ABSTRACT
The intermolecular asymmetric Stetter reaction is an almost unexplored transformation for biocatalysts. Previously reported thiamine diphosphate (ThDP)-dependent PigD from Serratia marcescens is the first enzyme identified to catalyze the Stetter reaction of α,ß-unsaturated ketones (Michael acceptor substrates) and α-keto acids. PigD is involved in the biosynthesis of the potent cytotoxic agent prodigiosin. Here, we describe the investigation of two new ThDP-dependent enzymes, SeAAS from Saccharopolyspora erythraea and HapD from Hahella chejuensis. Both show a high degree of homology to the amino acid sequence of PigD (39 and 51 %, respectively). The new enzymes were heterologously overproduced in Escherichia coli, and the yield of soluble protein was enhanced by co-expression of the chaperone genes groEL/ES. SeAAS and HapD catalyze intermolecular Stetter reactions in vitro with high enantioselectivity. The enzymes possess a characteristic substrate range with respect to Michael acceptor substrates. This provides support for a new type of ThDP-dependent enzymatic activity, which is abundant in various species and not restricted to prodigiosin biosynthesis in different strains. Moreover, PigD, SeAAS, and HapD are also able to catalyze asymmetric carbon-carbon bond formation reactions of aldehydes and α-keto acids, resulting in 2-hydroxy ketones.
Subject(s)
Carboxylic Acids/metabolism , Coenzymes/metabolism , Enzymes/metabolism , Gammaproteobacteria/enzymology , Ketones/metabolism , Saccharopolyspora/enzymology , Thiamine Pyrophosphate/metabolism , Aldehydes/metabolism , Cloning, Molecular , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Enzymes/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Gammaproteobacteria/genetics , Gammaproteobacteria/metabolism , Gene Expression , Molecular Sequence Data , Saccharopolyspora/genetics , Saccharopolyspora/metabolism , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Substrate SpecificityABSTRACT
Mycobacterium tuberculosis (Mtb) and Rhodococcus jostii RHA1 have similar cholesterol catabolic pathways. This pathway contributes to the pathogenicity of Mtb. The hsaAB cholesterol catabolic genes have been predicted to encode the oxygenase and reductase, respectively, of a flavin-dependent mono-oxygenase that hydroxylates 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione (3-HSA) to a catechol. An hsaA deletion mutant of RHA1 did not grow on cholesterol but transformed the latter to 3-HSA and related metabolites in which each of the two keto groups was reduced: 3,9-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-17-one (3,9-DHSA) and 3,17-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9-one (3,17-DHSA). Purified 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione 4-hydroxylase (HsaAB) from Mtb had higher specificity for 3-HSA than for 3,17-DHSA (apparent k(cat)/K(m) = 1000 +/- 100 M(-1) s(-1) versus 700 +/- 100 M(-1) s(-1)). However, 3,9-DHSA was a poorer substrate than 3-hydroxybiphenyl (apparent k(cat)/K(m) = 80 +/- 40 M(-1) s(-1)). In the presence of 3-HSA the K(m)(app) for O(2) was 100 +/- 10 microM. The crystal structure of HsaA to 2.5-A resolution revealed that the enzyme has the same fold, flavin-binding site, and catalytic residues as p-hydroxyphenyl acetate hydroxylase. However, HsaA has a much larger phenol-binding site, consistent with the enzyme's substrate specificity. In addition, a second crystal form of HsaA revealed that a C-terminal flap (Val(367)-Val(394)) could adopt two conformations differing by a rigid body rotation of 25 degrees around Arg(366). This rotation appears to gate the likely flavin entrance to the active site. In docking studies with 3-HSA and flavin, the closed conformation provided a rationale for the enzyme's substrate specificity. Overall, the structural and functional data establish the physiological role of HsaAB and provide a basis to further investigate an important class of monooxygenases as well as the bacterial catabolism of steroids.
Subject(s)
Bacterial Proteins/metabolism , Cholesterol/metabolism , Flavins/metabolism , Mixed Function Oxygenases/metabolism , Mycobacterium tuberculosis/enzymology , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Binding Sites , Biocatalysis , Crystallography, X-Ray , Gas Chromatography-Mass Spectrometry , Gene Deletion , Hydroxylation , Kinetics , Mixed Function Oxygenases/chemistry , Mixed Function Oxygenases/genetics , Mixed Function Oxygenases/isolation & purification , Models, Molecular , Phenotype , Protein Structure, SecondaryABSTRACT
Oxysterols from steroid autooxidation have numerous harmful effects, but their biodegradation is poorly understood. Microarrays were used to study mineralization of the most common oxysterol, 7-ketocholesterol (7KC), by Rhodococcus jostii RHA1. Growth on 7KC versus growth on cholesterol resulted in 363 differentially expressed genes, including upregulation of two large gene clusters putatively encoding steroid catabolism. Despite this difference, 7KC degradation required key genes involved in cholesterol degradation, indicating a common catabolic route.
