Eric L. Carter

Significance: Rev-Erbβ is a heme-responsive transcription factor. Yet, the critical role of heme redox chemistry in regulating the protein’s heme occupancy and function as a repressor and gas sensor remains elusive. We show that the heme in Rev-Erbβ exists in the Fe ³⁺ -state to be replete under the limiting labile heme pool in cellulo. However, binding and sensing gases require Fe ²⁺ -heme. We show that signaling gases can drive Fe ³⁺ -heme reduction by electrochemically coupling heme reduction to the tight binding of CO/NO to Fe ²⁺ -heme. Our work describes a mechanism to explain how and why Fe ³⁺ -hemoproteins like Rev-Erbβ are ideal for in cellulo gas signaling.

Urease from Klebsiella aerogenes is composed of three subunits (UreA–UreB–UreC) that assemble into a (UreABC)₃ quaternary structure. UreC harbors the dinuclear nickel active site, whereas the functions of UreA and UreB remain unknown. UreD and UreF accessory proteins previously were suggested to reposition UreB and increase the level of exposure of the nascent urease active site, thus facilitating metallocenter assembly. In this study, cells were engineered to separately produce (UreAC)₃ or UreB, and the purified proteins were characterized. Monomeric UreB spontaneously binds to the trimeric heterodimer of UreA and UreC to form (UreABC*)₃ apoprotein, as shown by gel filtration chromatography, integration of electrophoretic gel band intensities, and mass spectrometry. Similar to the authentic urease apoprotein, the active enzyme is produced by incubation of (UreABC*)₃ with Ni²⁺ and bicarbonate. Conversely, UreBΔ1–19, lacking the 19-residue potential hinge and tether to UreC, does not form a complex with (UreAC)₃ and yields negligible levels of the active enzyme when incubated under activation conditions with (UreAC)₃. Comparison of activities and nickel contents for (UreAC)₃, (UreABC*)₃, and (UreABC)₃ samples treated with Ni²⁺ and bicarbonate and then desalted indicates that UreB facilitates efficient incorporation of the metal into the active site and protects the bound metal from chelation. Amylose resin pull-down studies reveal that MBP-UreD (a fusion of maltose binding protein with UreD) forms complexes with (UreABC)₃, (UreAC)₃, and UreB in vivo, but not in vitro. By contrast, MBP-UreD does not form an in vivo complex with UreBΔ1–19. The soluble MBP-UreD–UreF–UreG complex binds in vitro to (UreABC)₃, but not to (UreAC)₃ or UreB. Together, these data demonstrate that UreB facilitates the interaction of urease with accessory proteins during metallocenter assembly, with the N-terminal hinge and tether region being specifically required for this process. In addition to its role in urease activation, UreB enhances the stability of UreC against proteolytic cleavage.

Helicobacter mustelae , a gastric pathogen of ferrets, synthesizes a distinct iron-dependent urease in addition to its archetypical nickel-containing enzyme. The iron-urease is oxygen-labile, with the inactive protein exhibiting a methemerythrin-like electronic spectrum. Significantly, incubation of the oxidized protein with dithionite under anaerobic conditions leads to restoration of activity and bleaching of the spectrum. Structural analysis of the oxidized species reveals a dinuclear iron metallocenter bridged by a lysine carbamate, closely resembling the traditional nickel-urease active site. Although the iron-urease is less active than the nickel-enzyme, its activity allows H. mustelae to survive the carnivore’s low-nickel gastric environment.

Assembly of the Klebsiella aerogenes urease metallocenter requires four accessory proteins, UreD, UreE, UreF, and UreG, to effectively deliver and incorporate two Ni2+ ions into the nascent active site of the urease apoprotein (UreABC). Each accessory protein has been purified and characterized with the exception of UreD due to its insolubility when it is overproduced in recombinant cells. In this study, a translational fusion was made between the maltose binding protein (MBP) and UreD, with the resulting MBP-UreD found to be soluble in Escherichia coli cell extracts and able to complement a DeltaureD-urease cluster in this host microorganism. MBP-UreD was purified as a large multimer (> 670 kDa) that bound approximately 2.5 Ni2+ ions (K(d) of approximately 50 microM, where K(d) is the dissociation constant) per UreD protomer according to equilibrium dialysis measurements. Zn2+ directly competes with 10-fold higher affinity (approximately 4 Zn2+ ions per protomer; K(d) of 5 microM) for the Ni2+ binding sites. MBP pulldown experiments demonstrated that the UreD domain of MBP-UreD formed in vivo complexes with UreF, UreG, UreF plus UreG, or UreABC when these proteins were overproduced in the same E. coli cells. In addition, a UreABC-(MBP-UreD)-UreFG complex was observed in cells producing all urease components. Comparative in vitro binding experiments with purified proteins demonstrated an approximate 1:1 binding ratio between the UreD domain of MBP-UreD and the UreF domain of the UreEF fusion, only weak or transient interaction between MBP-UreD and UreG, and no binding with UreABC. These studies are the first to describe the properties of purified UreD, and they extend our understanding of its binding partners both in vitro and in the cell.

The abg locus of the Escherichia coli chromosome includes three genes encoding proteins (AbgA, AbgB, and AbgT) that enable uptake and utilization of the folate breakdown product, p -aminobenzoyl-glutamate (PABA-GLU). We report on the purification and characterization of the p -aminobenzoyl-glutamate hydrolase (PGH) holoenzyme encoded by abgA and abgB . One-step purification was accomplished using a plasmid carrying abgAB with a hexahistidine tag on the carboxyl terminus of AbgB and subsequent metal affinity chromatography (MAC). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed two subunits (∼53-kDa and ∼47-kDa proteins) of the expected masses of AbgB and AbgA; N-terminal sequencing confirmed the subunit identification, and amino acid analysis yielded a 1:1 ratio of the subunits. Size exclusion chromatography coupled with light-scattering analysis of purified PGH revealed a predominant molecular mass of 206 kDa and a minor component of 400 to 500 kDa. Both peaks contained PGH activity, and SDS-PAGE revealed that fractions containing activity were composed of both AbgA and AbgB. MAC-purified PGH was highly stimulated by manganese chloride. Kinetic analysis of MAC-purified PGH revealed a K _m value for PABA-GLU of 60 ± 0.08 μM and a specific activity of 63,300 ± 600 nmol min ⁻¹ mg ⁻¹ . Folic acid and a variety of dipeptides served as poor substrates of PGH. This locus of the E. coli chromosome may encode a portion of a folate catabolism pathway.

Urease, the first enzyme to be crystallized, contains a dinuclear nickel metallocenter that catalyzes the decomposition of urea to produce ammonia, a reaction of great agricultural and medical importance. Several mechanisms of urease catalysis have been proposed on the basis of enzyme crystal structures, model complexes, and computational efforts, but the precise steps in catalysis and the requirement of nickel versus other metals remain unclear. Purified bacterial urease is partially activated via incubation with carbon dioxide plus nickel ions; however, in vitro activation also has been achieved with manganese and cobalt. In vivo activation of most ureases requires accessory proteins that function as nickel metallochaperones and GTP-dependent molecular chaperones or play other roles in the maturation process. In addition, some microorganisms control their levels of urease by metal ion-dependent regulatory mechanisms.