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Article

The Crystal Structure of NAD(P)H Oxidase from Lactobacillus sanfranciscensis: Insights into the Conversion of O₂ into Two Water Molecules by the Flavoenzyme^†^,^‡

Supporting Info

George T. Lountos,^§ Rongrong Jiang,^Δ William B. Wellborn, Tracey L. Thaler,^§ Andreas S. Bommarius,*^§ and Allen M. Orville*^§^#

School of Chemistry and Biochemistry, School of Chemical and Biomolecular Engineering, and the Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia 30332

Biochemistry, 2006, 45 (32), pp 9648–9659

DOI: 10.1021/bi060692p

Publication Date (Web): July 20, 2006

†

This work was supported by the Georgia Tech Research Corporation, the Office of the Vice Provost for Research, Georgia Institute of Technology, an American Chemical Society Petroleum Research Fund type G grant (40310-G4), and an American Heart Association Grant in Aid (0555286B) to A.M.O. Support was provided to A.S.B. by the Georgia Tech Research Corporation and the Office of Vice Provost for Research. G.T.L. was supported in part by a U.S. Department of Education GAANN fellowship. W.B.W. was supported by the Undergraduate Research Scholar Program of the NSF.

‡

The atomic coordinates and structure factors have been deposited in the Protein Data Bank with the corresponding file names 2cdu and 2cdusf.

School of Chemistry and Biochemistry and the Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology.

Current address: Macromolecular Crystallography Laboratory, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD 21702-1201.

School of Chemical and Biomolecular Engineering and the Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology.

Current address: School of Chemical and Biomedical Engineering, Nanyang Technological University, 16 Nanyang Drive, Singapore 637722.

Current address: Biocatalytics, Inc., 129 Hill Ave., Suite 103, Pasadena, CA 91106-1955.

To whom correspondence should be addressed. A.M.O.: e-mail, amorv@bnl.gov; phone, 631-344-4739; fax, 631-344-2741. A.S.B.: e-mail, andreas.bommarius@chbe.gatech.edu; phone, 404-385-1334; fax, 404-894-2291.

Current address: Biology Department, Brookhaven National Laboratory, Upton, NY 11973.

Abstract

The FAD-dependent NAD(P)H oxidase from Lactobacillus sanfrancisensis (L.san-Nox2) catalyzes the oxidation of 2 equivalents of either NADH or NADPH and reduces 1 equivalent of O₂ to yield 2 equivalents of water. During steady-state turnover only 0.5% of the reducing equivalents are detected in solution as hydrogen peroxide, suggesting that it is not released from the enzyme after the oxidation of the first equivalent of NAD(P)H and reaction with O₂. Here we report the crystal structure of L.san-Nox2 to 1.8 Å resolution. The enzyme crystallizes as a dimer with each monomer consisting of a FAD binding domain (residues 1−120), a NAD(P)H binding domain (residues 150−250), and a dimerization domain (residues 325−451). The electron density for the redox-active Cys42 residue located adjacent to the si-face FAD is consistent with oxidation to the sulfenic acid (Cys-SOH) state. The side chain of Cys42 is also observed in two conformations; in one the sulfenic acid is hydrogen bonded to His10 and in the other it hydrogen bonds with the FAD O2‘ atom. Surprisingly, the NAD(P)H binding domains each contain an ADP ligand as established by electron density maps and MALDI-TOF analysis of the ligands released from heat-denatured enzyme. The ADP ligand copurifies with the enzyme, and its presence does not inhibit enzyme activity. Consequently, we hypothesize that either NADPH or NADH substrates bind via a long channel that extends from the enzyme exterior and terminates at the FAD re-face. A homology model of the NADH oxidase from Lactococcus lactis (L.lac-Nox2) was also generated using the crystal structure of L.san-Nox2, which reveals several important similarities and differences between the two enzymes. HPLC analysis of ligands released from denatured L.lac-Nox2 indicates that it does not bind ADP, which correlates with the specificity of the enzyme for oxidation of NADH.