Structural basis for the biosynthesis of the CN ligand of [NiFe] hydrogenase

[NiFe] hydrogenases carry a NiFe(CN)2CO center at the active site, catalyzing the reversible H2oxidation. The complex NiFe center is biosynthesized and inserted into the enzyme by six specific maturation proteins: Hyp proteins (HypABCDEF). HypE and HypF are involved in biosynthesis of cyanide ligands, which are attached to the Fe atom in the NiFe center. First, HypF catalyzes a transfer reaction of the carbamoyl moiety of carbamoylphosphate to the C-terminal cysteine residue of HypE. Then, HypE catalyzes an ATP-dependent dehydration of the carbamoylated C-terminal cysteine of HypE to thiocyanate. Although structures of HypE proteins have been determined, there has been no structural evidence to explain how HypE dehydrates thiocarboxamide into thiocyanate. In order to elucidate the catalytic mechanism of HypE, we have determined the crystal structures of the carbamoylated and cyanated states of HypE from Thermococcus kodakarensis in complex with nucleotides at 1.53 Å and 1.64 Å resolution, respectively [1]. Carbamoylation of the C-terminal cysteine (Cys338) of HypE by chemical modification is clearly observed in the present structures. A conserved glutamate residue (Glu272) is close to the thiocarboxamide nitrogen atom of Cys338. However, the configuration of Glu272 is less favorable for proton abstraction. On the other hand, the thiocarboxamide oxygen atom of Cys338 interacts with a conserved lysine residue (Lys134) through a water molecule. Interestingly, a conserved arginine residue makes close contact with Lys134 and lowers the pKa of Lys134, suggesting that Lys134 functions as a proton acceptor. These observations suggest that the dehydration of thiocarboxamide into thiocyanate is catalyzed by a two-step deprotonation process, in which Lys134 and Glu272 function as the first and second bases, respectively.

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Structures of c-di-GMP/cGAMP degrading phosphodiesterase VcEAL: identification of a novel conformational switch and its implication

Biochemical Journal ◽

10.1042/bcj20190399 ◽

2019 ◽

Vol 476 (21) ◽

pp. 3333-3353 ◽

Cited By ~ 3

Author(s):

Malti Yadav ◽

Kamalendu Pal ◽

Udayaditya Sen

Keyword(s):

Active Site ◽

Metal Binding ◽

Metal Ion ◽

Triosephosphate Isomerase ◽

Catalytic Mechanism ◽

Degradation Mechanism ◽

Structural Basis ◽

Conserved Residues ◽

Phosphodiester Bond ◽

Cyclic Dinucleotides

Cyclic dinucleotides (CDNs) have emerged as the central molecules that aid bacteria to adapt and thrive in changing environmental conditions. Therefore, tight regulation of intracellular CDN concentration by counteracting the action of dinucleotide cyclases and phosphodiesterases (PDEs) is critical. Here, we demonstrate that a putative stand-alone EAL domain PDE from Vibrio cholerae (VcEAL) is capable to degrade both the second messenger c-di-GMP and hybrid 3′3′-cyclic GMP–AMP (cGAMP). To unveil their degradation mechanism, we have determined high-resolution crystal structures of VcEAL with Ca2+, c-di-GMP-Ca2+, 5′-pGpG-Ca2+ and cGAMP-Ca2+, the latter provides the first structural basis of cGAMP hydrolysis. Structural studies reveal a typical triosephosphate isomerase barrel-fold with substrate c-di-GMP/cGAMP bound in an extended conformation. Highly conserved residues specifically bind the guanine base of c-di-GMP/cGAMP in the G2 site while the semi-conserved nature of residues at the G1 site could act as a specificity determinant. Two metal ions, co-ordinated with six stubbornly conserved residues and two non-bridging scissile phosphate oxygens of c-di-GMP/cGAMP, activate a water molecule for an in-line attack on the phosphodiester bond, supporting two-metal ion-based catalytic mechanism. PDE activity and biofilm assays of several prudently designed mutants collectively demonstrate that VcEAL active site is charge and size optimized. Intriguingly, in VcEAL-5′-pGpG-Ca2+ structure, β5–α5 loop adopts a novel conformation that along with conserved E131 creates a new metal-binding site. This novel conformation along with several subtle changes in the active site designate VcEAL-5′-pGpG-Ca2+ structure quite different from other 5′-pGpG bound structures reported earlier.

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Structure based protein engineering to confer selectivity of guanine deaminase

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s205327331409562x ◽

2014 ◽

Vol 70 (a1) ◽

pp. C437-C437

Author(s):

Aruna Bitra ◽

Ruchi Anand

Keyword(s):

Crystal Structures ◽

Active Site ◽

Catalytic Mechanism ◽

Catalytic Efficiency ◽

Structural Basis ◽

Active Site Residues ◽

X Ray ◽

Secondary Activity ◽

Guanine Deaminase ◽

Differential Selectivity

Guanine deaminases (GDs) are important enzymes involved in both purine metabolism and nucleotide anabolism pathways. Here we present the molecular and catalytic mechanism of NE0047 and use the information obtained to engineer specific enzyme activities. NE0047 from Nitrosomonas europaea was found to be a high fidelity guanine deaminase (catalytic efficiency of 1.2 × 105 M–1 s–1). However; it exhibited secondary activity towards the structurally non-analogous triazine based compound ammeline. The X-ray structure of NE0047 in the presence of the substrate analogue 8-azaguanine help establish that the enzyme exists as a biological dimer and both the proper closure of the C-terminal loop and cross talk via the dimeric interface is crucial for conferring catalytic activity. It was further ascertained that the highly conserved active site residues Glu79 and Glu143 facilitate the deamination reaction by serving as proton shuttles. Moreover, to understand the structural basis of dual substrate specificity, X-ray structures of NE0047 in complex with a series of nucleobase analogs, nucleosides and substrate ammeline were determined. The crystal structures demonstrated that any substitutions in the parent substrates results in the rearrangement of the ligand in a catalytically unfavorable orientation and also impede the closure of catalytically important loop, thereby abrogating activity. However, ammeline was able to adopt a catalytically favorable orientation which, also allowed for proper loop closure. Based on the above knowledge of the crystal structures and the catalytic mechanism, the active site was subsequently engineered to fine-tune NE0047 activity. The mutated versions of the enzyme were designed so that they can function either exclusively as a GD or serve as specific ammeline deaminases. For example, mutations in the active site E143D and N66A confer the enzyme to be an unambiguous GD with no secondary activity towards ammeline. On the other hand, the N66Q mutant of NE0047 only deaminates ammeline. Additionally, a series of crystal structures of the mutant versions were solved that shed light on the structural basis of this differential selectivity.

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Structural Basis of Hydrogenotrophic Methanogenesis

Annual Review of Microbiology ◽

10.1146/annurev-micro-011720-122807 ◽

2020 ◽

Vol 74 (1) ◽

pp. 713-733

Author(s):

Seigo Shima ◽

Gangfeng Huang ◽

Tristan Wagner ◽

Ulrich Ermler

Keyword(s):

Transfer Reaction ◽

Catalytic Mechanism ◽

Methanogenic Archaea ◽

Energy Coupling ◽

Structural Basis ◽

Biomass Degradation ◽

Hydrogenotrophic Methanogenesis ◽

Sufficient Energy ◽

Ion Gradient ◽

Electron Reduction

Most methanogenic archaea use the rudimentary hydrogenotrophic pathway—from CO2 and H2 to methane—as the terminal step of microbial biomass degradation in anoxic habitats. The barely exergonic process that just conserves sufficient energy for a modest lifestyle involves chemically challenging reactions catalyzed by complex enzyme machineries with unique metal-containing cofactors. The basic strategy of the methanogenic energy metabolism is to covalently bind C1 species to the C1 carriers methanofuran, tetrahydromethanopterin, and coenzyme M at different oxidation states. The four reduction reactions from CO2 to methane involve one molybdopterin-based two-electron reduction, two coenzyme F420–based hydride transfers, and one coenzyme F430–based radical process. For energy conservation, one ion-gradient-forming methyl transfer reaction is sufficient, albeit supported by a sophisticated energy-coupling process termed flavin-based electron bifurcation for driving the endergonic CO2 reduction and fixation. Here, we review the knowledge about the structure-based catalytic mechanism of each enzyme of hydrogenotrophic methanogenesis.

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A glutamate/aspartate switch controls product specificity in a protein arginine methyltransferase

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1525783113 ◽

2016 ◽

Vol 113 (8) ◽

pp. 2068-2073 ◽

Cited By ~ 32

Author(s):

Erik W. Debler ◽

Kanishk Jain ◽

Rebeccah A. Warmack ◽

You Feng ◽

Steven G. Clarke ◽

...

Keyword(s):

Active Site ◽

Catalytic Mechanism ◽

Structural Basis ◽

Titration Calorimetry ◽

Type I ◽

Histone H4 ◽

Active Site Residues ◽

Protein Arginine Methyltransferase ◽

Arginine Methyltransferase ◽

Product Specificity

Trypanosoma brucei PRMT7 (TbPRMT7) is a protein arginine methyltransferase (PRMT) that strictly monomethylates various substrates, thus classifying it as a type III PRMT. However, the molecular basis of its unique product specificity has remained elusive. Here, we present the structure of TbPRMT7 in complex with its cofactor product S-adenosyl-l-homocysteine (AdoHcy) at 2.8 Å resolution and identify a glutamate residue critical for its monomethylation behavior. TbPRMT7 comprises the conserved methyltransferase and β-barrel domains, an N-terminal extension, and a dimerization arm. The active site at the interface of the N-terminal extension, methyltransferase, and β-barrel domains is stabilized by the dimerization arm of the neighboring protomer, providing a structural basis for dimerization as a prerequisite for catalytic activity. Mutagenesis of active-site residues highlights the importance of Glu181, the second of the two invariant glutamate residues of the double E loop that coordinate the target arginine in substrate peptides/proteins and that increase its nucleophilicity. Strikingly, mutation of Glu181 to aspartate converts TbPRMT7 into a type I PRMT, producing asymmetric dimethylarginine (ADMA). Isothermal titration calorimetry (ITC) using a histone H4 peptide showed that the Glu181Asp mutant has markedly increased affinity for monomethylated peptide with respect to the WT, suggesting that the enlarged active site can favorably accommodate monomethylated peptide and provide sufficient space for ADMA formation. In conclusion, these findings yield valuable insights into the product specificity and the catalytic mechanism of protein arginine methyltransferases and have important implications for the rational (re)design of PRMTs.

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Structural basis of [NiFe] hydrogenase maturation by Hyp proteins

Biological Chemistry ◽

10.1515/hsz-2012-0197 ◽

2012 ◽

Vol 393 (10) ◽

pp. 1089-1100 ◽

Cited By ~ 25

Author(s):

Satoshi Watanabe ◽

Daisuke Sasaki ◽

Taiga Tominaga ◽

Kunio Miki

Keyword(s):

Hydrogen Production ◽

Dynamic Process ◽

Active Site ◽

The Other ◽

Structural Basis ◽

Structural Studies ◽

Hydrogenase Maturation ◽

Recent Advances

Abstract [NiFe] hydrogenases catalyze reversible hydrogen production/consumption. The active site of [NiFe] hydrogenases contains a complex NiFe(CN)2CO center, and the biosynthesis/maturation of these enzymes is a complex and dynamic process, primarily involving six Hyp proteins (HypABCDEF). HypA and HypB are involved in the Ni insertion, whereas the other four Hyp proteins (HypCDEF) are required for the biosynthesis, assembly and insertion of the Fe(CN)2CO group. Over the last decades, a large number of functional and structural studies on maturation proteins have been performed, revealing detailed functions of each Hyp protein and the framework of the maturation pathway. This article will focus on recent advances in structural studies of the Hyp proteins and on mechanistic insights into the [NiFe] hydrogenase maturation.

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Insight into the Function of Active Site Residues in the Catalytic Mechanism of Human Ferrochelatase

Biochemical Journal ◽

10.1042/bcj20210460 ◽

2021 ◽

Author(s):

Amy E. Medlock ◽

Wided Najahi-Missaoui ◽

Mesafint T. Shiferaw ◽

Angela N. Albetel ◽

William N. Lanzilotta ◽

...

Keyword(s):

Active Site ◽

Metal Binding ◽

Metal Ion ◽

Catalytic Mechanism ◽

Structural Data ◽

Active Site Residues ◽

Proton Abstraction ◽

Product Release ◽

Iron Delivery ◽

High Level

Ferrochelatase catalyzes the insertion of ferrous iron into a porphyrin macrocycle to produce the essential cofactor, heme. In humans this enzyme not only catalyzes the terminal step, but also serves a regulatory step in the heme synthesis pathway. Over a dozen crystal structures of human ferrochelatase have been solved and many variants have been characterized kinetically. In addition, hydrogen deuterium exchange, resonance Raman, molecular dynamics, and high level quantum mechanic studies have added to our understanding of the catalytic cycle of the enzyme. However, an understanding of how the metal ion is delivered and the specific role that active site residues play in catalysis remain open questions. Data are consistent with metal binding and insertion occurring from the side opposite from where pyrrole proton abstraction takes place. To better understand iron delivery and binding as well as the role of conserved residues in the active site, we have constructed and characterized a series of enzyme variants. Crystallographic studies as well as rescue and kinetic analysis of variants were performed. Data from these studies are consistent with the M76 residue playing a role in active site metal binding and formation of a weak iron protein ligand being necessary for product release. Additionally, structural data support a role for E343 in proton abstraction and product release in coordination with a peptide loop composed of Q302, S303 and K304 that act a metal sensor.

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Covalent reaction of cerulenin at the active site of acyl-CoA reductase of Photobacterium phosphoreum

Biochemistry and Cell Biology ◽

10.1139/o89-024 ◽

1989 ◽

Vol 67 (2-3) ◽

pp. 163-167 ◽

Cited By ~ 7

Author(s):

Lee Wall ◽

Edward Meighen

Keyword(s):

Active Site ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Cysteine Residue ◽

The Other ◽

Photobacterium Phosphoreum ◽

Acyl Transferase ◽

Coa Reductase ◽

Myristoyl Coa

Inhibition of bioluminescence in Photobacterium phosphoreum by cerulenin has been demonstrated to be due to a specific inactivation of the acyl-CoA reductase subunit of the fatty acid reductase complex required for synthesis of the aldehyde substrate for the luminescent reaction. In contrast, the activities of the other luminescence-related enzymes, acyl-protein synthetase, acyl-transferase, and luciferase, were unaffected by cerulenin. Myristoyl-CoA, but not NADPH, protected the acyl-CoA reductase against cerulenin inhibition. Cerulenin blocked the acylation of the reductase with myristoyl-CoA and the reaction with N-ethylmaleimide. A shift in mobility of the reductase polypeptide on sodium dodecyl sulfate – polyacrylamide gel electrophoresis occurred after reaction with cerulenin, a shift which could be blocked by reaction with N-ethylmaleimide. These results demonstrate that cerulenin blocks aldehyde synthesis by covalent reaction with the acyl-CoA reductase and indicate that the reaction may occur at a cysteine residue involved in the formation of the acyl–reductase intermediate.Key words: bioluminescence, cerulenin, acyl-CoA reductase.

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Structure and possible mechanism of the CcbJ methyltransferase fromStreptomyces caelestis

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s139900471303397x ◽

2014 ◽

Vol 70 (4) ◽

pp. 943-957 ◽

Cited By ~ 9

Author(s):

Jacob Bauer ◽

Gabriela Ondrovičová ◽

Lucie Najmanová ◽

Vladimír Pevala ◽

Zdeněk Kameník ◽

...

Keyword(s):

Active Site ◽

Transfer Reaction ◽

Docking Studies ◽

The Other ◽

Class I ◽

Wild Type ◽

Native Form ◽

Docking Simulations ◽

Methyl Transfer ◽

Mad Phasing

TheS-adenosyl-L-methionine (SAM)-dependent methyltransferase CcbJ fromStreptomyces caelestiscatalyzes one of the final steps in the biosynthesis of the antibiotic celesticetin, methylation of the N atom of its proline moiety, which greatly enhances the activity of the antibiotic. Since several celesticetin variants exist, this enzyme may be able to act on a variety of substrates. The structures of CcbJ determined by MAD phasing at 3.0 Å resolution, its native form at 2.7 Å resolution and its complex withS-adenosyl-L-homocysteine (SAH) at 2.9 Å resolution are reported here. Based on these structures, three point mutants, Y9F, Y17F and F117G, were prepared in order to study its behaviour as well as docking simulations of both CcbJ–SAM–substrate and CcbJ–SAH–product complexes. The structures show that CcbJ is a class I SAM-dependent methyltransferase with a wide active site, thereby suggesting that it may accommodate a number of different substrates. The mutation results show that the Y9F and F117G mutants are almost non-functional, while the Y17F mutant has almost half of the wild-type activity. In combination with the docking studies, these results suggest that Tyr9 and Phe117 are likely to help to position the substrate for the methyl-transfer reaction and that Tyr9 may also facilitate the reaction by removing an H+ion. Tyr17, on the other hand, seems to operate by helping to stabilize the SAM cofactor.

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Structural Basis of Specific Glucoimidazole and Mannoimidazole Binding by Os3BGlu7

Biomolecules ◽

10.3390/biom10060907 ◽

2020 ◽

Vol 10 (6) ◽

pp. 907

Author(s):

Bodee Nutho ◽

Salila Pengthaisong ◽

Anupong Tankrathok ◽

Vannajan Sanghiran Lee ◽

James R. Ketudat Cairns ◽

...

Keyword(s):

Molecular Dynamics ◽

Binding Energy ◽

Active Site ◽

The Other ◽

Strong Preference ◽

Structural Basis ◽

Active Site Residue ◽

Weakly Bound ◽

Sugar Moiety ◽

Dynamics Simulations

β-Glucosidases and β-mannosidases hydrolyze substrates that differ only in the epimer of the nonreducing terminal sugar moiety, but most such enzymes show a strong preference for one activity or the other. Rice Os3BGlu7 and Os7BGlu26 β-glycosidases show a less strong preference, but Os3BGlu7 and Os7BGlu26 prefer glucosides and mannosides, respectively. Previous studies of crystal structures with glucoimidazole (GIm) and mannoimidazole (MIm) complexes and metadynamic simulations suggested that Os7BGlu26 hydrolyzes mannosides via the B2,5 transition state (TS) conformation preferred for mannosides and glucosides via their preferred 4H3/4E TS conformation. However, MIm is weakly bound by both enzymes. In the present study, we found that MIm was not bound in the active site of crystallized Os3BGlu7, but GIm was tightly bound in the −1 subsite in a 4H3/4E conformation via hydrogen bonds with the surrounding residues. One-microsecond molecular dynamics simulations showed that GIm was stably bound in the Os3BGlu7 active site and the glycone-binding site with little distortion. In contrast, MIm initialized in the B2,5 conformation rapidly relaxed to a E3/4H3 conformation and moved out into a position in the entrance of the active site, where it bound more stably despite making fewer interactions. The lack of MIm binding in the glycone site in protein crystals and simulations implies that the energy required to distort MIm to the B2,5 conformation for optimal active site residue interactions is sufficient to offset the energy of those interactions in Os3BGlu7. This balance between distortion and binding energy may also provide a rationale for glucosidase versus mannosidase specificity in plant β-glycosidases.

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Reaction of threonine synthase with the substrate analogue 2-amino-5-phosphonopentanoate: implications into the proton transfer at the active site

The Journal of Biochemistry ◽

10.1093/jb/mvz100 ◽

2019 ◽

Vol 167 (4) ◽

pp. 357-364

Author(s):

Yasuhiro Machida ◽

Takeshi Murakawa ◽

Akiko Sakai ◽

Mitsuo Shoji ◽

Yasuteru Shigeta ◽

...

Keyword(s):

Catalytic Reaction ◽

Active Site ◽

Catalytic Mechanism ◽

The Other ◽

Amino Group ◽

Threonine Synthase ◽

Spectral Changes ◽

Proton Migration ◽

Insight Into

Abstract Threonine synthase catalyses the conversion of O-phospho-l-homoserine and a water molecule to l-threonine and has the most complex catalytic mechanism among the pyridoxal 5′-phosphate-dependent enzymes. In order to study the less-characterized earlier stage of the catalytic reaction, we studied the reaction of threonine synthase with 2-amino-5-phosphonopentanoate, which stops the catalytic reaction at the enamine intermediate. The global kinetic analysis of the triphasic spectral changes showed that, in addition to the theoretically expected pathway, the carbanion is rapidly reprotonated at Cα to form an aldimine distinct from the external aldimine directly formed from the Michaelis complex. The Kd for the binding of inhibitor to the enzyme decreased with increasing pH, showing that the 2-amino-group-unprotonated form of the ligand binds to the enzyme. On the other hand, the rate constants for the proton migration steps within the active site are independent of the solvent pH, indicating that protons are shared by the active dissociative groups and are not exchanged with the solvent during the course of catalysis. This gives an insight into the role of the phosphate group of the substrate, which may increase the basicity of the ε-amino group of the catalytic lysine residue in the active site.

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