The Crystal Structure of D-Threonine Aldolase from Alcaligenes xylosoxidans Provides Insight into a Metal Ion Assisted PLP-Dependent Mechanism

Abstract

Threonine aldolases catalyze the pyridoxal phosphate (PLP) dependent cleavage of threonine into glycine and acetaldehyde and play a major role in the degradation of this amino acid. In nature, L- as well as D-specific enzymes have been identified, but the exact physiological function of D-threonine aldolases (DTAs) is still largely unknown. Both types of enantio-complementary enzymes have a considerable potential in biocatalysis for the stereospecific synthesis of various β-hydroxy amino acids, which are valuable building blocks for the production of pharmaceuticals. While several structures of L-threonine aldolases (LTAs) have already been determined, no structure of a DTA is available to date. Here, we report on the determination of the crystal structure of the DTA from Alcaligenes xylosoxidans (AxDTA) at 1.5 Å resolution. Our results underline the close relationship of DTAs and alanine racemases and allow the identification of a metal binding site close to the PLP-cofactor in the active site of the enzyme which is consistent with the previous observation that divalent cations are essential for DTA activity. Modeling of AxDTA substrate complexes provides a rationale for this metal dependence and indicates that binding of the β-hydroxy group of the substrate to the metal ion very likely activates this group and facilitates its deprotonation by His193. An equivalent involvement of a metal ion has been implicated in the mechanism of a serine dehydratase, which harbors a metal ion binding site in the vicinity of the PLP cofactor at the same position as in DTA. The structure of AxDTA is completely different to available structures of LTAs. The enantio-complementarity of DTAs and LTAs can be explained by an approximate mirror symmetry of crucial active site residues relative to the PLP-cofactor.

Crystallization and X-ray analysis ofD-threonine aldolase fromChlamydomonas reinhardtii

“…The recent publication of the crystal structure of a bacterial DTA from A. xylosoxidans identified a metal-binding site in the active site, which was consistent with previous observations that divalent metal ions are essential for DTA activity (PDB entry 4v15; Uhl et al, 2015). However, the current limited structural information on DTAs makes it difficult to understand the structural differences between bacterial and eukaryotic DTAs and to develop stereoselective d-or d-allothreonine aldolases.…”

“…Previous studies indicated that the bacterial DTAs from A. xylosoxidans and Arthrobacter sp. were active as monomers (Liu, Odani et al, 2000;Kataoka et al, 1997), but the crystal structure of DTA from A. xylosoxidans was predicted to be a stable dimer in solution (Uhl et al, 2015). Our results indicate that the assembly of CrDTA molecules was similar to that of bacterial DTAs.…”

“…The structure was solved by the molecular-replacement method using MOLREP (Vagin & Teplyakov, 2010) with the structure of DTA from A. xylosoxidans (PDB entry 4v15), which shares 40% identity with CrDTA, as a search model (Uhl et al, 2015). The results of molecular replacement showed a clear solution.…”

Synthesis and Deployment of an Elusive Fluorovinyl Cation Equivalent: Access to Quaternary α-(1′-Fluoro)vinyl Amino Acids as Potential PLP Enzyme Inactivators

“…The classic member of this family is L-serine hydroxymethyl transferase (SHMT), 79 involved in the one-carbon cycle associated with pyrimidine biosynthesis, a three-enzyme pathway in which the other two enzymes are targeted by the clinical chemotherapeutics 5-fluorouracil (thymidylate synthase) and methotrexate (dihydrofolate reductase - DHFR). More recently, PLP dependent aldolases for L-threonine, 80 D-threonine, 81 phenylserine 82 and α-methylserine, 83 with a number of these demonstrating considerable side chain tolerance. 84 Thus, the chemistry presented here is expected to be enabling tool across a broad spectrum of chemical biological studies.…”

Threonine aldolases: perspectives in engineering and screening the enzymes with enhanced substrate and stereo specificities

“…The first crystal structure of d -specific TAs was published recently by Gruber et al for the d TA from Alcaligenes xylosoxidans (axDTA) (Uhl et al 2015 ). DTA belongs to the alanine racemase family of PLP-dependent enzymes (fold type III), and its structure is different from l -specific TAs.…”