Tetartohedral twinning in IDI-2 from<i>Thermus thermophilus</i>: crystallization under anaerobic conditions

Ruyck, Jérôme de; Schubert, Heidi; Janczak, Matthew W.; Poulter, C. Dale

doi:10.1107/s2053230x14002143

Cited by 3 publications

(2 citation statements)

References 12 publications

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“…Thus, the large differences between the kinetic parameters K M FMNH 2 and K i deazaFMNH 2 measured in the presence of IPP and the thermodynamic parameters K D FMNH 2 and K D deazaFMNH 2 measured in the absence of IPP indicate that IDI-2 binds flavins more tightly in the presence of the IPP substrate, perhaps as the result of an IPP-induced conformational change in the enzyme that persists after IPP (or DMAPP) has been released. Although no structures have been reported for apoIDI-2, crystal structures of IDI-2·FMN red show changes in their quaternary structure and an enhancement in resolution upon binding IPP.…”

Section: Discussionmentioning

confidence: 90%

Kinetic and Binding Studies of Streptococcus pneumoniae Type 2 Isopentenyl Diphosphate:Dimethylallyl Diphosphate Isomerase

Janczak

Poulter

2016

Biochemistry

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Type 2 isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI-2) converts isopentenyl diphosphate (IPP) to dimethylallyl diphosphate (DMAPP), the two fundamental building blocks of isoprenoid molecules. IDI-2 is found in many species of bacteria and is a potential antibacterial target since this isoform is non-homologous to the type 1 enzyme in Homo sapiens. IDI-2 requires a reduced flavin mononucleotide to form the catalytically active ternary complex, IDI-2·FMNH2·IPP. For IDI-2 from the pathogenic bacterium Streptococcus pneumoniae, the flavin can be treated kinetically as a dissociable cosubstrate in incubations with IPP and excess NADH. Under these conditions, the enzyme follows a modified sequential ordered mechanism where FMN adds before IPP. Interestingly, the enzyme shows sigmoidal behavior when incubated with IPP and NADH with varied concentrations of FMN in aerobic conditions. In contrast, sigmoidal behavior is not seen in incubations under anaerobic conditions where FMN is reduced to FMNH2 before the reaction is initiated by addition of IPP. Stopped-flow experiments revealed that FMN, whether bound to IDI-2 or without enzyme in solution, is slowly reduced in a pseudo-first-order reaction upon addition of excess NADH (k(red)(FMN) = 5.7 × 10(-3) s(-1) and k(red)(IDI-2·FMN) = 2.8 × 10(-3) s(-1)), while reduction of the flavin is rapid upon addition of NADH to a mixture of IDI-2·FMN, and IPP (k(red)(IDI-2·FMN·IPP) = 8.9 s(-1)). Similar experiments with dithionite as the reductant gave k(red)(FMN) = 221 s(-1) and k(red)(IDI-2·FMN) = 411 s(-1). Dithionite reduction of FMN in the IDI-2·FMN and IPP mixture was biphasic with k(red)(IDI-2·FMN·IPP (fast)) = 326 s(-1) and k(red)(IDI-2·FMN·IPP (slow)) = 6.9 s(-1) The pseudo-first-order rate constant for the slow component was similar to those for NADH reduction of the flavin in the IDI-2·FMN and IPP mixture and may reflect a rate-limiting conformational change in the enzyme.

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Section: Discussionmentioning

confidence: 90%

Kinetic and Binding Studies of Streptococcus pneumoniae Type 2 Isopentenyl Diphosphate:Dimethylallyl Diphosphate Isomerase

Janczak

Poulter

2016

Biochemistry

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“…These properties were attributed to a change in the conformation of the homotetramer upon binding of IPP to one of the subunits. Changes in the conformation of the homotetramer upon IPP binding were also detected by crystallography. ,, …”

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confidence: 99%

Construction of Functional Monomeric Type 2 Isopentenyl Diphosphate:Dimethylallyl Diphosphate Isomerase

2016

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Type 2 isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI-2) catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in the isoprenoid biosynthetic pathway. The enzyme from Streptomyces pneumoniae (spIDI-2) is a homotetramer in solution with behavior, including a substantial increase in the rate of FMN reduction by NADPH in the presence of IPP, suggesting that substrate binding at one subunit alters the kinetic and binding properties of another. We now report the construction of catalytically active monomeric spIDI-2. The monomeric enzyme contains a single-point mutation (N37A) and a six-residue C-terminal deletion that preserves the secondary structure of the subunits in the wild-type (wt) homotetramer. UV-vis spectra of the enzyme-bound flavin mononucleotide (FMN) cofactor in FMNox, FMNred, and FMNred·IPP/DMAPP states are the same for monomeric and wt homotetrameric spIDI-2. The mutations in monomeric IDI-2 lower the melting temperature of the protein by 20 °C and reduce the binding affinities of FMN and IDI by 40-fold but have a minimal effect on kcat. Stopped-flow kinetic studies of monomeric spIDI-2 showed that the rate of reduction of FMN by NADH (k = 1.64 × 10(-3) s(-1)) is substantially faster when IPP is added to the monomeric enzyme (k = 0.57 s(-1)), similar to behavior seen for wt-spIDI-2. Our results indicate that cooperative interactions among subunits in the wt homotetramer are not responsible for the increased rate of reduction of spIDI-2·FMN by NADH, and two possible scenarios for the enhancement are suggested.

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Identifying and Overcoming Crystal Pathologies: Disorder and Twinning

Thompson

2017

Methods in Molecular Biology

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Macromolecular crystals are prone to a number of pathologies that result from aberrant molecular packing. Two common pathologies encountered in macromolecular crystals are rigid-body disorder and twinning. When a crystal displays one of these pathologies, its diffraction pattern is altered in a way that generally complicates structure determination. The severity of the underlying abnormalities varies from case to case, and sometimes the resulting alterations to the diffraction pattern are immediately obvious, while at other times they may go entirely unnoticed. Structure determination from a crystal that suffers from disorder or twinning may or may not be possible, depending on the specific nature of the pathology, and on how the data are handled. This chapter provides an introduction to these pathologies, with an emphasis on providing guidelines for identifying and overcoming them when they pose a threat to successful structure determination.

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Tetartohedral twinning in IDI-2 fromThermus thermophilus: crystallization under anaerobic conditions

Cited by 3 publications

References 12 publications

Kinetic and Binding Studies of Streptococcus pneumoniae Type 2 Isopentenyl Diphosphate:Dimethylallyl Diphosphate Isomerase

Kinetic and Binding Studies of Streptococcus pneumoniae Type 2 Isopentenyl Diphosphate:Dimethylallyl Diphosphate Isomerase

Construction of Functional Monomeric Type 2 Isopentenyl Diphosphate:Dimethylallyl Diphosphate Isomerase

Identifying and Overcoming Crystal Pathologies: Disorder and Twinning

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