Molecular and Mechanistic Characterization of PddB, the First PLP-Independent 2,4-Diaminobutyric Acid Racemase Discovered in an Actinobacterial D-Amino Acid Homopolymer Biosynthesis

Abstract

We recently disclosed that the biosynthesis of antiviral γ-poly-D-2,4-diaminobutyric acid (poly-D-Dab) in Streptoalloteichus hindustanus involves an unprecedented cofactor independent stereoinversion of Dab catalyzed by PddB, which shows weak homology to diaminopimelate epimerase (DapF). Enzymological properties and mechanistic details of this enzyme, however, had remained to be elucidated. Here, through a series of biochemical characterizations, structural modeling, and site-directed mutageneses, we fully illustrate the first Dab-specific PLP-independent racemase PddB and further provide an insight into its evolution. The activity of the recombinant PddB was shown to be optimal around pH 8.5, and its other fundamental properties resembled those of typical PLP-independent racemases/epimerases. The enzyme catalyzed Dab specific stereoinversion with a calculated equilibrium constant of nearly unity, demonstrating that the reaction catalyzed by PddB is indeed racemization. Its activity was inhibited upon incubation with sulfhydryl reagents, and the site-directed substitution of two putative catalytic Cys residues led to the abolishment of the activity. These observations provided critical evidence that PddB employs the thiolate-thiol pair to catalyze interconversion of Dab isomers. Despite the low levels of sequence similarity, a phylogenetic analysis of PddB indicated its particular relevance to DapF among PLP-independent racemases/epimerases. Secondary structure prediction and 3D structural modeling of PddB revealed its remarkable conformational analogy to DapF, which in turn allowed us to predict amino acid residues potentially responsible for the discrimination of structural difference between diaminopimelate and its specific substrate, Dab. Further, PddB homologs which seemed to be narrowly distributed only in actinobacterial kingdom were constantly encoded adjacent to the putative poly-D-Dab synthetase gene. These observations strongly suggested that PddB could have evolved from the primary metabolic DapF in order to organize the biosynthesis pathway for the particular secondary metabolite, poly-D-Dab. The present study is on the first molecular characterization of PLP-independent Dab racemase and provides insights that could contribute to further discovery of unprecedented PLP-independent racemases.

Keywords: 2; 4-diaminobutyric acid; D-amino acid; PLP-independent; amino acid racemase; biosynthesis; diaminopimelate epimerase; homo poly-amino acid; secondary metabolite.

FIGURE 1

The biosynthesis of Poly-D-Dab. (A) Periphery of the genes involved in the Poly-D-Dab biosynthesis in S. hindustanus NBRC15115. The genes coding for enzymes responsible for the biosynthesis of Poly-D-Dab (PddA to PddD) are highlighted in blue. (B) Poly-D-Dab biosynthesis, which partially shares the primary metabolic lysine biosynthetic diaminopimelate pathway. Racemization of Dab catalyzed by secondary metabolic PddB and epimerization of diaminopimelate catalyzed by primary metabolic DapF are comparably shown. Ask, aspartate kinase; Asd, aspartate-semialdehyde dehydrogenase; LysA, meso-diaminopimelic acid decarboxylase.

FIGURE 2

Purification and physicochemical characterization of PddB. (A) Purity of wild-type PddB and its mutants evaluated in this study. The purified proteins were analyzed on SDS-PAGE and stained with Coomassie Brilliant Blue R-250. Lane 1, molecular weight marker; lane 2, wild-type PddB; lane 3, C101A mutant; lane 4, C233S mutant. (B) Elution behavior of PddB on size-exclusion chromatography. (C) Absorption spectrum of PddB.

FIGURE 3

LC-MS analysis of Dab racemase reactions catalyzed by PddB. (A) Stereoinversions from 20 mM of L-Dab (black arrowheads) and from D-Dab (white arrowheads) monitored at 340 nm are shown the in upper and lower panels, respectively. All peaks presented exhibited 707.3 m/z as (M + H)⁺, which corresponded to Dab doubly labeled with FDLA, and no singly labeled Dab was detected. (B) Transition of the stereoinversion reactions from 20 mM of L-Dab (closed circle with a solid line) and from D-Dab (open circle with a dotted line) calculated based on the LC chromatograms are shown in the upper and lower panels, respectively.

FIGURE 4

Amino acid sequence and structural models of PddB. (A) Amino acid sequence alignment of PddB and DapF from Haemophilus influenzae (Hi-DapF) with their secondary structural comparison. Helices and sheets predicted by the Dictionary of Secondary Structure of Proteins (DSSP) algorithm are marked as blue tubes and green arrows, respectively. In the Hi-DapF sequence, amino acid residues which have been reported to interact with the substrate via hydrogen bonds or a salt bridge are highlighted in red. (B) 3D structure of Hi-DapF in complex with an irreversible inhibitor LL-AziDAP (PDB ID: 2GKE). (C) 3D structural model of PddB. The model was built in the SWISS-MODEL server using the structure of Hi-DapF as a template.

FIGURE 5

Proposed reaction mechanism of PddB that employs the thiolate-thiol pair in the active site. The two catalytic Cys residues interconvert L-Dab and D-Dab through a transient planar carbanion intermediate as indicated within parentheses. This intermediate can be derived from either isomer. Fundamentally, the base (thiolate) deprotonates the substrate C^α, which can then be protonated via the acid (thiol) of the other Cys residue. The arrows represent electronic rearrangements relating to the racemization.

FIGURE 6

Phylogenetic tree analysis of the selected PLP-independent racemases/epimerases. The tree was generated in Genetyx software with the Unweighted Pair Group Method using the arithmetic Average (UPGMA) method. Ba-GluR, glutamate racemase from Bacillus anthracis; Ec-GluR, glutamate racemase from Escherichia coli; Ls-AspR, aspartate racemase from Lactobacillus sakei; Tl-AspR, aspartate racemase from Thermococcus litoralis; Hi-DapF, DapF from Haemophilus influenzae; Ec-DapF, DapF from E. coli; Cd-ProR, proline racemase from Clostridioides difficile; Tl-ProR, proline racemase from T. litoralis; Ab-HypE, L-hydroxyproline 2-epimerase from Azospirillum brasilense.

FIGURE 7

Predictive active site model for PddB. (A) Schematic representation of interactions between the non-reacting stereocenter of LL-AziDap and the six amino acid residues within the active-site cavity of Hi-DapF (PDB ID: 2GKE). Hydrogen bonds and a salt bridge are shown in blue dotted lines and brown dotted line, respectively. (B) Active site model for PddB generated in the SWISS-MODEL server using the structure of Hi-DapF as a template. Configuration of amino acid residues corresponding to the six active site residues in Hi-DapF are schematically shown.