.

scholarly journals Crystal Structure of Butyrate Kinase 2 from Thermotoga maritima, a Member of the ASKHA Superfamily of Phosphotransferases

2009 ◽  
Vol 191 (8) ◽  
pp. 2521-2529 ◽  
Author(s):  
Jiasheng Diao ◽  
Miriam S. Hasson
Keyword(s):  
Crystal Structure ◽  
Substrate Specificity ◽  
Active Site ◽  
Thermotoga Maritima ◽  
Structural Basis ◽  
Acetate Kinase ◽  
Phosphoryl Transfer ◽  
Atp Binding ◽  
Binding Motif ◽  
Butyrate Kinase

ABSTRACT The enzymatic transfer of phosphoryl groups is central to the control of many cellular processes. One of the phosphoryl transfer mechanisms, that of acetate kinase, is not completely understood. Besides better understanding of the mechanism of acetate kinase, knowledge of the structure of butyrate kinase 2 (Buk2) will aid in the interpretation of active-site structure and provide information on the structural basis of substrate specificity. The gene buk2 from Thermotoga maritima encodes a member of the ASKHA (acetate and sugar kinases/heat shock cognate/actin) superfamily of phosphotransferases. The encoded protein Buk2 catalyzes the phosphorylation of butyrate and isobutyrate. We have determined the 2.5-Å crystal structure of Buk2 complexed with (β,γ-methylene) adenosine 5′-triphosphate. Buk2 folds like an open-shelled clam, with each of the two domains representing one of the two shells. In the open active-site cleft between the N- and C-terminal domains, the active-site residues consist of two histidines, two arginines, and a cluster of hydrophobic residues. The ATP binding region of Buk2 in the C-terminal domain consists of abundant glycines for nucleotide binding, and the ATP binding motif is similar to those of other members of the ASKHA superfamily. The enzyme exists as an octamer, in which four disulfide bonds form between intermolecular cysteines. Sequence alignment and structure superposition identify the simplicity of the monomeric Buk2 structure, a probable substrate binding site, the key residues in catalyzing phosphoryl transfer, and the substrate specificity differences among Buk2, acetate, and propionate kinases. The possible enzyme mechanisms are discussed.

The biosynthesis of cyclopropane fatty acids. I. Feeding experiments with oleic acid-9,10-d2, oleic acid-8,8,11,11-d4, and L-methionine-methyl-d3

1981 ◽  
Vol 59 (5) ◽  
pp. 828-838 ◽  
Author(s):  
Peter H. Buist ◽  
David B. Maclean
Keyword(s):  
Oleic Acid ◽  
Fatty Acid ◽  
Double Bond ◽  
Cyclopropane Ring ◽  
Octadecenoic Acid ◽  
Cyclopropane Fatty Acid ◽  
Feeding Experiments ◽  
Cyclopropane Fatty Acids ◽  
Methylene Unit ◽  
Methylene Group

cis-9-Octadecenoic acid-9,10-d2 and cis-9-octadecenoic acid-8,8,11,11-d4 and L-methionine-methyl-d3 were administered to cultures of Lactobacillus plantarum and converted by the organism to the corresponding cyclopropyl compounds. The first experiment showed that no scrambling or loss of label occurred when a methylene unit was added across the double bond of the labelled substrate. In the second experiment, again, no loss or scrambling of label occurred, a result which ruled out any mechanism for biological cyclopropanation involving allylic activation of the double bond. The third experiment yielded a biosynthetic cyclopropane fatty acid containing deuterium located exclusively at the methylene group of the cyclopropane ring. Up to 17% of a d1-species accompanied the major dideuterated compound. The lone deuterium in the d1-cyclopropane fatty acid occupies both positions of the methylene group to the same extent. It has been shown that the extent to which d1-cyclopropane fatty acid is produced increases with cell growth. The extent of exchange was similar in the cyclopropanation of both the 9,10-and 11,12-isomers of cis-octadecenoic acid.

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