Structure of alanine dehydrogenase from Archaeoglobus: active site analysis and relation to bacterial cyclodeaminases and mammalian mu crystallin.

Biotechnology Division of the National Institute of Standards and Technology, Gaithersburg, MD 20899-8312, USA.
Journal of Molecular Biology (Impact Factor: 3.96). 10/2004; 342(1):119-30. DOI: 10.1016/j.jmb.2004.06.090
Source: PubMed

ABSTRACT The hyperthermophilic archaeon Archaeoglobus fulgidus contains an L-Ala dehydrogenase (AlaDH, EC that is not homologous to known bacterial dehydrogenases and appears to represent a previously unrecognized archaeal group of NAD-dependent dehydrogenases. The gene (Genbank; TIGR AF1665) was annotated initially as an ornithine cyclodeaminase (OCD) on the basis of strong homology with the mu crystallin/OCD protein family. We report the structure of the NAD-bound AF1665 AlaDH (AF-AlaDH) at 2.3 A in a C2 crystal form with the 70 kDa dimer in the asymmetric unit, as the first structural representative of this family. Consistent with its lack of homology to bacterial AlaDH proteins, which are mostly hexameric, the archaeal dimer has a novel structure. Although both types of AlaDH enzyme include a Rossmann-type NAD-binding domain, the arrangement of strands in the C-terminal half of this domain is novel, and the other (catalytic) domain in the archaeal protein has a new fold. The active site presents a cluster of conserved Arg and Lys side-chains over the pro-R face of the cofactor. In addition, the best ordered of the 338 water molecules in the structure is positioned well for mechanistic interaction. The overall structure and active site are compared with other dehydrogenases, including the AlaDH from Phormidium lapideum. Implications for the catalytic mechanism and for the structures of homologs are considered. The archaeal AlaDH represents an ancient and previously undescribed subclass of Rossmann-fold proteins that includes bacterial ornithine and lysine cyclodeaminases, marsupial lens proteins and, in man, a thyroid hormone-binding protein that exhibits 30% sequence identity with AF1665.

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    ABSTRACT: L-Ornithine cyclodeaminase (OCD) is involved in L-proline biosynthesis and catalyzes the unique deaminating cyclization of L-ornithine to L-proline via a Δ1-pyrroline-2-carboxyrate (Pyr2C) intermediate. Although this pathway functions in only a few bacteria, many archaea possess OCD-like genes (proteins), among which only AF1665 protein (gene) from Archaeoglobus fulgidus has been characterized as an NAD+-dependent L-alanine dehydrogenase (AfAlaDH). However, the physiological role of OCD-like proteins from archaea has been unclear. Recently, we revealed that Pyr2C reductase, involved in trans-3-hydroxy-L-proline (T3LHyp) metabolism of bacteria, belongs to the OCD protein superfamily and catalyzes only the reduction of Pyr2C to L-proline (no OCD activity) [FEBS Open Bio (2014) 4, 240-250]. In this study, based on bioinformatics analysis, we assumed that the OCD-like gene from Thermococcus litoralis DSM 5473 is related to T3LHyp and/or proline metabolism (TlLhpI). Interestingly, TlLhpI showed three different enzymatic activities: AlaDH; N-methyl-L-alanine dehydrogenase; Pyr2C reductase. Kinetic analysis suggested strongly that Pyr2C is the preferred substrate. In spite of their similar activity, TlLhpI had a poor phylogenetic relationship to the bacterial and mammalian reductases for Pyr2C and formed a close but distinct subfamily to AfAlaDH, indicating convergent evolution. Introduction of several specific amino acid residues for OCD and/or AfAlaDH by site-directed mutagenesis had marked effects on both AlaDH and Pyr2C reductase activities. The OCC_00387 gene, clustered with the TlLhpI gene on the genome, encoded T3LHyp dehydratase, homologous to the bacterial and mammalian enzymes. To our knowledge, this is the first report of T3LHyp metabolism from archaea.
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    ABSTRACT: trans-4-Hydroxy-L-proline (T4LHyp) and trans-3-hydroxy-L-proline (T3LHyp) occur mainly in collagen. A few bacteria can convert T4LHyp to α-ketoglutarate, and we previously revealed a hypothetical pathway consisting of four enzymes at the molecular level (J Biol Chem (2007) 282, 6685-6695; J Biol Chem (2012) 287, 32674-32688). Here, we first found that Azospirillum brasilense has the ability to grow not only on T4LHyp but also T3LHyp as a sole carbon source. In A. brasilense cells, T3LHyp dehydratase and NAD(P)H-dependent Δ1-pyrroline-2-carboxylate (Pyr2C) reductase activities were induced by T3LHyp (and D-proline and D-lysine) but not T4LHyp, and no effect of T3LHyp was observed on the expression of T4LHyp metabolizing enzymes: a hypothetical pathway of T3LHyp→Pyr2C→L-proline was proposed. Bacterial T3LHyp dehydratase, encoded to LhpH gene, was homologous with the mammalian enzyme. On the other hand, Pyr2C reductase encoded to LhpI gene was a novel member of ornithine cyclodeaminase/μ-crystallin superfamily, differing from known bacterial protein. Furthermore, the LhpI enzymes of A. brasilense and another bacterium showed several different properties, including substrate and coenzyme specificities. T3LHyp was converted to proline by the purified LhpH and LhpI proteins. Furthermore, disruption of LhpI gene from A. brasilense led to loss of growth on T3LHyp, D-proline and D-lysine, indicating that this gene has dual metabolic functions as a reductase for Pyr2C and Δ1-piperidine-2-carboxylate in these pathways, and that the T3LHyp pathway is not linked to T4LHyp and L-proline metabolism.
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