Role of ammonia-lyases in the synthesis of the dithiomethylamine ligand during [FeFe]-hydrogenase maturation

Abstract

The generation of an active [FeFe]-hydrogenase requires the synthesis of a complex metal center, the H-cluster, by three dedicated maturases: the radical S-adenosyl-l-methionine (SAM) enzymes HydE and HydG, and the GTPase HydF. A key step of [FeFe]-hydrogenase maturation is the synthesis of the dithiomethylamine (DTMA) bridging ligand, a process recently shown to involve the aminomethyl-lipoyl-H-protein from the glycine cleavage system, whose methylamine group originates from serine and ammonium. Here we use functional assays together with electron paramagnetic resonance and electron-nuclear double resonance spectroscopies to show that serine or aspartate together with their respective ammonia-lyase enzymes can provide the nitrogen for DTMA biosynthesis during in vitro [FeFe]-hydrogenase maturation. We also report bioinformatic analysis of the hyd operon, revealing a strong association with genes encoding ammonia-lyases, suggesting important biochemical and metabolic connections. Together, our results provide evidence that ammonia-lyases play an important role in [FeFe]-hydrogenase maturation by delivering the ammonium required for dithiomethylamine ligand synthesis.

Keywords: GTPase; HydF; [FeFe]-hydrogenase; ammonia lyase; ammonium; dithiomethylamine; glycine cleavage system; hyd operon.

Figure 1

[FeFe]-hydrogenase from Clostridium pasteurianum (3C8Y) and its active site H-cluster (inset). Atom color, identity: rust, iron; yellow, sulfur; blue, nitrogen; red, oxygen; gray, carbon.

Figure 2

Schematic depiction of the current model for [FeFe]-hydrogenase (HydA) maturation by the maturases HydG, HydE, and HydF, as well as components of the GCS including H-protein, T-protein, and SHMT, which together convert the HydA^ΔEFGcontaining only the [4Fe-4S]_Hcluster to the holo-[FeFe]-hydrogenase.

Figure 3

2K Q-band¹⁵N Mims ENDOR spectra centered at the¹⁵N Larmor frequency and taken at g = 2.01 of mature HydA. A, Maturation in the presence of ¹⁵NH₄⁺ (lower two spectra) results in ¹⁵N incorporation into DTMA regardless of the presence of lysate, while ¹⁵N incorporation from ¹³C,¹⁵N-serine (top two spectra) occurs only in the presence of lysate, indicating a role for an enzyme in the lysate. The bottom spectrum in panel (A) is reproduced from reference (14). (∗) represents harmonics of the ¹³C ENDOR response (not shown) from ¹³C incorporated in the DTMA from the doubly labeled serine; (↓) represents Mims ‘holes’ (see Experimental Procedures). B, maturation in the presence of ¹⁵N-aspartate and AspA, or ¹⁵N-serine and SdaA, results in the incorporation of ¹⁵N into DTMA, as revealed by ¹⁵N ENDOR spectra.

Figure 4

Select [FeFe]-hydrogenase operons. A, organisms containing the aspA gene. B, organisms without the aspA gene. aspA: aspartate ammonia-lyase gene, sdaA: serine ammonia-lyase gene, ilvA: threonine ammonia-lyase gene. An ‘x’ in the table indicates the corresponding gene is not found.

Figure 5

Genomic context analysis for HydF. Top, sequence similarity network showing the HydF protein set. The proteins with at least one of the Pfam domains of interest are shown as teal “V’s” and those proteins that have at least one of the hyd gene Pfam domains, but no other Pfam domain of interest are shown as pale purple circles. Each cluster is named with the predominant annotations of proteins in that cluster. Figure created using Cytoscape and a perfuse force-directed layout algorithm. Bottom, a table showing the genes of interest and their associated Pfam domain, the number of nodes for which at least one of those Pfam domains was identified as a genomic neighbor within the ± 10 and ± 20 ORF of hydF, and the percentage of the total number of nodes represented.

Figure 6

Specific activities of HydA matured under defined conditions using the maturases HydE, HydF, and HydG in the presence of different sources of ammonium. The source of ammonium is indicated by the color (purple, ammonium chloride; blue, aspartate, and AspA; red, serine, and SdaA). Assays were conducted in triplicate; individual results are shown as transparent colored shapes with the mean as a colored line and the error bars are shown in black. Assay conditions are as described in the supplemental methods.

Figure 7

Role of ammonia-lyases in HydA maturation.