Earlier in vivo studies have shown that the sequential action of the IspG and IspH proteins is essential for the reductive transformation of 2C-methyl-D-erythritol 2,4-cyclodiphosphate into dimethylallyl diphosphate and isopentenyl diphosphate via 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate. A recombinant fusion protein comprising maltose binding protein and IspG protein domains was purified from a recombinant Escherichia coli strain. The purified protein failed to transform 2C-methyl-D-erythritol 2,4-cyclodiphosphate into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate, but catalytic activity could be restored by the addition of crude cell extract from an ispG-deficient E. coli mutant. This indicates that auxiliary proteins are required, probably as shuttles for redox equivalents. On activation by photoreduced 10-methyl-5-deazaisoalloxazine, the recombinant protein catalyzed the formation of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate from 2C-methyl-D-erythritol 2,4-cyclodiphosphate at a rate of 1 nmol⅐min ؊1 ⅐mg ؊1 . Similarly, activation by photoreduced 10-methyl-5-deaza-isoalloxazine enabled purified IspH protein to catalyze the conversion of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate into a 6:1 mixture of isopentenyl diphosphate and dimethylallyl diphosphate at a rate of 0.4 mol⅐min ؊1 ⅐mg ؊1 . IspH protein could also be activated by a mixture of flavodoxin, flavodoxin reductase, and NADPH at a rate of 3 nmol⅐min ؊1 ⅐mg ؊1 . The striking similarities of IspG and IspH protein are discussed, and plausible mechanistic schemes are proposed for the two reactions.

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.

An open reading frame (Acc. no. P50740) on the Bacillus subtilis chromosome extending from bp 184 997-186 043 with similarity to the idi-2 gene of Streptomyces sp. CL190 specifying type II isopentenyl diphosphate isomerase was expressed in a recombinant Escherichia coli strain. The recombinant protein with a subunit mass of 39 kDa was purified to apparent homogeneity by column chromatography. The protein was shown to catalyse the conversion of dimethylallyl diphosphate into isopentenyl diphosphate and vice versa at rates of 0.23 and 0.63 lmolAEmg . NADPH is required under aerobic but not under anaerobic assay conditions. The enzyme is related to a widespread family of (S)-a-hydroxyacid oxidizing enzymes including flavocytochrome b 2 and L-lactate dehydrogenase and was shown to catalyse the formation of [2,[3][4][5][6][7][8][9][10][11][12][13] C 2 ]lactate from [2,[3][4][5][6][7][8][9][10][11][12][13] C 2 ]pyruvate, albeit at a low rate of 1 nmolAEmg. Putative genes specifying type II isopentenyl diphosphate isomerases were found in the genomes of Archaea and of certain eubacteria but not in the genomes of fungi, animals and plants. The analysis of the occurrence of idi-1 and idi-2 genes in conjunction with the mevalonate and nonmevalonate pathway in 283 completed and unfinished prokaryotic genomes revealed 10 different classes. Type II isomerase is essential in some important human pathogens including Staphylococcus aureus and Enterococcus faecalis where it may represent a novel target for anti-infective therapy.

Regioselective transformations are highly desirable in organic synthesis, since they allow for the differentiation between two or more (chemically identical) reactive centers, [1] which is otherwise only possible by employing sophisticated and laborious protection strategies. [2] However, protectinggroup-free strategies are superior and have received outstanding merits for their successes. [3,4] Although the regioselective amination of, for example, alkanes, [5] allylic systems, [6] or indoles, [7] has been recently described, the regioselective asymmetric bioamination of diketones has not yet been reported, to the best of our knowledge. For example, diketones, such as 1,5-diketo compounds, may serve as possible precursors for a chiral piperidine scaffold. [8] Consequently, we chose 2,6-diketones 1 as model substrates to investigate the possible asymmetric regioselective amination employing w-transaminases (w-TAs; Scheme 1). [9,10] Various (S)-and (R)-stereoselective w-transaminases were tested initially for the transformation of diketone 1 a at a substrate concentration of 50 mm (Table 1). Five w-TAs (Chromobacterium violaceum, [10i, 11] Bacillus megaterium, [10i, 12] (R)-Arthrobacter, [10b] Aspergillus terreus, and Hyphomonas neptunium [10b,h] ) out of six showed perfect regioselectivity for the differentiation between the two keto groups. Hence, the amination occurred exclusively at the sterically less demanding w-1 ketone moiety, leading to the amino ketone 2 a, while the w-3 position remained untouched. The intermediate amino ketone 2 a spontaneously cyclized, finally giving D1piperideine 4 a. Only the w-TA from Vibrio fluvialis [9h, 13] (entry 3) showed diminished regioselectivity, since regioisomer 5 a was formed in minor quantities (5-7 %) along with regioisomer 4 a at high conversion. Notably, the corresponding diamine was never detected in any experiment.Using alanine as amine donor led to the formation of pyruvate as a by-product, which was removed/recycled to alanine through the use of an alanine dehydrogenase (AlaDH) system. In all cases, perfect conversions were achieved for this system. When removing pyruvate by reduction to lactate through the use of a lactate dehydrogenase (LDH) system, the conversions varied from 56-98 %. The Scheme 1. Regioselective amination of various 1,5-diketones. Table 1: Asymmetric reductive amination of diketone 1 a. [a] AlaDH system LDH system Entry w-TA conv [%] 4 a [%] ee 4 a [%] 5 a [%] conv [%] 4 a [%] ee 4 a [%] 5 a [%] 1 C. violaceum > 99 > 99 > 99 (S) < 0.1 93 93 > 99 (S) < 0.1 2 B. megaterium > 99 83 [b] > 99 (S) < 0.1 98 98 > 99 (S) < 0.1 3 V. fluvialis > 99 93 > 99 (S) 7 98 92 > 99 (S) 6 4 (R)-Arthrobacter > 99 87 [b] > 99 (R) < 0.1 63 63 > 99 (R) < 0.1 5 A. terreus > 99 > 99 > 99 (R) < 0.1 56 56 > 99 (R) < 0.1 6 H. neptunium > 99 89 [b] > 99 (R) < 0.1 78 78 > 99 (R) < 0.1 [a] Conversions/compositions and ee values were determined by GC-FID analysis. Reaction conditions: diketone 1 a (50 mm), lyophilized E. coli cells containing the overexpressed w-T...