Sulfoxide synthases are non-heme iron enzymes that catalyze oxidative carbon-sulfur bond formation between cysteine derivatives and N-a-trimethylhistidine as a key step in the biosynthesis of thiohistidines. The complex catalytic mechanism of this enzyme reaction has emerged as the controversial subject of several biochemical and computational studies. These studies all used the structure of the g-glutamyl cysteine utilizing sulfoxide synthase, MthEgtB from Mycobacterium thermophilum (EC 1.14.99.50), as a structural basis. To provide an alternative model system we have solved the crystal structure of CthEgtB from Chloracidobacterium thermophilum (EC 1.14.99.51) that utilizes cysteine as a sulfur donor. This structure reveals a completely different configuration of active site residues that are involved in oxygen binding and activation. Furthermore, comparison of the two EgtB structures enables a classification of all ergothioneine biosynthetic EgtBs into five sub-types, each characterized by unique active-site features. This active site diversity provides an excellent platform to examine the catalytic mechanism of sulfoxide synthases by comparative enzymology, but also raises the question as to why so many different solutions to the same biosynthetic problem have emerged.

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Sulfoxide synthases are non-hemei ron enzymes that participatei nt he biosynthesis of thiohistidines, such as ergothioneine ando vothiol A. The sulfoxide synthase EgtB from Chloracidobacterium thermophilum (CthEgtB) catalyzes oxidative coupling between the side chains of N-a-trimethyl histidine( TMH) and cysteine (Cys) in ar eaction that entails complete reduction of molecular oxygen,c arbon-sulfur (CÀ S) and sulfur-oxygen (SÀO) bond formationa sw ell as carbon-hydrogen (CÀH) bond cleavage. In this report, we show that CthEgtB and other bacterial sulfoxide synthases cannote fficiently accept selenocysteine (SeCys) as as ubstrate in place of cysteine. In contrast, the sulfoxide synthase from the filamentous fungus Chaetomium thermophilum (CthEgt1) catalyzes CÀSa nd CÀSe bond formation at almost equale fficiency.W ed iscuss evidence suggesting that this functional differenceb etween bacterial and fungal sulfoxide synthases emerges from different modes of oxygen activation.[a] K.

N-Acetylmannosamine kinase (EC 2.7.1.60) is involved in the catabolism of sialic acid for many bacterial pathogens implicated in human disease such as Escherichia coli, Staphylococcus aureus, Vibrio cholerae and V. vulnificus. Interestingly, some human commensals and bacterial pathogens can scavenge sialic acids from their surrounding environment and degrade them as a source of carbon, nitrogen and energy. This process requires a cluster of genes known as the 'Nan-Nag cluster', which have proven to be essential for S. aureus growth on sialic acids, suggesting that the pathway is a viable antimicrobial drug target. The enzyme N-acetylmannosamine kinase is involved in the catabolism of sialic acid, transferring a phosphate group from adenosine-5 0 -triphosphate to the C6 position of N-acetylmannosamine to generate N-acetylmannosamine-6-phosphate. The gene was cloned into an appropriate expression vector; recombinant protein was expressed in E. coli BL21 (DE3) cells and purified via anionexchange chromatography, hydrophobic interaction chromatography and sizeexclusion chromatography. Purified N-acetylmannosamine kinase was screened for crystallization. The best crystal diffracted to a resolution of beyond 2.6 Å in space group P2. Understanding the structural nature of this enzyme from methicillin-resistant S. aureus will provide insights necessary for the development of future antimicrobials.