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The X-ray susceptibility of the lysine-pyridoxal-5′-phosphate Schiff base in Bacillus alcalophilus phosphoserine aminotransferase has been investigated using crystallographic data collected at 100 K to 1.3 Å resolution, complemented by on-line spectroscopic studies. X-rays induce deprotonation of the internal aldimine, changes in the Schiff base conformation, displacement of the cofactor molecule, and disruption of the Schiff base linkage between pyridoxal-5′-phosphate and the Lys residue. Analysis of the 'undamaged' structure reveals a significant chemical strain on the internal aldimine bond that leads to a pronounced geometrical distortion of the cofactor. However, upon crystal exposure to the X-rays, the strain and distortion are relaxed and eventually diminished when the total absorbed dose has exceeded 4.7 × 106 Gγ. Our data provide new insights into the enzymatic activation of pyridoxal-5′-phosphate and suggest that special care should be taken while using macromolecular crystallography to study details in strained active sites. [ABSTRACT FROM AUTHOR]

The crystal structure of the vitamin B6-dependent enzyme phosphoserine aminotransferase from the obligatory alkaliphile Bacillus alcalophilus has been determined at 1.08 Å resolution. The model was refined to an R-factor of 11.7% ( Rfree = 13.9%). The enzyme displays a narrow pH optimum of enzymatic activity at pH 9.0. The final structure was compared to the previously reported structure of the mesophilic phosphoserine aminotransferase from Escherichia coli and to that of phosphoserine aminotransferase from a facultative alkaliphile, Bacillus circulans subsp. alkalophilus. All three enzymes are homodimers with each monomer comprising a two-domain architecture. Despite the high structural similarity, the alkaliphilic representatives possess a set of distinctive structural features. Two residues directly interacting with pyridoxal-5′-phosphate are replaced, and an additional hydrogen bond to the O3′ atom of the cofactor is present in alkaliphilic phosphoserine aminotransferases. The number of hydrogen bonds and hydrophobic interactions at the dimer interface is increased. Hydrophobic interactions between the two domains in the monomers are enhanced. Moreover, the number of negatively charged amino acid residues increases on the solvent-accessible molecular surface and fewer hydrophobic residues are exposed to the solvent. Further, the total amount of ion pairs and ion networks is significantly reduced in the Bacillus enzymes, while the total number of hydrogen bonds is increased. The mesophilic enzyme from Escherichia coli contains two additional β-strands in a surface loop with a third β-strand being shorter in the structure. The identified structural features are proposed to be possible factors implicated in the alkaline adaptation of phosphoserine aminotransferase. [ABSTRACT FROM AUTHOR]

S-Ribosylhomocysteinase (LuxS) cleaves the thioether bond in S-ribosylhomocysteine to produce homocysteine and 4,5-dihydroxy-2,3-pentanedione. This reaction serves the dual purposes of detoxification of S-adenosylhomocysteine and production of type 2 quorum sensing molecule. Recent research has shown that LuxS uses Fe2+ to catalyze an internal redox reaction, shifting the ribose carbonyl group from its C1 to C3 position. Subsequent β-elimination completes this highly unusual reaction. LuxS and other enzymes on the same pathway may provide a novel class of antibacterial drug targets. [Copyright &y& Elsevier]

The serine hydroxymethyltransferase from Bacillus subtilis (bsSHMT) and B. stearothermophilus (bstSHMT) are both homodimers and share ∼77% sequence identity; however, they show very different thermal stabilities and unfolding pathways. For investigating the role of N- and C-terminal domains in stability and unfolding of dimeric SHMTs, we have swapped the structural domains between bs- and bstSHMT and generated the two novel chimeric proteins bsbstc and bstbsc, respectively. The chimeras had secondary structure, tyrosine, and pyridoxal-5′-phosphate microenvironment similar to that of the wild-type proteins. The chimeras showed enzymatic activity slightly higher than that of the wild-type proteins. Interestingly, the guanidium chloride (GdmCl)-induced unfolding showed that unlike the wild-type bsSHMT, which undergoes dissociation of native dimer into monomers at low guanidium chloride (GdmCl) concentration, resulting in a non-cooperative unfolding of enzyme, its chimera bsbstc, having the C-terminal domain of bstSHMT was resistant to low GdmCl concentration and showed a GdmCl-induced cooperative unfolding from native dimer to unfolded monomer. In contrast, the wild-type dimeric bstSHMT was resistant to low GdmCl concentration and showed a GdmCl-induced cooperative unfolding, whereas its chimera bstbsc, having the C- terminal domain of bsSHMT, showed dissociation of native dimer into monomer at low GdmCl concentration and a GdmCl-induced non-cooperative unfolding. These results clearly demonstrate that the C-terminal domain of dimeric SHMT plays a vital role in stabilization of the oligomeric structure of the native enzyme hence modulating its unfolding pathway. [ABSTRACT FROM AUTHOR]

This study describes the comparative analysis of two insect recombinant aminotransferases, Aedes aegypti 3-hydroxykynurenine (3-HK) transaminase/alanine glyoxylate aminotransferase (Ae-HKT/AGT) and Drosophila melanogaster serine pyruvate aminotransferase (Dm-Spat), which share 52% identity in their amino acid sequences. Both enzymes showed AGT activity. In addition, Ae-HKT/AGT is also able to catalyze the transamination of 3-HK or kynurenine with glyoxylate, pyruvate or oxaloacetate as the amino acceptor. Kinetic analysis and other data suggest that Ae-HKT/AGT plays a critical role in mosquito tryptophan catabolism by detoxifying 3-HK and that Dm-Spat is primarily involved in glyoxylate detoxification. [Copyright &y& Elsevier]

The eightfold (βα) barrel structure, first observed in triose-phosphate isomerase, occurs ubiquitously in nature. It is nearly always an enzyme and most often involved in molecular or energy metabolism within the cell. In this review we bring together data on the sequence, structure and function of the proteins known to adopt this fold. We highlight the sequence and functional diversity in the 21 homologous superfamilies, which include 76 different sequence families. In many structures, the barrels are “mixed and matched” with other domains generating additional variety. Global and local structure-based alignments are used to explore the distribution of the associated functional residues on this common structural scaffold. Many of the substrates/co-factors include a phosphate moiety, which is usually but not always bound towards the C-terminal end of the sequence. Some, but not all, of these structures, exhibit a structurally conserved “phosphate binding motif”. In contrast metal-ligating residues and catalytic residues are distributed along the sequence. However, we also found striking structural superposition of some of these residues. Lastly we consider the possible evolutionary relationships between these proteins, whose sequences are so diverse that even the most powerful approaches find few relationships, yet whose active sites all cluster at one end of the barrel. This extreme example of the “one fold-many functions” paradigm illustrates the difficulty of assigning function through a structural genomics approach for some folds. [Copyright &y& Elsevier]

Two new parameters, I and C, are introduced for the quantitative evaluation of functional chimeras: I ( impact) and C ( context dependence) are the free energy difference and sum, respectively, of the effects on a given property measured in forward and retro chimeras. The forward chimera is made by substitution of a part 'a' from ensemble A into the analogous position of homologous ensemble B ( SB → A). The C value is a measure of the interaction of the interrogated position with its surroundings, whereas I is an expression of the quantitative importance of the probed position. Both I and C vary with the evaluated property, for example, kinetics, binding, thermostability, and so forth. The retro chimera is the reverse substitution of the analogous part 'b' from B into A, SA → B. The I and C values derived from original data for forward and retro mutations in aspartate and tyrosine aminotransferase, from literature data for quasi domain exchange in oncomodulin and for the interaction of Tat with bovine and human TAR are evaluated. The most salient derived conclusions are, first, that Thr 109 (AATase) or Ser 109 (TATase) is an important discriminator for dicarboxylic acid selectivity by these two enzymes ( I < −2.9 kcal/mol). The T109S mutation in AATase produces a nearly equal and opposite effect to S109T in TATase ( C < 0.4 kcal/mol). Second, an I value of 5.5 kcal/mol describes the effects of mirror mutations D94S (site 1) and S55D (site 2) in the Ca2+ binding sites of oncomodulin on Ca2+ affinity. The second mirror set, G98D (site 1) and D59G (site 2), yields a smaller impact ( I = −3.4 kcal/mol) on Ca2+ binding; however, the effect is significantly more nearly context independent ( C = −0.6 versus C = −2.7 kcal/mol). Third, the stem and loop regions of HIV and BIV TAR are predominantly responsible for the species specific interaction with BIV Tat65-81 ( I = −1.5 to −1.6 kcal/mol), whereas I = 0.1 kcal/mol for bulge TAR chimeras. The C values are from −0.3 to −1.2 kcal/mol. The analysis described should have important applications to protein design. [ABSTRACT FROM AUTHOR]

We report a patient with anti-epileptic treatment refractory neonatal seizures responsive to pyridoxine. Biochemical analysis revealed normal markers for antiquitin deficiency and also mutation analysis of the ALDH7A1 (Antiquitin) gene was negative. Mutation analysis of the PNPO gene revealed a novel, homozygous, presumed pathogenic mutation (c.481C > T; p.(Arg161Cys)). Measurements of B6 vitamers in a CSF sample after pyridoxine administration revealed elevated pyridoxamine as the only metabolic marker for PNPO deficiency. With pyridoxine monotherapy the patient is seizure free and neurodevelopmental outcome at the age of 14 months is normal.