Reply to Kiser: Dioxygen binding in NOV1 crystal structures

Abstract

In PNAS (1) Kiser has expressed some skepticism about the identity of the active-site dioxygen molecule and suggests that the density is better modeled with two water molecules at partial occupancy. In response, we have performed an extended analysis with the following results. There are currently 321 entries for proteins containing dioxygen (PDB OXY) in the Protein Data Bank (PDB). As might be expected, the electron density ranges from highly symmetric to highly irregular. For example, naphthalene 1,2-dioxygenase [PDB ID code 1O7N (2)], cited by Kiser as a comparable enzyme, has a dioxygen bound in a sideon manner to an iron in almost exactly the same orientation as the NOV1 structures with and without substrate (3). Like NOV1, the density surrounding the dioxygen is not completely symmetric, and there are many more examples in the PDB. Oxygen atoms from a dioxygen species that are in identical chemical environments have more regular density and similar B-factors, although the opposite is also true. In apo-NOV1 (PDB ID code 5J53), a solvent molecule is 2.4 Å from the oxygen atom that is most closely bound to the iron, and it may perturb the oxygen's position. This may also address Kiser's second critique, which is that the two oxygens have different B-factors and the oxygen closer to the iron has the high B-factor. Naphthalene 1,2-dioxygenase (PDB ID code 1O7N) also has a higher B-factor for the oxygen bound more closely to the iron, and a survey of OXY PDB entries shows that the B-factors are often quite different. Finally, Kiser reports that refinement of individual oxygen atoms or an unrestrained dioxygen results in O-O distances of ∼1.8 Å, which is longer than the expected 1.2 Å. However, as has been pointed out by others, unrestrained refinement should only be used for ultra-high-resolution structures with observations to parameters ratios of 10 or greater (4). Below that ratio, the uncertainty will be very large, and the results may be meaningless. To demonstrate this, we refined a number of related dioxygen-bound structures this way (Table 1). Naphthalene 1,2-dioxygenase (PDB ID code 1O7N) (2) and homogentisate 1,2-dioxygenase (PDB ID code 3ZDS) (5) both bind dioxygen similarly to NOV1. They refine to distances that are much higher (∼1.8 Å) or lower (∼0.9 Å) than dioxygen. When we performed the same refinement on apo-NOV1 (PDB ID code 5J53), we did not obtain the distances reported by Kiser. They were only slightly larger than standard dioxygen (Table 1), and we note that when the occupancy of the dioxygen in apo-NOV1 (PDB ID code 5J53) is lowered the distances converge on 1.2 Å (Table 1) and the B-factors also decrease. The occupancies of the dioxygen were refined in both NOV1 structures, but at the data resolutions involved B-factors and occupancies are highly correlated (4). Therefore, it is possible that the dioxygen could be modeled with an occupancy slightly lower than the refined value of 0.9. In summary, our original paper and the analyses presented here show that Kiser's statements in no way pre...

Evolutionary aspects and enzymology of metazoan carotenoid cleavage oxygenases

“…Dioxygen was observed in two crystal structures (VP14 and NOV1), with or without substrate and binding of substrate did not cause significant conformational changes to the enzymes [22,53]. The question of the presence of dioxygen in NOV1 structure is still open to discussion in the literature [56,57]. Substrate binding in the stilbenoid-cleaving NOV2, CAO1 and apocarotenoid ACO oxygenases did not induce a significant electronic change in the iron as observed by Mossbauer spectroscopy, nor did it induce significant protein structural changes and therefore such gating mechanisms are not likely to apply for these enzymes [54].…”

Caulobacter segnis Dioxygenase CsO2: A Practical Biocatalyst for Stilbenoid Ozonolysis

“…The resulting plasmid was then inserted in E. coli BL21 (DE3) cells, for a classic expression mediated by isopropyl‐β‐D‐thiogalactopiranoside (IPTG) under the control of T7 promoter, producing a largely insoluble fraction [19] . Improvements have been observed through co‐expression with the chaperonin gene groEL and the co‐chaperonin gene groES (plasmid pGRO, Takara Bio), although better results were obtained using pETDuet‐1 to yield a gene product without the His‐tag, completely losing the possibility for a rapid purification via conventional ion affinity chromatography [17] . A change of the vector system (pET26b(+)), with consequent change of the position of the His‐tag (from C‐ to N‐terminus), gave a stable and soluble CsO2 in good yields (50 mg starting from 3.0 g of wet cell paste) (Figure S1).…”

“…Solubility of organic substrates is often a major issue in aqueous biotransformations; therefore, the influence of different co‐solvents on CsO2 activity was studied (Table S1). Acetone maintained the same initial enzymatic activity, which was negatively affected by the other water‐miscible solvents, including DMSO which was previously used for CsO2‐catalyzed oxidation of 4‐vinylguiacol [17–20] …”

“…The search for CCD homologues led to the discovery of enzymes that, albeit not able to cleave C=C of carotenoids, cleave α‐β double bond of stilbene derivatives (lignostilbene dioxygenases, LSDs) [15] . One of the first LSDs was found in Sphingomonas paucimobilis , [16] and subsequently an LSD from Novosphingobium aromaticivorans (NOV1) was characterized [17] . NOV1 is able to catalyze the cleavage of double bonds of a wide range of stilbene‐like compounds with a 4′‐OH group.…”

Mechanistic insights into dioxygen activation by a manganese corrole complex: a broken-symmetry DFT study

“…[3][4][5][6] Notably, ironoxygen species have been trapped and characterized spectroscopically in some dioxygen-activating heme and nonheme enzymes. [7][8][9][10] Although, various metal-oxygen intermediates have been successfully synthesized in biomimetic studies, 11 these synthetic metal-oxygen complexes were usually generated using articial oxidants, such as iodosylbenzene (PhIO), NaOCl, peracids, and H 2 O 2 . [12][13][14] As such, signicant efforts have been made to use O 2 as the source of oxidant in biomimetic oxidation reaction over the last few decades.…”