The structure of GDP-4-keto-6-deoxy-D-mannose-3-dehydratase: a unique coenzyme B6-dependent enzyme

L-colitose is a 3,6-dideoxysugar found in the O-antigens of some Gram-negative bacteria such as Escherichia coli and in marine bacteria such as Pseudoalteromonas tetraodonis. The focus of this investigation, GDP-4-keto-6-deoxy-D-mannose-3-dehydratase, catalyzes the third step in colitose production, which is the removal of the hydroxyl group at C3' of GDP-4-keto-6-deoxymannose. It is an especially intriguing PLP-dependent enzyme in that it acts as both a transaminase and a dehydratase. Here we present the first X-ray structure of this enzyme isolated from E. coli Strain 5a, type O55:H7. The two subunits of the protein form a tight dimer with a buried surface area of approximately 5000 A2. This is a characteristic feature of the aspartate aminotransferase superfamily. Although the PLP-binding pocket is formed primarily by one subunit, there is a loop, delineated by Phe 240 to Glu 253 in the second subunit, that completes the active site architecture. The hydrated form of PLP was observed in one of the enzyme/cofactor complexes described here. Amino acid residues involved in anchoring the cofactor to the protein include Gly 56, Ser 57, Asp 159, Glu 162, and Ser 183 from one subunit and Asn 248 from the second monomer. In the second enzyme/cofactor complex reported, a glutamate ketimine intermediate was found trapped in the active site. Taken together, these two structures, along with previously reported biochemical data, support the role of His 188 as the active site base required for catalysis.     

The B6 database: a tool for the description and classification of vitamin B6-dependent enzymatic activities and of the corresponding protein families

Background -  

Enzymes that depend on vitamin B6 (and in particular on its metabolically active form, pyridoxal 5'-phosphate, PLP) are of great relevance to biology and medicine, as they catalyze a wide variety of biochemical reactions mainly involving amino acid substrates. Although PLP-dependent enzymes belong to a small number of independent evolutionary lineages, they encompass more than 160 distinct catalytic functions, thus representing a striking example of divergent evolution. The importance and remarkable versatility of these enzymes, as well as the difficulties in their functional classification, create a need for an integrated source of information about them.     

Mechanisms and structures of vitamin B(6)-dependent enzymes involved in deoxy sugar biosynthesis

PLP is well-regarded for its role as a coenzyme in a number of diverse enzymatic reactions. Transamination, deoxygenation, and aldol reactions mediated by PLP-dependent enzymes enliven and enrich deoxy sugar biosynthesis, endowing these compounds with unique structures and contributing to their roles as determinants of biological activity in many natural products. The importance of deoxy aminosugars in natural product biosynthesis has spurred several recent structural investigations of sugar aminotransferases. The structure of a PMP-dependent enzyme catalyzing the C-3 deoxygenation reaction in the biosynthesis of ascarylose was also determined. These studies, and the crystal structures they have provided, offer a wealth of new insights regarding the enzymology of PLP/PMP-dependent enzymes in deoxy sugar biosynthesis. In this review, we consider these recent achievements in the structural biology of deoxy sugar biosynthetic enzymes and the important implications they hold for understanding enzyme catalysis and natural product biosynthesis in general. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.     

Mechanism of B12-dependent ribonucleotide reductase

Summary Ribonucleotide reductase from L. leichmmannii catalyzes cleavage of the carbon cobalt bond of AdoCbl homolytically in a kinetically competent fashion. This cleavage triggers a chain of events which results in cleavage of the 3C-H bond of the nucleotide substrate followed by cleavage of the 2 carbon hydroxyl bond. Involvement of a radical cation has been suggested as a possible mechanism by which this unusual reduction reaction might occur. Furthermore, cleavage of the 3 carbon hydrogen bond of [3-³H]NTP resulted in no ³H release to solvent and no ³H recovered in AdoCbl. These results were interpreted to mean that in this system AdoCbl does not serve as a H abstractor, but rather as a radical chain initiator. A protein residue on the RTPR is postulated to carry out the actual H abstraction from the substrate.These results and the conclusions drawn from them are further supported by recent experiments using [3-³H]CIUTP. Incubation of RTPR with [3-³H]CIUTP resulted in release of ³H₂O, uracil, PPP_i, formation of Coll and 5 deoxyadenosine. The ³H₂O release confirms the enzyme's ability to cleave the 3C-H bond of a nucleotide analog. Furthermore, little if any ³H was recovered in the 5 deoxyadenosine and the rate of ³H₂O release from [3³H]CIUTP was 12 times faster than the rate of ³H₂O release from [5-³H]AdoCbl. These observations support the conclusions drawn from data with the normal substrate; ie, AdoCbl serves as a radical chain initiator rather than a direct H abstractor from substrate.     

IL-4 promotes Stat6-dependent survival of autoreactive B cells in vivo without inducing autoantibody production

Persistent cross-linking of hen egg lysozyme (HEL)-specific B cell membrane Ig (mIg) in double transgenic mice that express soluble HEL as a self Ag (HEL-Ig mice) decreases B cell mIgM expression, responsiveness, and life span. Because in vitro treatment with IL-4 inhibits T cell apoptosis through a Stat6-independent mechanism, increases mIg expression, and suppresses activation-induced B cell death, we studied IL-4 effects on B cell mIg expression, survival, and Ab secretion in Stat6-sufficient and deficient HEL-Ig mice. IL-4 treatment nearly normalized B cell number and greatly increased the percentage of mature B cells in HEL-Ig mice, but failed to normalize mIgM expression or spontaneous LPS-induced IgM secretion. IL-4 effects on B cell survival and maturation were CD4(+) T cell independent, but Stat6 dependent, and did not involve receptor editing. IL-4 had to be present while B cells were generated to have a detectable effect on autoreactive B cell survival; however, the survival of B cells generated in the presence of IL-4 was substantially increased even after IL-4 was withdrawn. These observations suggest that: 1) activation-induced B cell death and anergy are independent processes; 2) B cells that survive to maturity develop increased resistance to Ag-induced deletion; and 3) IL-4 promotes B and T cell survival through different mechanisms.     

Biosynthesis of xanthurenic acid 8-O-beta-D-glucoside in Drosophila. Characterization of the xanthurenic acid:UDP-glucosyltransferase activity

Xanthurenic acid 8-glucoside is a side metabolite of the tryptophan-xanthommatin pathway in Drosophila. From 3-hydroxykynurenine, two biosynthetic pathways can be envisaged, one via xanthurenic acid, and another via 3-O-glucoside of 3-hydroxykynurenine. In this report evidence is presented to show that the synthesis takes place via xanthurenic acid. (a) We have demonstrated that the Drosophila melanogaster vermilion purple mutant (unable to synthesize 3-hydroxykynurenine) synthesizes xanthurenic acid 8-glucoside when fed with xanthurenic acid; and (b) the activities required for its synthesis via xanthurenic acid have been found (3-hydroxykynurenine transaminase and xanthurenic acid:UDP-glucosyltransferase). This is the first time that a UDP-glucosyltransferase activity that utilizes xanthurenic acid has been demonstrated. The enzyme in crude extracts from Drosophila sordidula shows the following characteristics. (a) It has optimal activity at 35 degrees C at pH 7.1 (in buffer Tris-HCl), and in the presence of a divalent cation (Mg2+ or Mn2+); (b) the activity is inhibited by xanthurenic acid (above 1.5 mM), UDP, D-gluconic acid 1,5-lactone, and Triton X-100; (c) it is localized in both the microsomal and the soluble fractions; (d) the specific activity is two times higher in heads than in bodies; and (e) the activity is enhanced in flies fed with phenobarbital.     

4-Hydroxybutyric aciduria

Recent work has led to the discovery of six patients with 4-hydroxybutyric acid-uria, a severe hereditary human pathology characterized by the accumulation of a compound of known neuropharmacologic activity in body fluids. The enzymatic deficiency has been localized to succinic semialdehyde dehydrogenase, one of two enzymes involved in the metabolism of the neurotransmitter GABA. The disease is inherited as an autosomal recessive trait, and carrier detection has been accomplished by quantification of intermediate enzyme activities. The clinical, enzymatic and pharmacologic characteristics of this disease are reviewed here.