Inositol-1-phosphate Synthase fromArchaeoglobus fulgidusIs a Class II Aldolase

Abstract

A gene putatively identified as the Archaeoglobus fulgidus inositol-1-phosphate synthase (IPS) gene was overexpressed to high level (about 30-40% of total soluble cellular proteins) in Escherichia coli. The recombinant protein was purified to homogeneity by heat treatment followed by two column chromatographic steps. The native enzyme was a tetramer of 168 +/- 4 kDa (subunit molecular mass of 44 kDa). At 90 degrees C the K(m) values for glucose-6-phosphate and NAD(+) were estimated as 0.12 +/- 0.04 mM and 5.1 +/- 0.9 microM, respectively. Use of (D)-[5-(13)C]glucose-6-phosphate as a substrate confirmed that the stereochemistry of the product of the IPS reaction was L-myo-inositol-1-phosphate. This archaeal enzyme, with the highest activity at its optimum growth temperature among all IPS reported (k(cat) = 9.6 +/- 0.4 s(-1) with an estimated activation energy of 69 kJ/mol), was extremely heat stable. However, the most unique feature of A. fulgidus IPS was that it absolutely required divalent metal ions for activity. Zn(2+) and Mn(2+) were the best activators with K(D) approximately 1 microM, while NH(4)(+) (a critical activator for all the other characterized IPS enzymes) had no effect on the enzyme. These properties suggested that this archaeal IPS was a class II aldolase. In support of this, stoichiometric reduction of NAD(+) to NADH could be followed spectrophotometrically when EDTA was present along with glucose-6-phosphate.

Structural and Enzymatic Analysis of MshA from Corynebacterium glutamicum

“…Production of 1-L-Ins-1-P-The gene for inositol-1-phosphate synthase from the hyperthermophile Archaeoglobus fulgidus (Af_INO) was cloned as described by a previous group (39). Briefly, the Af_INO gene was PCR-amplified from genomic DNA and ligated into pET23a(ϩ) vector utilizing Nde1 and HindIII restriction sites.…”

Biochemical Characterization of WbpA, a UDP-N-acetyl-d-glucosamine 6-Dehydrogenase Involved in O-antigen Biosynthesis in Pseudomonas aeruginosa PAO1

“…It is interesting to note that neither AlgD nor Ugd, which perform similar reactions on nucleotide activated hexoses, require NH 4 ϩ or K ϩ for activity, although this does not eliminate the possibility that NH 4 ϩ are K ϩ are directly involved in the active site of WbpA. An example of this is 1-L-myo-inositol-1-phosphate (MIP) synthase from yeast compared with MIP synthase from Archaebacteria fulgidus, both of which catalyze the conversion of D-glucose 6-phosphate to MIP (39). Only the yeast MIP is activated by NH 4 ϩ , and the crystal structure of this enzyme suggests the involvement of NH 4 ϩ in the reaction mechanism of this enzyme (40).…”

Occurrence of myo-inositol-1-phosphate phosphatase in pteridophytes: characteristics of the enzyme from the reproductive pinnules of Dryopteris filix-mas (L.) Schott

“…Generation of free myo-inositol may be blocked if MIPP is not active. The MIPS reaction has been reported to occur in a large number of living systems namely archea (Chen et al, 2000), bacteria (Bachhawat and Mande, 2000), protozoa (Lohia et al, 1999), lower plants (Donahue and Henry, 1981a,b;Escamilla et al, 1982;Dasgupta et al, 1984;RayChoudhury et al, 1997;Benaroya et al, 2004, Chhetri et al, 2005, higher plants (Loewus and Loewus, 1971;Loewus et al, 1978;Ogunyemi et al, 1978;Adhikari et al, 1987;Johnson and Sussex, 1995;Johnson and Wang, 1996;Chun et al, 2003), and animals (Pittner and Hoffmann-Ostenhof, 1976;Maeda and Eisenberg Jr., 1980;Majumder, 1983, 1988;Chiu et al, 2003). In pteridophytes, Benaroya et al (2004) and Chettri et al (2005Chettri et al ( , 2006 have recently documented the occurrence and characterization of L-myo-inositol-1-phosphate synthase.…”