Metabolic bifunctionality of Rv0812 couples folate and peptidoglycan biosynthesis in Mycobacterium tuberculosis.

Comparative sequence analysis has enabled the annotation of millions of genes from organisms across the evolutionary tree. However, this approach has inherently biased the annotation of phylogenetically ubiquitous, rather than species-specific, functions. The ecologically unusual pathogen Mycobacterium tuberculosis (Mtb) has evolved in humans as its sole reservoir and emerged as the leading bacterial cause of death worldwide. However, the physiological factors that define Mtb's pathogenicity are poorly understood. Here, we report the structure and function of a protein that is required for optimal in vitro fitness and bears homology to two distinct enzymes, Rv0812. Despite diversification of related orthologues into biochemically distinct enzyme families, rv0812 encodes a single active site with aminodeoxychorismate lyase and D-amino acid transaminase activities. The mutual exclusivity of substrate occupancy in this active site mediates coupling between nucleic acid and cell wall biosynthesis, prioritizing PABA over D-Ala/D-Glu biosynthesis. This bifunctionality reveals a novel, enzymatically encoded fail-safe mechanism that may help Mtb and other bacteria couple replication and division.

Fe-S cluster biogenesis in Gram-positive bacteria: SufU is a zinc-dependent sulfur transfer protein.

The biosynthesis of Fe-S clusters in Bacillus subtilis and other Gram-positive bacteria is catalyzed by the SufCDSUB system. The first step in this pathway involves the sulfur mobilization from the free amino acid cysteine to a sulfur acceptor protein SufU via a PLP-dependent cysteine desulfurase SufS. In this reaction scheme, the formation of an enzyme S-covalent intermediate is followed by the binding of SufU. This event leads to the second half of the reaction where a deprotonated thiol of SufU promotes the nucleophilic attack onto the persulfide intermediate of SufS. Kinetic analysis combined with spectroscopic methods identified that the presence of a zinc atom tightly bound to SufU (Ka = 10(17) M(-1)) is crucial for its structural and catalytic competency. Fe-S cluster assembly experiments showed that despite the high degree of sequence and structural similarity to the ortholog enzyme IscU, the B. subtilis SufU does not act as a standard Fe-S cluster scaffold protein. The involvement of SufU as a dedicated agent of sulfur transfer, rather than as an assembly scaffold, in the biogenesis of Fe-S clusters in Gram-positive microbes indicates distinct strategies used by bacterial systems to assemble Fe-S clusters.

Protected sulfur transfer reactions by the Escherichia coli Suf system.

The first step in sulfur mobilization for the biosynthesis of Fe-S clusters under oxidative stress and iron starvation in Escherichia coli involves a cysteine desulfurase SufS. Its catalytic reactivity is dependent on the presence of a sulfur acceptor protein, SufE, which acts as the preferred substrate for this enzyme. Kinetic analysis of the cysteine:SufE sulfurtransferase reaction of the E. coli SufS that is partially protected from reducing agents, such as dithiothreitol and glutathione, was conducted. Under these conditions, the reaction displays a biphasic profile in which the first phase involves a fast sulfur transfer reaction from SufS to SufE. The accumulation of persulfurated/polysulfurated forms of SufE accounts for a second phase of the slow catalytic turnover rate. The presence of the SufBCD complex enhances the activity associated with the second phase, while modestly inhibiting the activity associated with the initial sulfur transfer from SufS to SufE. Thus, the rate of sulfur transfer from SufS to the final proposed SufBCD Fe-S cluster scaffold appears to be dependent on the availability of the final sulfur acceptor. The use of a stronger reducing agent [tris(2-carboxyethyl)phosphine hydrochloride] elicited the maximal activity of the SufS-SufE reaction and surpassed the stimulatory effect of SufBCD. This concerted sulfur trafficking path involving sequential transfer from SufS to SufE to SufBCD guarantees the protection of intermediates at a controlled flux to meet cellular demands encountered under conditions detrimental to thiol chemistry and Fe-S cluster metabolism.