Identification of a stable complex between a [NiFe]-hydrogenase catalytic subunit and its maturation protease

Abstract

Salmonella enterica serovar Typhimurium has the ability to use molecular hydrogen as a respiratory electron donor. This is facilitated by three [NiFe]-hydrogenases termed Hyd-1, Hyd-2, and Hyd-5. Hyd-1 and Hyd-5 are homologous oxygen-tolerant [NiFe]-hydrogenases. A critical step in the biosynthesis of a [NiFe]-hydrogenase is the proteolytic processing of the catalytic subunit. In this work, the role of the maturation protease encoded within the Hyd-5 operon, HydD, was found to be partially complemented by the maturation protease encoded in the Hyd-1 operon, HyaD. In addition, both maturation proteases were shown to form stable complexes, in vivo and in vitro, with the catalytic subunit of Hyd-5. The protein-protein interactions were not detectable in a strain that could not make the enzyme metallocofactor.

Keywords: Salmonella enterica; [NiFe]-hydrogenase, protein-protein interactions; anaerobic respiration; bacterial hydrogen metabolism; metalloenzyme biosynthesis.

Figure 1

Cross‐talk between HydD and HyaD during the biosynthesis of Hyd‐5. Salmonella enterica strains LT2a (parental strain); LB03 (up‐regulated, His‐tagged Hyd‐5); LB03T (ΔtatABC); MAS05 producing HA‐tagged HydB (LB03_HA); MAS06 (ΔhypD _HA); MAS07 (ΔhydD _HA); MAS08 (ΔhyaD _HA); MAS09 (ΔhyaD ΔhydD _HA); MAS09 complemented with pBAD‐HyaD (ΔhyaD ΔhydD _HA/HyaD); and MAS09 complemented with pBADH‐ydD (ΔhyaD ΔhydD _HA/HydD) were all grown anaerobically before being harvested, washed, and analyzed. (A) Immunodetection of the large subunit HydB_HA. Whole‐cell protein samples were separated by SDS/PAGE (8% w/v acrylamide with a Bis‐Tris buffer system), blotted onto nitrocellulose, and challenged with an anti‐HA serum to detect the HydB_HA protein. Each lane was loaded with 60 μg of total protein. The position of unprocessed (u) and processed (p) large subunits are indicated along with molecular weight markers. (B) Immunodetection of the small subunit HydA_HIS. Proteins from crude cell extracts were resolved by SDS/PAGE using 12% w/v acrylamide in Tris‐glycine buffer system. Immunoblots were revealed with antisera against the His tag to detect HydA_HIS. Each lane was loaded with 120 μg of total protein. The position of unprocessed (u) and processed (p) small subunits are indicated along with molecular weight markers. (C) Rocket immunoelectrophoresis of periplasmic fractions. Periplasmic protein samples were prepared by sucrose/lysozyme/EDTA treatment of cells and 2 μg of protein was electrophoresed through 1% (w/v) agarose containing 3 μL of an anti‐Hyd‐5 serum. Plates were then incubated at 37 °C under a H₂ atmosphere with benzyl viologen and tetrazolium red.

Figure 2

Genetic analysis of HydD‐HydB and HyaD‐HydB interactions using a bacterial two‐hybrid assay. Interaction of HydD or HyaD with HydB and two truncated forms of HydB: HydB_T1, containing the mature form of HydB; or HydB_T2, containing a deletion of DNA encoding the C‐terminal 65 amino acid residues of HydB, was quantified by β‐galactosidase activity assays in extracts obtained from anaerobic cultures of (A) Escherichia coli MAE01 (ΔcyaA) or (B) MAE02 (ΔcyaA ΔhypF) strains. E. coli reporter strains transformed with the empty vectors, pUT18 (18) and pT25 (25), or pUT18‐NarG_ss (18NarG) and pT25‐NarJ (25NarJ), were included as negative or positive controls, respectively. Values, expressed as Miller units, are the average of three independent assays ± SE.

Figure 3

Biochemical analysis of the HydD‐HydB and HyaD‐HydB interactions by copurification experiments. Extracts from anaerobic cultures of Escherichia coli FTD147 (ΔhyaB, ΔhybC, ΔhycE) harboring (A) pQE80‐HydB‐HydD_HIS or (B) pQE80‐HydB‐HyaD_HIS derivative plasmids were applied to a 5 mL HisTrap‐HP IMAC column and eluted fractions were pooled and concentrated. Proteins were resolved in 12% acrylamide SDS/PAGE gels and stained with Instant Blue (left panels) or immunoblotted using an antiserum against His tag (right panels). The arrows indicate the band corresponding to HydB identified by mass spectrometry or HydD_HIS and HyaD_HIS identified by western immunoblot analysis. Numbers on the left margins of the panels indicate the position of the molecular weight standards (kDa). S, soluble fraction; FT, flow through; W, wash; E, eluate fraction.