Crystal Structures of Cystathionine ß-Synthase from Saccharomyces cerevisiae: One Enzymatic Step at a Time.

Cystathionine ß-synthase (CBS) is a key regulator of sulfur amino acid metabolism, taking homocysteine from the methionine cycle to the biosynthesis of cysteine via the trans-sulfuration pathway. CBS is also a predominant source of H2S biogenesis. Roles for CBS have been reported for neuronal death pursuant to cerebral ischemia, promoting ovarian tumor growth, and maintaining drug-resistant phenotype by controlling redox behavior and regulating mitochondrial bioenergetics. The trans-sulfuration pathway is well-conserved in eukaryotes, but the analogous enzymes have different enzymatic behavior in different organisms. CBSs from the higher organisms contain a heme in an N-terminal domain. Though the presence of the heme, whose functions in CBSs have yet to be elucidated, is biochemically interesting, it hampers UV-vis absorption spectroscopy investigations of pyridoxal 5'-phosphate (PLP) species. CBS from Saccharomyces cerevisiae (yCBS) naturally lacks the heme-containing N-terminal domain, which makes it an ideal model for spectroscopic studies of the enzymological reaction catalyzed and allows structural studies of the basic yCBS catalytic core (yCBS-cc). Here we present the crystal structure of yCBS-cc, solved to 1.5 Å. Crystal structures of yCBS-cc in complex with enzymatic reaction intermediates have been captured, providing a structural basis for residues involved in catalysis. Finally, the structure of the yCBS-cc cofactor complex generated by incubation with an inhibitor shows apparent off-pathway chemistry not normally seen with CBS.

Structure of a eukaryotic thiaminase I.

Thiaminases, enzymes that cleave vitamin B1, are sporadically distributed among prokaryotes and eukaryotes. Thiaminase I enzymes catalyze the elimination of the thiazole ring moiety from thiamin through substitution of the methylene group with a nitrogenous base or sulfhydryl compound. In eukaryotic organisms, these enzymes are reported to have much higher molecular weights than their bacterial counterparts. A thiaminase I of the single-celled amoeboflagellate Naegleria gruberi is the only eukaryotic thiaminase I to have been cloned, sequenced, and expressed. Here, we present the crystal structure of N. gruberi thiaminase I to a resolution of 2.8 Å, solved by isomorphous replacement and pseudo-two-wavelength multiwavelength anomalous diffraction and refined to an R factor of 0.231 (Rfree, 0.265). This structure was used to solve the structure of the enzyme in complex with 3-deazathiamin, a noncleavable thiamin analog and enzyme inhibitor (2.7 Å; R, 0.233; Rfree, 0.267). These structures define the mode of thiamin binding to this class of thiaminases and indicate the involvement of Asp272 as the catalytic base. This enzyme is able to use thiamin as a substrate and is active with amines such as aniline and veratrylamine as well as sulfhydryl compounds such as l-cysteine and ß-mercaptoethanol as cosubstrates. Despite significant differences in polypeptide sequence and length, we have shown that the N. gruberi thiaminase I is homologous in structure and activity to a previously characterized bacterial thiaminase I.

Crystal structure of Bacillus subtilis GabR, an autorepressor and transcriptional activator of gabT.

Bacillus subtilis GabR is a transcription factor that regulates gamma-aminobutyric acid (GABA) metabolism. GabR is a member of the understudied MocR/GabR subfamily of the GntR family of transcription regulators. A typical MocR/GabR-type regulator is a chimeric protein containing a short N-terminal helix-turn-helix DNA-binding domain and a long C-terminal pyridoxal 5'-phosphate (PLP)-binding putative aminotransferase domain. In the presence of PLP and GABA, GabR activates the gabTD operon, which allows the bacterium to use GABA as nitrogen and carbon sources. GabR binds to its own promoter and represses gabR transcription in the absence of GABA. Here, we report two crystal structures of full-length GabR from B. subtilis: a 2.7-Å structure of GabR with PLP bound and the 2.55-Å apo structure of GabR without PLP. The quaternary structure of GabR is a head-to-tail domain-swap homodimer. Each monomer comprises two domains: an N-terminal winged-helix DNA-binding domain and a C-terminal PLP-binding type I aminotransferase-like domain. The winged-helix domain contains putative DNA-binding residues conserved in other GntR-type regulators. Together with sedimentation velocity and fluorescence polarization assays, the crystal structure of GabR provides insights into DNA binding by GabR at the gabR and gabT promoters. The absence of GabR-mediated aminotransferase activity in the presence of GABA and PLP, and the presence of an active site configuration that is incompatible with stabilization of the GABA external aldimine suggest that a GabR aminotransferase-like activity involving GABA and PLP is not essential to its primary function as a transcription regulator.

Structure and mechanism of benzaldehyde dehydrogenase from Pseudomonas putida ATCC 12633, a member of the Class 3 aldehyde dehydrogenase superfamily.

Benzaldehyde dehydrogenase from Pseudomonas putida (PpBADH) belongs to the Class 3 aldehyde dehydrogenase (ALDH) family. The Class 3 ALDHs are unusual in that they are generally dimeric (rather than tetrameric), relatively non-specific and utilize both NAD+ and NADP+. To date, X-ray structures of three Class 3 ALDHs have been determined, of which only two have cofactor bound, both in the NAD+ form. Here we report the crystal structure of PpBADH in complex with NADP+ and a thioacyl intermediate adduct. The overall architecture of PpBADH resembles that of most other members of the ALDH superfamily, and the cofactor binding residues are well conserved. Conversely, the pattern of cofactor binding for the rat Class 3 ALDH differs from that of PpBADH and other ALDHs. This has been interpreted in terms of a different mechanism for the rat enzyme. Comparison with the PpBADH structure, as well as multiple sequence alignments, suggest that one of two conserved glutamates, at positions 215 (209 in rat) and 337 (333 in rat), would act as the general base necessary to hydrolyze the thioacyl intermediate. While the latter is the general base in the rat Class 3 ALDH, site-specific mutagenesis indicates that Glu215 is the likely candidate for PpBADH, a result more typical of the Class 1 and 2 ALDH families. Finally, this study shows that hydride transfer is not rate limiting, lending further credence to the suggestion that PpBADH is more similar to the Class 1 and 2 ALDHs than it is to other Class 3 ALDHs.

Structural basis for conformational plasticity of the Parkinson's disease-associated ubiquitin hydrolase UCH-L1.

The ubiquitin C-terminal hydrolase UCH-L1 (PGP9.5) comprises >1% of total brain protein but is almost absent from other tissues [Wilkinson, K. D., et al. (1989) Science 246, 670-673]. Mutations in the UCH-L1 gene have been reported to be linked to susceptibility to and protection from Parkinson's disease [Leroy, E., et al. (1998) Nature 395, 451-452; Maraganore, D. M., et al. (1999) Neurology 53, 1858-1860]. Abnormal overexpression of UCH-L1 has been shown to correlate with several forms of cancer [Hibi, K., et al. (1998) Cancer Res. 58, 5690-5694]. Because the amino acid sequence of UCH-L1 is similar to that of other ubiquitin C-terminal hydrolases, including the ubiquitously expressed UCH-L3, which appear to be unconnected to neurodegenerative disease, the structure of UCH-L1 and the effects of disease associated mutations on the structure and function are of considerable importance. We have determined the three-dimensional structure of human UCH-L1 at 2.4-A resolution by x-ray crystallography. The overall fold resembles that of other ubiquitin hydrolases, including UCH-L3, but there are a number of significant differences. In particular, the geometry of the catalytic residues in the active site of UCH-L1 is distorted in such a way that the hydrolytic activity would appear to be impossible without substrate induced conformational rearrangements.