CysG structure reveals tetrapyrrole-binding features and novel regulation of siroheme biosynthesis

Abstract

Sulfur metabolism depends on the iron-containing porphinoid siroheme. In Salmonella enterica, the S-adenosyl-L-methionine (SAM)-dependent bismethyltransferase, dehydrogenase and ferrochelatase, CysG, synthesizes siroheme from uroporphyrinogen III (uro'gen III). The reactions mediated by CysG encompass two branchpoint intermediates in tetrapyrrole biosynthesis, diverting flux first from protoporphyrin IX biosynthesis and then from cobalamin (vitamin B(12)) biosynthesis. We determined the first structure of this multifunctional siroheme synthase by X-ray crystallography. CysG is a homodimeric gene fusion product containing two structurally independent modules: a bismethyltransferase and a dual-function dehydrogenase-chelatase. The methyltransferase active site is a deep groove with a hydrophobic patch surrounded by hydrogen bond donors. This asymmetric arrangement of amino acids may be important in directing substrate binding. Notably, our structure shows that CysG is a phosphoprotein. From mutational analysis of the post-translationally modified serine, we suggest a conserved role for phosphorylation in inhibiting dehydrogenase activity and modulating metabolic flux between siroheme and cobalamin pathways.

Substrate interactions with human ferrochelatase

“…This observation is consistent with the high degree of porphyrin substrate specificity (20,33) and spectroscopic studies (34) on the enzyme. Similar observations have been made for another heme biosynthetic enzyme, uroporphyrinogen decarboxylase (28). In this case, the propionate groups are also held in place through ionic interactions with arginine residues and hydrogenbonding interactions with tyrosine and serine residues.…”

Chelatases: distort to select?

“…Heme, chlorophyll, cobalamin (vitamin B 12 ), siroheme and coenzyme F 430 belong to a family of prosthetic groups that are characterized by their tetrapyrrole-derived nature and contain a central, complexed metal ion: Fe 2+ in heme and siroheme, Mg 2+ in chlorophyll and bacterio-chlorophyll, Co 2+ in cobalamin, and Ni 2+ in coenzyme F 430 [5,6,8,9]. All of these molecules are derived from a common tetrapyrrole, uroporphyrinogen III ( Figure I), and require the function of specific enzymes or chelatases for the insertion of their metal ion ( Figure I).…”

“…On the basis of structural, functional and genetic studies and results from genome sequencing projects [6,24], three main classes of chelatases are recognized: (i) ATPdependent heterotrimeric chelatases, including protoporphyrin IX magnesiumchelatase (ChlH-I-D or BchlH-I-D) involved in chlorophyll or bacteriochlorophyll biosynthesis [9], and aerobic cobaltochelatase (CobN-S-T) associated with cobalamin biosynthesis [6]; (ii) ATP-independent chelatases, including protoporphyrin IX ferrochelatase (HemH) involved in heme biosynthesis [5,16,33], sirohydrochlorin ferrochelatase (SirB) associated with siroheme biosynthesis [6,7], and anaerobic sirohydrochlorin cobaltochelatases (CbiK and CbiX) involved in cobalamin biosynthesis [6]; and (iii) multifunctional homodimeric chelatases associated with siroheme biosynthesis, including siroheme synthase (CysG) [8], and precorrin-2 dehydrogenase and sirohydrochloride ferrochelatase (Met8p) in yeast [24]. The enzymes in the third class house two enzymatic activities: dehydrogenase and chelatase [8,24].…”

“…Metalated tetrapyrroles such as the cofactors and prosthetic groups heme, chlorophyll, cobalamin (vitamin B 12 ) and coenzyme F 430 have a centrally chelated metal ion and arise from a common tetrapyrrole, uroporphyrinogen III. The chemical nature of the chelated metal ion varies; for example, Fe 2+ is present in heme, Mg 2+ in chlorophyll, Co 2+ in cobalamin and Ni 2+ in coenzyme F 430 [5][6][7][8][9].…”

“…Essential to the catalytic insertion of metal ions into various tetrapyrroles is a divergent group of enzymes called chelatases [5][6][7][8][9], which ensure specificity of the metal ion in response to cellular requirements (Box 1). Although a model of the general reaction mechanism of porphyrin metalation has emerged [10][11][12][13][14][15], how chelatases bestow metal ion selectivity and specificity on porphyrin metalation remains unresolved.…”