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Structural studies of Pseudomonas and Chromobacterium ?-aminotransferases provide insights into their differing substrate specificity.

ABSTRACT: The crystal structures and inhibitor complexes of two industrially important ?-aminotransferase enzymes from Pseudomonas aeruginosa and Chromobacterium violaceum have been determined in order to understand the differences in their substrate specificity. The two enzymes share 30% sequence identity and use the same amino acceptor, pyruvate; however, the Pseudomonas enzyme shows activity towards the amino donor ?-alanine, whilst the Chromobacterium enzyme does not. Both enzymes show activity towards S-?-methylbenzylamine (MBA), with the Chromobacterium enzyme having a broader substrate range. The crystal structure of the P. aeruginosa enzyme has been solved in the holo form and with the inhibitor gabaculine bound. The C. violaceum enzyme has been solved in the apo and holo forms and with gabaculine bound. The structures of the holo forms of both enzymes are quite similar. There is little conformational difference observed between the inhibitor complex and the holoenzyme for the P. aeruginosa aminotransferase. In comparison, the crystal structure of the C. violaceum gabaculine complex shows significant structural rearrangements from the structures of both the apo and holo forms of the enzyme. It appears that the different rigidity of the protein scaffold contributes to the substrate specificity observed for the two ?-aminotransferases.

SUBMITTER: Sayer C

PROVIDER: S-EPMC3606037 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Structural studies of Pseudomonas and Chromobacterium ω-aminotransferases provide insights into their differing substrate specificity.

Sayer Christopher C Isupov Michail N MN Westlake Aaron A Littlechild Jennifer A JA

Acta crystallographica. Section D, Biological crystallography 20130314 Pt 4

The crystal structures and inhibitor complexes of two industrially important ω-aminotransferase enzymes from Pseudomonas aeruginosa and Chromobacterium violaceum have been determined in order to understand the differences in their substrate specificity. The two enzymes share 30% sequence identity and use the same amino acceptor, pyruvate; however, the Pseudomonas enzyme shows activity towards the amino donor β-alanine, whilst the Chromobacterium enzyme does not. Both enzymes show activity toward ...[more]

PMID: 23519665

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Project description:Aldosterone is a major mineralocorticoid hormone that plays a key role in the regulation of electrolyte balance and blood pressure. Excess aldosterone levels can arise from dysregulation of the renin-angiotensin-aldosterone system and are implicated in the pathogenesis of hypertension and heart failure. Aldosterone synthase (cytochrome P450 11B2, CYP11B2) is the sole enzyme responsible for the production of aldosterone in humans. Blocking of aldosterone synthesis by mediating aldosterone synthase activity is thus a recently emerging pharmacological therapy for hypertension, yet a lack of structural information has limited this approach. Here, we present the crystal structures of human aldosterone synthase in complex with a substrate deoxycorticosterone and an inhibitor fadrozole. The structures reveal a hydrophobic cavity with specific features associated with corticosteroid recognition. The substrate binding mode, along with biochemical data, explains the high 11?-hydroxylase activity of aldosterone synthase toward both gluco- and mineralocorticoid formation. The low processivity of aldosterone synthase with a high extent of intermediates release might be one of the mechanisms of controlled aldosterone production from deoxycorticosterone. Although the active site pocket is lined by identical residues between CYP11B isoforms, most of the divergent residues that confer additional 18-oxidase activity of aldosterone synthase are located in the I-helix (vicinity of the O(2) activation path) and loops around the H-helix (affecting an egress channel closure required for retaining intermediates in the active site). This intrinsic flexibility is also reflected in isoform-selective inhibitor binding. Fadrozole binds to aldosterone synthase in the R-configuration, using part of the active site cavity pointing toward the egress channel. The structural organization of aldosterone synthase provides critical insights into the molecular mechanism of catalysis and enables rational design of more specific antihypertensive agents.

Dataset Information

Structural studies of Pseudomonas and Chromobacterium ?-aminotransferases provide insights into their differing substrate specificity.

Publications

Structural studies of Pseudomonas and Chromobacterium ω-aminotransferases provide insights into their differing substrate specificity.

Similar Datasets

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