Refinement of 3D models of horseradish peroxidase isoenzyme C: Predictions of 2D NMR assignments and substrate binding sites

Abstract

In this study, two alternative three-dimensional (3D) models of horseradish peroxidase (HRP-C)-differing mainly in the structure of a long untemplated insertion-were refined, systematically assessed, and used to make predictions that can both guide and be tested by future experimental studies. A key first step in the model-building process was a procedure for multiple sequence alignment based on structurally conserved regions and key conserved residues, including those side chains providing ligands to the two Ca2+ binding sites. The model refinements reported here include (1) optimization of side-chain conformations; (3) addition of structural waters using a template-independent procedure; (2) structural refinement of the untemplated 34 amino acid insertion located between the F and G helices, using both energy criteria and NMR data; (4) unconstrained energy optimization of the refined models. Using these procedures, two refined structures of HRP-C were obtained, differing mainly in the conformation of this long insertion. The presence of residues in this insertion that could potentially interact with bound substrates suggests a functional role that may be related to the general ability of class III peroxidases to form stable 1:1 complexes with a variety of substrates. The structural validity of the models was systematically assessed by a variety of criteria. Most notably, the ProsaII z scores and Profiles 3D scores of the two HRP-C models indicated that they are significantly better than would be obtained by simple amino acid replacement, using any of the known structures as a template. These two 3D HRP-C models, were then used to predict candidate residues for the assignment of NOESY cross-peaks previously noted in 2D-NMR studies. Specifically, the residues known as Ile X, Phe A, Phe B, aliphatic residue Q, and Ile T. Candidate substrate binding sites were also identified and compared with experimentally based predictions. This work is timely because new X-ray structures are anticipated that will facilitate the validation of these procedures.

Chemical, Spectroscopic and Structural Investigation of the Substrate‐Binding Site in Ascorbate Peroxidase

“…The elution of the haem adducts was monitored at 400 nm in a silica tlow cell (Hellma, 80 PI) and Shimadzu 1602 spectrophotometer linked to a personal computer. The resultant peaks were identified by comparison with the haem adducts obtained from a sample of HRP treated in the same way and assigned previously (Ator and Ortiz de Montellano, 1987a;Gilfoyle et al, 1996). Percentage modification was based upon integration of the peaks by software from the manufacturer.…”

“…Samples of APX or HRP (0.25 ml, 25 pM) were allowed to turnover in the presence of phenylhydrazine (0.6 mM) and hydrogen peroxide (0.2 mM). The progress of enzyme inactivation was followed by means of an assay (Gilfoyle et al, 1996) was necessary. Samples of inactivated APX were brought immediately to pH 2.0 in 200 mM glycine containing 6 M guanidine hydrochloride (0.25 ml).…”

“…electronic absorption (Leigh et al, 1975;Schejter et al, 1976;Saxena et al, 1990;Paul and Ohlsson, 1978;Adak et al, 1995), vibrational (Kitagawa et al, 1983;Teroaka and Kitagawa, 1981 ;Smulevich et al, 1991 ;Holzbaur et al, 1996) and fluorescence (Fidy and Paul, 1989) spectroscopies, molecular modelling (Zhao et al, 1996), and chemical modification (Adak et al, 1996;Ator and Ortiz de Montellano, 1987a, b ;Bhattacharyya et al, 1993). NMR spectroscopy of HRP has been particularly informative in this context (Sakurada et al, 1986;Saxena et al, 1990;Schejter et al, 1976;Morishima et al, 1977;Morishima and Ogawa 1979;Leigh et al, 1975;La Mar et al, 2992Veitch, 1995;de Ropp et al, 1995), especially when combined with site-directed mutagenesis (Veitch et al, 1995 ;Gilfoyle et al, 1996).…”

“…NMR spectroscopy of HRP has been particularly informative in this context (Sakurada et al, 1986;Saxena et al, 1990;Schejter et al, 1976;Morishima et al, 1977;Morishima and Ogawa 1979;Leigh et al, 1975;La Mar et al, 2992Veitch, 1995;de Ropp et al, 1995), especially when combined with site-directed mutagenesis (Veitch et al, 1995 ;Gilfoyle et al, 1996). Substrates are bound within NOE distance of the haem protons and are known to interact with an unassigned phenylalanine residue, thought to be Phe179 or Phe221.…”

Metabolism of Bergamottin by Cytochromes P450 2B6 and 3A5

“…Waters were then introduced using in-house software that places waters based on topological and energy metrics (Zhao et al, 1996). The resulting initial structure with modeled waters was then energy minimized for 150 steps of steepest descents minimization followed by 3000 steps of conjugate gradient minimization.…”