Biochemical characterization of a dihydroneopterin aldolase used for methanopterin biosynthesis in methanogens

Abstract

The gene encoding 7,8-dihydroneopterin aldolase (DHNA) was recently identified in archaea through comparative genomics as being involved in methanopterin biosynthesis (V. Crécy-Lagard, G. Phillips, L. L. Grochowski, B. El Yacoubi, F. Jenney, M. W. Adams, A. G. Murzin, and R. H. White, ACS Chem. Biol. 7:1807-1816, 2012, doi:10.1021/cb300342u). Archaeal DHNA shows a unique secondary and quaternary structure compared with bacterial and plant DHNAs. Here, we report a detailed biochemical examination of DHNA from the methanogen Methanocaldococcus jannaschii. Kinetic studies show that M. jannaschii DHNA possesses a catalytic capability with a kcat/Km above 10(5) M(-1) s(-1) at 70°C, and at room temperature it exhibits a turnover number (0.07 s(-1)) comparable to bacterial DHNAs. We also found that this enzyme follows an acid-base catalytic mechanism similar to the bacterial DHNAs, except when using alternative catalytic residues. We propose that in the absence of lysine, which is considered to be the general base in bacterial DHNAs, an invariant water molecule likely functions as the catalytic base, and the strictly conserved His35 and Gln61 residues serve as the hydrogen bond partners to adjust the basicity of the water molecule. Indeed, substitution of either His35 or Gln61 causes a 20-fold decrease in kcat. An invariant Tyr78 is also shown to be important for catalysis, likely functioning as a general acid. Glu25 plays an important role in substrate binding, since replacing Glu25 by Gln caused a ≥25-fold increase in Km. These results provide important insights into the catalytic mechanism of archaeal DHNAs.

FIG 1

(A) The structure of tetrahydromethanopterin (H₄MPT); (B) biosynthesis of the pterin portion of methanopterin. DHNP, 7,8-dihydroneopterin; H₂HMP, 7,8-dihydro-6-hydroxymethylpterin; H₂HMP-PP, 7,8-dihydro-6-hydroxymethylpterin pyrophosphate.

FIG 2

Reaction mechanisms for class I, II, and III aldolases.

FIG 3

(A) pH dependence of k_cat/K_m for wild-type DHNA from M. jannaschii; (B) pH dependence of k_cat/K_m for the H35N and Y78F variants. Data were obtained by measuring the rate of product formation at various pHs by incubating 15 ng of wild-type DHNA (500 ng H35N or Y78F variant) in 25 mM TES-tricine-CAPS buffer from pH 5.9 to 10.5; at each pH, the enzyme reacted with various DHNP concentrations (1 to 200 μM) for 10 min at 70°C. The k_cat/K_m was calculated by fitting the data to the Michaelis-Menten equation, and the pH profiles of wild-type and H35N enzymes were fitted to equation 1, and the pH profile of the Y78F enzyme was fitted to equation 2.

FIG 4

(A) Comparison of bacterial (left) and archaeal (right) DHNAs at the levels of protein sequence and secondary structure assignment (blue arrows represent β-sheet structure, green helices represent α-helices; the secondary structure assignment was performed by Phyre2 [38]); (B) secondary structure (cyan highlights α-helices while hot pink represents β-sheet); (C) quaternary structure (the four different colors highlight four identical subunits). Two bacterial DHNA tetrameric rings “head to head” stack together and assemble into a hollow cylinder octamer. Only one tetrameric ring is shown here. For archaeal DHNA, four subunits assemble into a compact homotetramer instead. Red boxes indicate the locations of the active sites.

FIG 5

Comparison of the active sites of archaeal (A) and bacterial (B) DHNAs. In panel A, the protein coordinates are from the structure of M. jannaschii DHNA (PDB accession no. 2OGF). The product molecule H₂HMP (from PDB accession no. 1HQ2) was docked to the DHNA putative active site by Autodock Vina built-in Chimera (39, 40). Secondary structures and residues in green are from subunit A; those in orange are from subunit C; and Tyr 78, highlighted in yellow, is from subunit B. In panel B, coordinates are for the structure of bacterial DHNA from Vibrio cholerae O1 biovar El Tor strain N16961 (PDB accession no. 3O1K with aligned H₂HMP [PDB accession no. 2DHN] in the active site). Numbering reflects the DHNA from S. aureus. Secondary structures in green are from subunit C, and those in orange are from subunit A. Red spheres represent water molecules. Dotted lines indicate inferred polar contacts.