The Role of Active Site Glutamate Residues in Catalysis of Rhodobacter capsulatus Xanthine Dehydrogenase

Silke Leimkühler,Amy L Stockert,Kiyohiko Igarashi,Takeshi Nishino,Russ Hille

doi:10.1074/jbc.m405778200

Abstract

Xanthine dehydrogenase (XDH) from the bacterium Rhodobacter capsulatus catalyzes the hydroxylation of xanthine to uric acid with NAD+ as the electron acceptor. R. capsulatus XDH forms an (alphabeta)2 heterotetramer and is highly homologous to homodimeric eukaryotic xanthine oxidoreductases. Here we first describe reductive titration and steady state kinetics on recombinant wild-type R. capsulatus XDH purified from Escherichia coli, and we then proceed to evaluate the catalytic importance of the active site residues Glu-232 and Glu-730. The steady state and rapid reaction kinetics of an E232A variant exhibited a significant decrease in both kcat and kred as well as increased Km and Kd values as compared with the wild-type protein. No activity was determined for the E730A, E730Q, E730R, and E730D variants in either the steady state or rapid reaction experiments, indicating at least a 10(7) decrease in catalytic effectiveness for this variant. This result is fully consistent with the proposed role of this residue as an active site base that initiates catalysis.

Highlights

Xanthine dehydrogenase (XDH) from the bacterium Rhodobacter capsulatus catalyzes the hydroxylation of xanthine to uric acid with NAD؉ as the electron acceptor
We first describe reductive titration and steady state kinetics on recombinant wild-type R. capsulatus XDH purified from Escherichia coli, and we proceed to evaluate the catalytic importance of the active site residues Glu-232 and Glu-730
To obtain molybdenum-saturated R. capsulatus XDH, enzyme purified by nickel-nitrilotriacetic acid and Q-Sepharose chromatography was applied to a folate affinity column

Summary

EXPERIMENTAL PROCEDURES

Protein Purification—R. capsulatus XDH was purified using the procedure described by Leimkuhler et al [6] with some modifications. The inactive form of XDH was eluted from the affinity column with 20 mM Tris, 0.2 mM EDTA, pH 7.8, containing 10% pyrophosphate. The fully active form of XDH was eluted with 20 mM Tris, 0.2 mM EDTA, pH 7.8, containing 15% pyrophosphate and 20 mM salicylate. Both fractions were separately concentrated using ultrafiltration, gel filtered using a PD-10 gel filtration column (Amersham Biosciences), and stored in 20 mM Tris, 0.2 mM EDTA, 2.5 mM dithiothreitol, pH 7.8. For recombinant wild-type XDH, enzyme concentrations of 4 –5 ␮M were mixed in equal volumes of various concentrations of xanthine or NADH. Thiocyanate (complexed with iron) was quantitated by A460 using an SCN standard curve

RESULTS

XDH variant

FeSIIred KdSNADH

DISCUSSION

NDb ND ND ND