Metalloenzymes, crystal structure

Lieberman, R.L. and Rosenzweig, A.C. (2005) Crystal structure of a membrane-bound metalloenzyme that catalyses the biological oxidation of methane, Nature, 434, 177-182. 

Metalloenzymes, 33 40, see also Enzymes crystal structure, 44 230-258 DNA repair, 45 251 inhibitors, 36 40-42 molybdenum, 45 2, 53, 60-63 nuleic acid hydrolysis, 45 251-252 superoxide dismustases and, 45 130 zinc-containing, hard and soft acid-base behavior, 42 103-109 Metalloids... 

An increasing number of crystal structures of metalloenzymes have been reported, with more molecular structures of the metal active centers. We believe, however, that this is only the beginning of a new bioinorganic chemistry. The structural data provide more questions... 

The alcohol dehydrogenases are zinc metalloenzymes of broad specificity. They oxidize a wide range of aliphatic and aromatic alcohols to their corresponding aldehydes and ketones, using NAD+ as a coenzyme (see equation 16.1). The two most studied enzymes are those from yeast and horse liver. The crystal structures... 

The most important aspect of the study of Co(II) metalloenzymes is the possibility of using the metal ion as a functional, built-in reporter of the dynamics of the active site. The spectral and magnetic properties of Co (II) carbonic anhydrase have given valuable clues to the catalytic function of this enzyme. The recent studies of Co(II) alkaline phosphatase and Co (II) carboxypeptidase A indicate the general applicability of this approach to enzymes where the probe properties of the constitutive metal ion are poor. The comparison of the absorption spectra of these enzymes and low-molecular weight models have shown that the proteins provide irregular, and in some cases nearly tetrahedral environments. It is obvious, however, that a knowledge of the crystal structures of the enzymes is necessary before the full potential of this method can be exploited. 

Metalloenzymes pose a particular problem to both experimentalists and modelers. Crystal structures of metalloenzymes typically reveal only one state of the active site and the state obtained frequently depends on the crystallization conditions. In some cases, states probably not relevant to any aspect of the mechanism have been obtained, and in many cases it may not be possible to obtain states of interest, simply because they are too reactive. This is where molecular modeling can make a unique contribution and a recent study of urease provides a good example of what can be achieved119 1. A molecular mechanics study of urease as crystallized revealed that a water molecule was probably missing from the refined crystal structure. A conformational search of the active site geometry with the natural substrate, urea, bound led to the determination of a consensus binding model[I91]. Clearly, the urea complex cannot be crystallized because of the rate at which the urea is broken down to ammonia and, therefore, modeling approaches such as this represent a real contribution to the study of metalloenzymes. 

Fig. 7.2 Tlie crystal structure of mammalian Ser/Thr protein phosphatase-1, complexed with the toxin mycrocystin was determined at 2.1 A resolution. PPl has a single domain with a fold, distinct from that of the protein tyrosine phosphatases. The Ser/Thr protein phosphatase-1, is a metalloenzyme with two metal ions positioned at the active site with the help of a p-a-p-o-p scaffold. A dinuclear ion centre consisting of Mn2+ And Fe2+ g situated at the catalytic site that binds the phosphate moiety of the substrate. Ser/Thr phosphatases, PPl and PP2A, are inhibited by the membrane-permeable ocadaic acid and by cyclic hexapeptides, known as microcystins. The toxin molecule is depicted as a ball-and-stick structure. On the left and on the ri t, two different views of the same molecule are shown. Microcystin binds to three distinct regions of the phosphatase to the metaLbinding site, to a hydrophobic groove, and to the edge of a C-terminal groove in the vicinity of the active site. At the surface are binding sites for substrates and inhibitors. These ribbon models are reproduced vnth permission of the authors and Nature from ref. 9.

The molecular details of the action of metalloenzymes have begun to be elucidated in the past few years (42). Crystal structures for bovine carboxypeptidase A (43), thermolysin (44), and horse liver alcohol dehydrogenase (45) are now available, and chemical and kinetic studies have defined the role of zinc in substrate binding and catalysis. In fact, many of the significant features elucidating the mode of action of enzymes in general have been defined at the hands of zinc metalloenzymes. 

The crystal structure of the catalytic subunit of mammalian protein phosphatase-1 complexed with microcystin-LR has recently been determined at 2.1 A resolution (139). The metalloenzyme exhibits a fold unrelated to the known structures of catalytic subunits of tyrosine phosphatases. The two metal ions are positioned in a central p-a-P -a-P scaffold at the active site, from which three surface grooves protrude as potential binding sites for substrates and inhibitors. The C-terminus of the catalytic subunit is located at the end of one of these grooves, so that regulatory sequences following this domain may possibly modulate the function. This fold is expected to be closely preserved in the protein phosphatases 2A and 2B. 

Metalloenzymes containing non-heme iron centers are widespread in nature. Several members of this family isolated from mammals, plants, or bacteria, have now been structurally characterized (4). In addition to the crystal structures for the isolated resting states, an increasing amount of spectroscopic information has become available concerning the active sites of these iron enzymes (25-27). The non-heme iron enzymes perform a broad range of functions, but most important is their role in the activation of dioxygen for... 

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