IspH protein of Escherichia coli: studies on iron-sulfur cluster implementation and catalysis.

The ispH gene of Escherichia coli specifies an enzyme catalyzing the conversion of 1-hydroxy-2-methyl-2-(E)-butenyl diphosphate into a mixture of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in the nonmevalonate isoprenoid biosynthesis pathway. The implementation of a gene cassette directing the overexpression of the isc operon involved in the assembly of iron-sulfur clusters into an Escherichia coli strain engineered for ispH gene expression increased the catalytic activity of IspH protein anaerobically purified from this strain by a factor of at least 200. For maximum catalytic activity, flavodoxin and flavodoxin reductase were required in molar concentrations of 40 and 12 microM, respectively. EPR experiments as well as optical absorbance indicate the presence of a [3Fe-4S](+) cluster in IspH protein. Among 4 cysteines in total, the 36 kDa protein carries 3 absolutely conserved cysteine residues at the amino acid positions 12, 96, and 197. Replacement of any of the conserved cysteine residues reduced the catalytic activity by a factor of more than 70 000.

Stereochemical studies on the making and unmaking of isopentenyl diphosphate in different biological systems.

To investigate the unknown stereochemical course of the reaction catalyzed by the type-II isomerase, which interconverts isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), a sample of [1,2-(13)C2]-IPP stereospecifically labelled with 2H at C2 was prepared by incubating a D2O solution of (E)-4-hydroxy-3-methyl[1,2-(13)C2]but-2-enyl diphosphate with a recombinant IspH protein of Escherichia coli in the presence of NADH as a reducing agent and flavodoxin as well as flavodoxin reductase as auxiliary proteins. As monitored by 13C-NMR spectroscopy, treatment of the deuterated IPP with either type-I or type-II IPP isomerase resulted in the formation of DMAPP molecules retaining all the 2H label of the starting material. From the known stereochemical course of the type-I isomerase-catalyzed reaction, one has to conclude that the label introduced from D2O in the course of the IspH reaction resides specifically in the H(Si)-C2 position of IPP and that the two isomerases mobilize specifically the same H(Re)-C2 ligand of their common IPP substrate. The outcome of an additional experiment, in which unlabelled IPP was incubated in D2O with the type-II enzyme, demonstrates that the two isomerases also share the same preference in selecting for their reaction the (E)-methyl group of DMAPP.

Influence of compliance and smoking habits on the outcomes of supportive periodontal therapy (SPT) in a private practice.

PURPOSE: The purpose of this retrospective cohort study was to evaluate the effect of smoking habits and patient compliance on the outcomes of supportive periodontal therapy (SPT) (tooth loss and residual pockets defined by probing depth of > or = 5 mm) in a private practice situation. MATERIALS AND METHODS: Eighty-seven patients, who completed active periodontal treatment and then followed an SPT program for at least 5 years, were recruited from the patient pool of a private dental practice. After active periodontal therapy and at the follow-up examination 5-11 years later, pocket probing depths (PPD) and tooth loss were assessed, and the patients were divided into 4 subgroups based on their smoking history: non-smokers (NS); occasional smokers (OS); moderate smokers (S); and heavy smokers (HS). The patient cohort was also divided into 4 subgroups based on patient compliance (mean delay from the scheduled recall sessions): fully compliant (< 1 week); compliant within 1-3 weeks; compliant within 3-6 weeks; and not compliant (> 6 weeks). RESULTS: The mean tooth loss per patient and year ranged from 0.11 - 0.18 in the various subgroups with no significant differences between them. After a mean observation period of 7.3+/-1.5 years, the incidence of new sites with residual probing depth of > or =5 mm varied between 1.2% for the NS and 13.8% for the HS (p < 0.05,), and between 3.2% for the compliant and 5.8% for the non-compliant patients. CONCLUSION: Smoking habits significantly influenced the treatment outcomes of SPT, while compliance was less influential regarding the incidence of new residual pockets during 7.3 years of SPT.