Characterization of yeast homoserine dehydrogenase, an antifungal target: the invariant histidine 309 is important for enzyme integrity.

Fungal homoserine dehydrogenase (HSD) is required for the biosynthesis of threonine, isoleucine and methionine from aspartic acid, and is a target for antifungal agents. HSD from the yeast Saccharomyces cerevisiae was overproduced in Escherichia coli and 25 mg of soluble dimeric enzyme was purified per liter of cell culture in two steps. HSD efficiently reduces aspartate semialdehyde to homoserine (Hse) using either NADH or NADPH with kcat/Km in the order of 10(6-7) M(-1) x s(-1) at pH 7.5. The rate constant of the reverse direction (Hse oxidation) was also significant at pH 9.0 (kcat/Km approximately 10(4-5) M(-1) x s(-1)) but was minimal at pH 7.5. Chemical modification of HSD with diethyl pyrocarbonate (DEPC) resulted in a loss of activity that could be obviated by the presence of substrates. UV difference spectra revealed an increase in absorbance at 240 nm for DEPC-modified HSD consistent with the modification of two histidines (His) per subunit. Amino acid sequence alignment of HSD illustrated the conservation of two His residues among HSDs. These residues, His79 and His309, were substituted to alanine (Ala) using site directed mutagenesis. HSD H79A had similar steady state kinetics to wild type, while kcat/Km for HSD H309A decreased by almost two orders of magnitude. The recent determination of the X-ray structure of HSD revealed that His309 is located at the dimer interface [B. DeLaBarre, P.R. Thompson, G.D. Wright, A.M. Berghuis, Nat. Struct. Biol. 7 (2000) 238-244]. The His309Ala mutant enzyme was found in very high molecular weight complexes rather than the expected dimer by analytical gel filtration chromatography analysis. Thus the invariant His309 plays a structural rather than catalytic role in these enzymes.

Determinants of the nuclear localization of the heterodimeric DNA fragmentation factor (ICAD/CAD).

Programmed cell death or apoptosis leads to the activation of the caspase-activated DNase (CAD), which degrades chromosomal DNA into nucleosomal fragments. Biochemical studies revealed that CAD forms an inactive heterodimer with the inhibitor of caspase-activated DNase (ICAD), or its alternatively spliced variant, ICAD-S, in the cytoplasm. It was initially proposed that proteolytic cleavage of ICAD by activated caspases causes the dissociation of the ICAD/CAD heterodimer and the translocation of active CAD into the nucleus in apoptotic cells. Here, we show that endogenous and heterologously expressed ICAD and CAD reside predominantly in the nucleus in nonapoptotic cells. Deletional mutagenesis and GFP fusion proteins identified a bipartite nuclear localization signal (NLS) in ICAD and verified the function of the NLS in CAD. The two NLSs have an additive effect on the nuclear targeting of the CAD-ICAD complex, whereas ICAD-S, lacking its NLS, appears to have a modulatory role in the nuclear localization of CAD. Staurosporine-induced apoptosis evoked the proteolysis and disappearance of endogenous and exogenous ICAD from the nuclei of HeLa cells, as monitored by immunoblotting and immunofluorescence microscopy. Similar phenomenon was observed in the caspase-3-deficient MCF7 cells upon expressing procaspase-3 transiently. We conclude that a complex mechanism, involving the recognition of the NLSs of both ICAD and CAD, accounts for the constitutive accumulation of CAD/ICAD in the nucleus, where caspase-3-dependent regulation of CAD activity takes place.

Overproduction and characterization of a dimeric non-zinc glyoxalase I from Escherichia coli: evidence for optimal activation by nickel ions.

The ubiquitous glyoxalase system converts toxic alpha-keto aldehydes into their corresponding nontoxic 2-hydroxycarboxylic acids, utilizing glutathione (GSH) as a cofactor. The first enzyme in this system, glyoxalase I (GlxI), catalyzes the isomerization of the hemithioacetal formed nonenzymatically between GSH and cytotoxic alpha-keto aldehydes. To study the Escherichia coli GlxI enzyme, the DNA encoding this protein, gloA, was isolated and incorporated into the plasmid pTTQ18. Nucleotide sequencing of the gloA gene predicted a polypeptide of 135 amino acids and Mr of 14 919. The gloA gene has been overexpressed in E. coli and shown to encode for GlxI. An effective two-step purification protocol was developed, yielding 150-200 mg of homogeneous protein per liter of culture. Electrospray mass spectrometry confirmed the monomeric weight of the purified protein, while gel filtration analysis indicated GlxI to be a homodimer of 30 kDa. Zinc, the natural metal ion found in the Homo sapiens and Saccharomyces cerevisiae GlxI, had no effect on the activity of E. coli GlxI. In contrast, the addition of NiCl2 to the growth medium or to purified E. coli apo-GlxI greatly enhanced the enzymatic activity. Inductively coupled plasma and atomic absorption analyses indicated binding of only one nickel ion per dimeric enzyme, suggesting only one functional active site in this homodimeric enzyme. In addition, the apoprotein regained maximal activity with one molar equivalence of nickel chloride, indicative of tight metal binding. The effects of pH on the kinetics of the nickel-activated enzyme were also studied. This is the first example of a non-zinc activated GlxI whose maximal activation is seen with Ni2+.

Isolation and sequencing of a gene coding for glyoxalase I activity from Salmonella typhimurium and comparison with other glyoxalase I sequences.

The glyoxalase I gene (gloA) from Salmonella typhimurium has been isolated in Escherichia coli on a multi-copy pBR322-derived plasmid, selecting for resistance to 3 mM methylglyoxal on Luria-Bertani agar. The region of the plasmid which confers the methylglyoxal resistance in E. coli was sequenced. The deduced protein sequence was compared to the known sequences of the Homo sapiens and Pseudomonas putida glyoxalase I (GlxI) enzymes, and regions of strong homology were used to probe the National Center for Biotechnology Information protein database. This search identified several previously known glyoxalase I sequences and other open reading frames with unassigned function. The clustal alignments of the sequences are presented, indicating possible Zn2+ ligands and active site regions. In addition, the S. typhimurium sequence aligns with both the N-terminal half and the C-terminal half of the proposed GlxI sequences from Saccharomyces cerevisiae and Schizosaccharomyces pombe, suggesting that the structures of the yeast enzymes are those of fused dimers.

HPLC determination of D-glucaric acid in human urine.

An isocratic HPLC method has been developed for the direct measurement of D-glucaric acid in human urine. Pretreatment of urine with a boronic acid gel removed many interfering substances, including L-ascorbic acid and D-glucuronic acid. This method has a detection limit of 10 microM D-glucaric acid (approximately 7 mumoles/g creatinine). The run-to-run precisions were 9.1% and 7.7% at urinary D-glucaric acid concentrations of 41 and 219 mumoles/g creatinine, respectively. Urinary D-glucaric acid concentrations in normal adults were found to cover a range of 15 to 89 mumoles/g creatinine (mean = 47 mumoles/g creatinine). The sensitivity of this method in detecting abnormal elevations in D-glucaric acid was demonstrated through its ability to measure changes in urinary concentrations with time after ingestion of D-glucuronolactone.