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The DMSO Reductase Family of Microbial Molybdenum Enzymes Alastair G

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The DMSO Reductase Family of Microbial Molybdenum Enzymes Alastair G SHOWCASE ON RESEARCH The DMSO Reductase Family of Microbial Molybdenum Enzymes Alastair G. McEwan and Ulrike Kappler Centre for Metals in Biology, School of Molecular and Microbial Sciences, University of Queensland, QLD 4072 Molybdenum is the only element in the second row of led to a division of the oxotransferases into the sulfite transition metals which has a defined role in biology. It dehydrogenase and the dimethylsulfoxide (DMSO) exhibits redox states of (VI), (V) and (IV) within a reductase families (Fig. 1) (4). The Mo hydroxylases biologically-relevant range of redox potentials and is and oxotransferases can act either as dehydrogenases capable of catalysing both oxygen atom transfer and or reductases in catalysis. This reaction can be proton/electron transfer. Apart from nitrogenase, all summarised by the general scheme: + - enzymes containing molybdenum have an active site X + H2O D X=O + 2H + 2e composed of a molybdenum ion coordinated by one or During this process the Mo ion cycles between the two ene-dithiolate (dithiolene) groups that arise from (IV) and (VI) oxidation states with electrons being an unusual organic moiety known as the pterin transferred to or from an electron transfer partner or molybdenum cofactor or pyranopterin (1,2). The substrate. Experiments with xanthine dehydrogenase mononuclear molybdenum enzymes exhibit remarkable using 18O-labelled water have confirmed that the diversity of function and this is in part due to variations oxygen is incorporated into the product during at the Mo active site that are additional to the common substrate oxidation and this distinguishes the core structure. Prior to the appearance of X-ray crystal mononuclear molybdoenzymes from monoxygenases structures of molybdenum enzymes, EPR spectroscopy, where molecular oxygen rather than water acts as an X-ray absorption fine structure spectroscopy (EXAFS) oxygen atom donor (5). and biochemical analysis had identified differences The last decade has seen a resurgence of interest in Mo between the molybdenum hydroxylases, exemplified by enzymes as a consequence of the new structural xanthine dehydrogenase, and those enzymes that information and also because the remarkable mostly functioned as 'oxotransferases' (3). The former bioenergetic diversity of microorganisms is possess a cyanolysable sulfido group at the Mo active underpinned to a large degree by Mo enzymes (6). site, while the latter are insensitive to cyanide and may Molybdenum hydroxylases and examples of the sulfite possess oxo ligands (Fig. 1). Since 1995, X-ray crystal dehydrogenase family can be found in all three domains s t r u c t u r e s h a v e r a i s e d t h e u n d e r s t a n d i n g o f of life (4). In contrast, the DMSO reductase family molybdenum enzymes to a much higher level and have appears to be restricted to bacteria and archaea (5). Xanthine dehydrogenase family Sulfite dehydrogenase family F i g . 1 . S t r u c t u r e o f t h e m o l y b d e n u m c o f a c t o r s found in the 3 families of mononuclear molybdenum enzymes. O n l y t h e p y r a n o p t e r i n m o i e t i e s o f t h e o r g a n i c c o m p o n e n t o f t h e molybdenum cofactors are shown. For further details see http://metallo.scripps.edu/ PROMISE/MOCOMAIN. html DMSO dehydrogenase family Vol 35 No 3 December 2004 AUSTRALIAN BIOCHEMIST Page 17 DMSO Reductase: SHOWCASE ON Microbial Molybdenum Enzymes RESEARCH Table 1. Enzymes of the DMSO reductase family for which a crystal structure has been determined. Enzyme Type Reaction Source PDB DMSO reductase III (CH ) SO + 2e- + 2H+ " (CH ) S + H O Rhodobacter sphaeroides 1EU1 3 2 3 2 2 Rhodobacter capsulatus 4DMR R e s p i r a t o r y n i t r a t e II NO - + 2e- + 2H+ " NO - + H O Escherichia coli 1Q16 3 2 2 reductase (Nar) 1R27 P e r i p l a s m i c n i t r a t e I NO - + 2e- + 2H+ " NO - + H O Desulfovibrio desulfuricans 2NAP 3 2 2 reductase (Nap) TMAO reductase III (CH ) NO + 2e- + 2H+ " (CH ) N + H O Shewanella massilia 1TMO 3 3 3 3 2 Formate dehydrogenase-H I HCOOH " CO + 2e- + 2H+ Escherichia coli 1AA6 2 Formate dehydrogenase-N I HCOOH " CO + 2e- + 2H+ Escherichia coli 1KQF 2 Arsenite oxidase I AsO - + H O " AsO - + 2e- + 2H+ Alcaligenes faecalis 1G8K 2 2 3 M o l y b d e n u m E n z y m e s i n M i c r o b i a l referred to the PROMISE website (http://metallo.scripps. Respiratory Processes edu/PROMISE/MOCOMAIN.html, accessed October 2004). Two bioenergetic features which distinguish Crystal structures of seven molybdenum enzymes of prokaryotes from eukaryotes are the ability to grow the DMSO reductase family (Table 1) have revealed u n d e r a n o x i c c o n d i t i o n s u s i n g o x i d a t i v e that they have a similar tertiary structure. The phosphorylation (anaerobic respiration) and the ability prototype is DMSO reductase from Rhodobacter to generate energy by respiration using inorganic sphaeroides which contains about 40% α-helix and 20% compounds as electron donors (lithotrophy). Mo β-sheet and is composed of four domains that are enzymes of the DMSO reductase family are pivotal in folded around the Moco (8). Domains I, II and III these two bioenergetic processes (Table 1). Examples define a 'cup' shape with the Mo ion located at the of electron acceptors reduced by molybdenum base. Domain IV forms the bottom of the cup and enzymes in anaerobic respiration include nitrate, closes the cleft formed by the other domains. Domains DMSO, TMAO (trimethylamine-N-oxide), selenate, II and III form a series of hydrogen bond interactions arsenate, (per)chlorate, polysulfide and thiosulfate, with the guanosine moiety of the GMP attached to the while electron donors oxidised by molybdenum so-called P-pterin and Q-Pterin respectively, while enzymes during lithotrophic growth include nitrite, Domain IV forms extensive interactions with the arsenite and dimethylsulfide. pyranopterin moieties (Fig. 2). Structure of Molybdenum Enzymes of the Active Site Structure and Mechanism DMSO Reductase Family The four thiolates of the two pyranopterins In all enzymes of the DMSO reductase family it has coordinate the Mo ion in a distorted square planar been found that the Mo is ligated by four thiolates that structure, but additional ligands to the Mo form a arise from two pyranopterin molecules (Fig. 1). The trigonal cap and these are in part responsible for the pyranopterin is a reduced pterin derivative with a variation in the properties of Mo enzymes (Fig. 1). dihydroxybutyl side-chain containing a cis-(ene)- With the exception of arsenite oxidase, the Mo ion is dithiolate (dithiolene) group (1). With the exception of also coordinated by an amino acid side chain (2). The the Escherichia coli respiratory nitrate reductase (NarG) presence of different amino acid side chains in (7), crystal structures have shown that pyranopterin enzymes correlates with their phylogenetic relationship adopts a tryclic structure in which the pyran ring forms and this leads to their classification as Type I (cysteine an O-acetal linkage via a hydroxyl of the dihydroxybutyl or selenocysteine ligand), Type II (aspartate ligand) or side chain (2). In enzymes of the DMSO reductase Type III (serine ligand) (Table 1) (9). In addition to the family, the pyranopterin is in a dinucleotide form i.e. the amino acid ligand, each enzyme has an oxygen ligand pyranopterin phosphate is linked to a GMP molecule. In in the form of an oxo-, hydroxo- or aqua-group. One contrast, the pyranopterin in enzymes of the sulfite function of the amino acid ligand is to help 'tune' the dehydrogenase and xanthine dehydrogenase family is Mo centre to a redox potential which is appropriate for not in a dinucleotide form and ends with a single catalysis. It has been shown that in DMSO reductase phosphate residue. For further details of variations in mutation of the serine ligand to a cysteine results in a molybdenum cofactor (Moco) structure the reader is lowered mid-point potential of the MoV/IV couple Page 18 AUSTRALIAN BIOCHEMIST Vol 35 No 3 December 2004 SHOWCASE ON DMSO Reductase: RESEARCH Microbial Molybdenum Enzymes compared to wild-type enzyme, while the MoVI/V the distance from the Fe-S cluster associated with redox couple had a higher potential (10). This S147 C Domain I of the Mo-containing subunit to the edge of mutant form of DMSO reductase has almost no a pyranopterin is close enough (7Å in the case of activity towards S-oxides, and reduced activity respiratory nitrate reductase) for electron transfer at a towards some N-oxides, but higher activity towards rate sufficient for enzyme catalysis as defined by other N-oxides such as adenine N-oxide. Another Dutton's Ruler (15). For Type III enzymes (Table 1) important function of the amino acid side chains is to that lack this Fe-S cluster, the electron transfer control the bond length of the oxygen ligand to the pathway to the Mo ion is not known. The majority of Mo centre since Mo-S bonds exhibit a greater degree enzymes of the DMSO reductase family are connected of covalency which causes a weakening of Mo=O to respiratory or photosynthetic electron transfer bonds.
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