A new type of carboxysomal carbonic anhydrase in sulfur chemolithoautotrophs from alkaline environments.

Autotrophic bacteria are able to fix CO2 in a great diversity of habitats, even though this dissolved gas is relatively scarce at neutral pH and above. As many of these bacteria rely on CO2 fixation by ribulose 1,5-bisphospate carboxylase/oxygenase (RubisCO) for biomass generation, they must compensate for the catalytical constraints of this enzyme with CO2-concentrating mechanisms (CCMs). CCMs consist of CO2 and HCO3- transporters and carboxysomes. Carboxysomes encapsulate RubisCO and carbonic anhydrase (CA) within a protein shell and are essential for the operation of a CCM in autotrophic Bacteria that use the Calvin-Benson-Basham cycle. Members of the genus Thiomicrospira lack genes homologous to those encoding previously described CA, and prior to this work, the mechanism of function for their carboxysomes was unclear. In this paper, we provide evidence that a member of the recently discovered iota family of carbonic anhydrase enzymes (ιCA) plays a role in CO2 fixation by carboxysomes from members of Thiomicrospira and potentially other Bacteria. Carboxysome enrichments from Thiomicrospira pelophila and Thiomicrospira aerophila were found to have CA activity and contain ιCA, which is encoded in their carboxysome loci. When the gene encoding ιCA was interrupted in T. pelophila, cells could no longer grow under low-CO2 conditions, and CA activity was no longer detectable in their carboxysomes. When T. pelophila ιCA was expressed in a strain of Escherichia coli lacking native CA activity, this strain recovered an ability to grow under low CO2 conditions, and CA activity was present in crude cell extracts prepared from this strain. IMPORTANCE: Here, we provide evidence that iota carbonic anhydrase (ιCA) plays a role in CO2 fixation by some organisms with CO2-concentrating mechanisms; this is the first time that ιCA has been detected in carboxysomes. While ιCA genes have been previously described in other members of bacteria, this is the first description of a physiological role for this type of carbonic anhydrase in this domain. Given its distribution in alkaliphilic autotrophic bacteria, ιCA may provide an advantage to organisms growing at high pH values and could be helpful for engineering autotrophic organisms to synthesize compounds of industrial interest under alkaline conditions.

Kinetic and Structural Characterization of a Flavin-Dependent Putrescine N-Hydroxylase from Acinetobacter baumannii.

Acinetobacter baumannii is a Gram-negative opportunistic pathogen that causes nosocomial infections, especially among immunocompromised individuals. The rise of multidrug resistant strains of A. baumannii has limited the use of standard antibiotics, highlighting a need for new drugs that exploit novel mechanisms of pathogenicity. Disrupting iron acquisition by inhibiting the biosynthesis of iron-chelating molecules (siderophores) secreted by the pathogen is a potential strategy for developing new antibiotics. Here we investigated FbsI, an N-hydroxylating monooxygenase involved in the biosynthesis of fimsbactin A, the major siderophore produced by A. baumannii. FbsI was characterized using steady-state and transient-state kinetics, spectroscopy, X-ray crystallography, and small-angle X-ray scattering. FbsI was found to catalyze the N-hydroxylation of the aliphatic diamines putrescine and cadaverine. Maximum coupling of the reductive and oxidative half-reactions occurs with putrescine, suggesting it is the preferred (in vivo) substrate. FbsI uses both NADPH and NADH as the reducing cofactor with a slight preference for NADPH. The crystal structure of FbsI complexed with NADP+ was determined at 2.2 Å resolution. The structure exhibits the protein fold characteristic of Class B flavin-dependent monooxygenases. FbsI is most similar in 3D structure to the cadaverine N-hydroxylases DesB and DfoA. Small-angle X-ray scattering shows that FbsI forms a tetramer in solution like the N-hydroxylating monooxygenases of the SidA/IucD/PvdA family. A model of putrescine docked into the active site provides insight into substrate recognition. A mechanism for the catalytic cycle is proposed where dehydration of the C4a-hydroxyflavin intermediate is partially rate-limiting, and the hydroxylated putrescine product is released before NADP+.

Methods for biochemical characterization of flavin-dependent N-monooxygenases involved in siderophore biosynthesis.

Siderophores are essential molecules released by some bacteria and fungi in iron-limiting environments to sequester ferric iron, satisfying metabolic needs. Flavin-dependent N-hydroxylating monooxygenases (NMOs) catalyze the hydroxylation of nitrogen atoms to generate important siderophore functional groups such as hydroxamates. It has been demonstrated that the function of NMOs is essential for virulence, implicating these enzymes as potential drug targets. This chapter aims to serve as a resource for the characterization of NMO's enzymatic activities using several biochemical techniques. We describe assays that allow for the determination of steady-state kinetic parameters, detection of hydroxylated amine products, measurement of the rate-limiting step(s), and the application toward drug discovery efforts. While not exhaustive, this chapter will provide a foundation for the characterization of enzymes involved in siderophore biosynthesis, allowing for gaps in knowledge within the field to be addressed.