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How Many Ways to Craft a Cofactor?

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How Many Ways to Craft a Cofactor? Commentary How many ways to craft a cofactor? Judith P. Klinman* Departments of Chemistry and Molecular and Cell Biology, University of California, Berkeley, CA 94720 tandard textbooks define enzymatic two electrons, forming hydrogen peroxide Scofactors as low molecular weight as a final product (10). The tryptophan- structures that are separate from and can derived cofactors, which are found in the bind reversibly to their cognate protein. periplasmic space of Gram-negative bac- As with all established paradigms, change teria, catalyze electron transfer, one at a is almost guaranteed, and work over the time to an exogenous acceptor (11). The last decade has forced us to expand our presence of an active site metal ion, definition of cofactor to include structures Cu(II), in the TPQ- and LTQ-containing that are derived from the protein itself. proteins had originally led to the proposal Early studies of ribonucleotide reductase of a redox role for the metal ion in cofac- had indicated the presence of a protein- tor reoxidation. Recent studies implicate, derived tyrosyl radical as the storage site instead, a nonmetal binding site for O for the free radical that initiates the con- 2 version of ribonucleotides to deoxyribo- with the role of the active site metal being nucleotides (1). This was followed by the stabilization of reduced oxygen interme- equally unorthodox finding of a protein- diates (12). In contrast, the proteins con- bound glycyl radical in select anaerobic taining TTQ (13) and CTQ must exclude proteins (2). More complex posttransla- O2, directing electrons to their external tionally derived redox cofactors appeared acceptor. The structure by Datta et al. (4) on the scene in l990, with the discovery of shows how this intramolecular electron the tyrosine-derived cofactor TPQ in a Fig. 1. Quino-cofactors derived from protein- transfer may occur in the CTQ-containing eukaryotic copper amine oxidase (3) (Fig. bound tyrosine (TPQ, LTQ) and tryptophan (TTQ, quinohemoprotein, identifying an N- 1). The field of quino-cofactors has turned CTQ). terminal heme with a solvent-accessible out to be structurally rich, with variants edge and a buried heme closer to the CTQ ␥ being reported that are formed from tryp- philic sink, analogous to the cofactor pyr- cofactor on the subunit. tophan as well as tyrosine. In a recent idoxal phosphate, thereby increasing the The discovery of each of the structures issue of PNAS, Datta et al. (4) amaze us acidity of the ␣-proton of substrate (Fig. in Fig. 1 has generated some serious head further with a new quino-cofactor derived 2). In each case where a structure is avail- scratching as to how such structures may from the cross-linking of oxidized trypto- able for a quino-protein, an aspartic acid arise. Studies of the biogenesis of TPQ are phan and cysteine and designated CTQ side chain has been found to lie in close the most developed, indicating that addi- (Fig. 1). proximity to the cofactor, thereby impli- tion of copper ion and O2 are sufficient to This exciting discovery follows an ear- cating this residue as the catalytic base for generate TPQ from tyrosine (14, 15). An lier report of a cofactor in which a tryp- proton abstraction (8, 9). The x-ray struc- x-ray structure of an unprocessed protein, tophyl quinone is cross-linked to a second in which the active site Cu(II) has been tryptophan to form TTQ (5) (Fig. 1) and ture for the CTQ containing quinohemo- is related to the finding that tyrosine- protein amine dehydrogenase by Datta et replaced by the nonredox metal Zn(II), based quinone cofactors also have been al. (4) repeats this theme, showing aspar- shows the hydroxyl group of the precursor observed to be cross-linked to a second tic acid (Asp-33) as the singly charged tryosine complexed to the metal (16). amino acid side chain in proximity to the Kinetic and spectroscopic studies indicate amino acid, i.e., the lysine tyrosyl quinone ␥ quino-cofactor on the subunit. Remark- that binding of O2 is required for the in lysyl oxidase, LTQ (6) (Fig. 1). What is ␥ remarkable is the lack of both sequence ably, the side chain of Asp-33 is held in formation of a tyrosine-copper charge and structural homology among the ty- place by a covalent, thioether linkage at its transfer complex during productive bio- ␤ rosyl-containing and tryptophyl quinone- -carbon to the sulfur of a cysteine (Cys- genesis, implicating O2 as the trigger for ␥ containing proteins. It appears that nature 78 ). This theme of thioether formation is the initiation of biogenesis (17, 18). A has found multiple pathways to generate repeated at two other positions within the comparison of the precursor, zinc-con- ␥ cofactors that are chemically and mecha- subunit. taining structure to the mature, copper- nistically similar. In the mechanism shown in Fig. 2, the and TPQ-containing protein indicates The extensive mechanistic work on protein-bound cofactor has been con- only small and subtle structural changes TPQ has served as a guide to understand- verted to a reduced, aminoquinol form, (16). The TPQ-containing proteins are ing the catalytic role of the protein- which must be recycled to the initial oxi- truly dual-function enzymes, catalyzing derived quinones (7). In all cases, the dized cofactor to complete the catalytic both the production of their own cofactor substrates of these diverse enzymes are cycle. A divergence between the trypto- primary amines that are capable of cova- phan- and tyrosine-derived cofactors is lent adduct formation with the quinone the pathway for aminoquinol reoxidation. See companion article on page 14268 in issue 25 of volume functional group of cofactor. The resulting In the case of TPQ and LTQ, molecular 98. covalent complex contributes an electro- oxygen is the acceptor of two protons and *E-mail: [email protected]. 14766–14768 ͉ PNAS ͉ December 18, 2001 ͉ vol. 98 ͉ no. 26 www.pnas.org͞cgi͞doi͞10.1073͞pnas.011602498 Downloaded by guest on September 26, 2021 Fig. 2. Mechanism for reductive half-reaction of TPQ enzymes. and amine oxidation without significant oxyadenosyl radical may be critical, for tho-quinone is a consequence of the pro- structural rearrangement. example, in the production of the three tein active site structure in which the The proteins that contain the trypto- thioether linkages found to stabilize the quinone has evolved and is not driven by phan-derived cofactors are conspicuous mature quinohemoprotein amine dehy- the requirements for catalytic activity. by their lack of bound, redox active metal drogenase structure. A separate protein This raises the possibility of additional, ions as well as an absence of O2 binding may catalyze the production of an inter- alternate side chains, e.g., a histidine or during catalytic turnover. In the case of mediate, tryptophyl quinone, that would the carboxylates of aspartate and gluta- both TTQ and CTQ, exogenous proteins then be susceptible to nucleophilic attack mate, functioning as cross-linking agents. are almost certainly involved in cofactor by a neighboring Cys (33 ␥). In this man- Melville et al. (29) addressed this question formation. The lack, thus far, of x-ray data ner the tryptophan-cysteine cross-link by preparing a model for the carboxylate for the precursor forms of either a TTQ- seen in CTQ would arise from heterolytic analog of TPQ and LTQ, in which an ester COMMENTARY or CTQ-containing enzyme leaves open as opposed to homolytic chemistry (cf. ref. linkage replaces the hydroxyl group and the possibility of large structural differ- 26). A similar process has been proposed lysyl side chain, respectively, at the 6 po- ences between the precursor and mature to explain the structures seen in the ty- sition of the ring (refer to Fig. 3 for forms of protein. A recent x-ray study of rosine-derived cofactors, TPQ and LTQ numbering). The first hint that the car- unprocessed galactose oxidase (19), a pro- (6) (Fig. 3). boxylate ester derivative may be unsuit- tein that contains a cross-linked cofactor A compelling question in this field con- able as a cofactor was its hydrolytic insta- derived from cysteine and tyrosine in its cerns the mechanistic imperatives for the bility near neutral pH. More significantly, mature form (20), indicates significant production of derivatized quinones as co- the redox potential for the carboxylate structural differences that include the factors. In the case of the cysteine tyrosyl ester derivative is elevated to ϩ133 mV vs. presence of an N-terminal peptide that is radical seen in galactose oxidase, the SCE (29), in contrast to the redox poten- cleaved in the process of cofactor produc- thioether at the ortho position of the tials for models of cofactors known to ϭ tion (21). tyrosyl radical does not appear to alter the occur in protein active sites [e.g., Em Ϫ ϭ Datta et al. (4) identify four ORFs for redox potential appreciably (27) and may 150 mV for TPQ at pH 6.8 (30), Em Ϫ ϭ quinohemoprotein, three of which encode play primarily a structural role. In the case 182 for LTQ at pH 7.0 (6), and Em the subunits of the amine dehydrogenase. of the various quinones (Fig. 1), it is Ϫ150 for TTQ at pH 6.8 (31)]. This small A fourth ORF may provide the key to the conceivable that the requisite chemistry range of redox potentials for TPQ, LTQ, biogenesis of CTQ, showing weak but (Fig. 2) could be performed with underi- and TTQ model compounds suggests that significant homology to proteins that be- vatized ortho-quinones (e.g., 2 in Fig. 3) a potential of Ϫ150 to Ϫ180 mV is linked long to a newly identified superfamily (28).
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