Monoclinic form of isopentenyl diphosphate isomerase: a case of polymorphism in biomolecular crystals

Type 1 isopentenyl diphosphate isomerase (IDI-1) in a new crystal form

Highlighting the factors governing transglycosylation in the GH5_5 endo-1,4-β-glucanase RBcel1

Transglycosylating glycoside hydrolases (GHs) offer great potential for the enzymatic synthesis of oligosaccharides. Although knowledge is progressing, there is no unique strategy to improve the transglycosylation yield. Obtaining efficient enzymatic tools for glycan synthesis with GHs remains dependent on an improved understanding of the molecular factors governing the balance between hydrolysis and transglycosylation. This enzymatic and structural study of RBcel1, a transglycosylase from the GH5_5 subfamily isolated from an uncultured bacterium, aims to unravel such factors. The size of the acceptor and donor sugars was found to be critical since transglycosylation is efficient with oligosaccharides at least the size of cellotetraose as the donor and cellotriose as the acceptor. The reaction pH is important in driving the balance between hydrolysis and transglycosylation: hydrolysis is favored at pH values below 8, while transglycosylation becomes the major reaction at basic pH. Solving the structures of two RBcel1 variants, RBcel1_E135Q and RBcel1_Y201F, in complex with ligands has brought to light some of the molecular factors behind transglycosylation. The structure of RBcel1_E135Q in complex with cellotriose allowed a +3 subsite to be defined, in accordance with the requirement for cellotriose as a transglycosylation acceptor. The structure of RBcel1_Y201F has been obtained with several transglycosylation intermediates, providing crystallographic evidence of transglycosylation. The catalytic cleft is filled with (i) donors ranging from cellotriose to cellohexaose in the negative subsites and (ii) cellobiose and cellotriose in the positive subsites. Such a structure is particularly relevant since it is the first structure of a GH5 enzyme in complex with transglycosylation products that has been obtained with neither of the catalytic glutamate residues modified.info:eu-repo/semantics/publishe

Glycoside hydrolase family 5: structural snapshots highlighting the involvement of two conserved residues in catalysis

The ability of retaining glycoside hydrolases (GHs) to transglycosylate is inherent to the double-displacement mechanism. Studying reaction intermediates, such as the glycosyl-enzyme intermediate (GEI) and the Michaelis complex, could provide valuable information to better understand the molecular factors governing the catalytic mechanism. Here, the GEI structure of RBcel1, an endo-1,4-β-glucanase of the GH5 family endowed with transglycosylase activity, is reported. It is the first structure of a GH5 enzyme covalently bound to a natural oligosaccharide with the two catalytic glutamate residues present. The structure of the variant RBcel1_E135A in complex with cellotriose is also reported, allowing a description of the entire binding cleft of RBcel1. Taken together, the structures deliver different snapshots of the double-displacement mechanism. The structural analysis revealed a significant movement of the nucleophilic glutamate residue during the reaction. Enzymatic assays indicated that, as expected, the acid/base glutamate residue is crucial for the glycosylation step and partly contributes to deglycosylation. Moreover, a conserved tyrosine residue in the −1 subsite, Tyr201, plays a determinant role in both the glycosylation and deglycosylation steps, since the GEI was trapped in the RBcel1_Y201F variant. The approach used to obtain the GEI presented here could easily be transposed to other retaining GHs in clan GH-A.info:eu-repo/semantics/publishe

Structure and mechanism of action of isopentenylpyrophosphate-dimethylallylpyrophosphate isomerase

We have obtained the three-dimensional X-ray crystallographic structure of a C67A mutant Escherichia coli isopentenylpyrophosphate-dimethylallylpyrophosphate isomerase (EC 5.3.3.2) complexed with the bromohydrin of isopentenylpyrophosphate, at 1.93 Å resolution. The overall backbone fold is very similar to that obtained previously for the wild-type enzyme in the presence of a divalent metal cation (Mn2+ or Mg2+). However, in the new structure, there are two metal binding sites, not just one. The first metal binding site is occupied by Mn2+, coordinated to three histidine and two glutamate residues, while the second is occupied by Mg2+, coordinated to two bromohydrin-ligand phosphate oxygens, the carbonyl oxygen of A67, a carboxyl oxygen of E87, and two water molecules. The C3 hydroxyl group of the bromohydrin inhibitor is involved in a short hydrogen bond to the carboxyl group of E116, one of the two Mn-bound glutamates. The structure obtained is consistent with a mechanism of action of the enzyme in which the carboxyl group of E116 protonates the double bond in isopentenylpyrophosphate, forming a carbocation, followed by removal of a C2 proton by the thiolate of C67, in the wild-type enzyme. The inhibition of the enzyme by a wide variety of other potent inhibitors is also readily explained on the basis of the bromohydrin inhibitor structure. Copyright © 2003 American Chemical Society.Journal ArticleResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, P.H.S.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Identification of a bifunctional enzyme MnmC involved in the biosynthesis of a hypermodified uridine in the wobble position of tRNA

The gene encoding the bifunctional enzyme MnmC that catalyzes the two last steps in the biosynthesis of 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U) in tRNA has been previously mapped at about 50 min on the Escherichia coli K12 chromosome, but to date the identity of the corresponding enzyme has not been correlated with any of the known open reading frames (ORFs). Using the protein fold-recognition approach, we predicted that the 74-kDa product of the yfcK ORF located at 52.6 min and annotated as “putative peptidase” comprises a methyltransferase domain and a FAD-dependent oxidoreductase domain. We have cloned, expressed, and purified the YfcK protein and demonstrated that it catalyzes the formation of mnm(5)s(2)U in tRNA. Thus, we suggest to rename YfcK as MnmC

The open reading frame TTC1157 of Thermus thermophilus HB27 encodes the methyltransferase forming N²-methylguanosine at position 6 in tRNA.

N(2)-methylguanosine (m(2)G) is found at position 6 in the acceptor stem of Thermus thermophilus tRNA(Phe). In this article, we describe the cloning, expression, and characterization of the T. thermophilus HB27 methyltransferase (MTase) encoded by the TTC1157 open reading frame that catalyzes the formation of this modified nucleoside. S-adenosyl-L-methionine is used as donor of the methyl group. The enzyme behaves as a monomer in solution. It contains an N-terminal THUMP domain predicted to bind RNA and contains a C-terminal Rossmann-fold methyltransferase (RFM) domain predicted to be responsible for catalysis. We propose to rename the TTC1157 gene trmN and the corresponding protein TrmN, according to the bacterial nomenclature of tRNA methyltransferases. Inactivation of the trmN gene in the T. thermophilus HB27 chromosome led to a total absence of m(2)G in tRNA but did not affect cell growth or the formation of other modified nucleosides in tRNA(Phe). Archaeal homologs of TrmN were identified and characterized. These proteins catalyze the same reaction as TrmN from T. thermophilus. Individual THUMP and RFM domains of PF1002 from Pyrococcus furiosus were produced. These separate domains were inactive and did not bind tRNA, reinforcing the idea that the THUMP domain acts in concert with the catalytic domain to target a particular position of the tRNA molecule.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Preliminary structural studies of Escherichia coli isopentenyl diphosphate isomerase

Escherichia coli isopentenyl diphosphate isomerase, an enzyme catalyzing a key step in isoprenoid biosynthesis, has been produced in selenomethionyl form. The protein was purified and crystallized by the hanging-drop vapour-diffusion method. Crystals display trigonal symmetry, with unit-cell parameters a = b = 71.3, c = 61.7 A, and diffract to 1.45 A resolution.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jFLWNAinfo:eu-repo/semantics/publishe