A Flavin-dependent Monooxygenase from Mycobacterium tuberculosis Involved in Cholesterol Catabolism

Abstract

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-). However, 3,9-DHSA was a poorer substrate than 3-hy-). In the presence of 3-HSA the K mapp for O 2 was 100 ؎ 10 M. The crystal structure of HsaA to 2.5-Å 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°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. Mycobacterium tuberculosis (Mtb),6 the most devastating infectious agent of mortality worldwide (1), remains a primary public health threat due to synergy with the human immunodeficiency virus and the emergence of extensively drug-resistant strains. Mtb has the unusual ability to survive and replicate in the hostile environment of the macrophage (2, 3) utilizing largely unknown mechanisms. Genes essential to intracellular survival have been identified using genome-wide mutagenesis (4). A cluster of such genes was subsequently predicted to specify cholesterol catabolism (5). Targeted gene deletion studies subsequently indicated that cholesterol catabolism occurs throughout infection, contributing to the virulence of Mtb (6 -8). Although the exact role of cholesterol in infection remains unclear, the gene deletion studies are consistent with the high concentrations of cholesterol found within caseating granulomas and the observed congregation of bacteria around cholesterol foci (9).The cholesterol catabolic pathway of Mtb is very similar to that of other pathogenic and environmental actinomycetes (10), as initially predicted in Rhodococcus jostii RHA1 (5). Sidechain degradation is initiate...

Interactions with the Substrate Phenolic Group Are Essential for Hydroxylation by the Oxygenase Component of p-Hydroxyphenylacetate 3-Hydroxylase

“…Ser-146 interactions with the substrate are not mandatory for hydroxylation but assist in positioning the enzyme-bound HPA in an optimum geometry for hydroxylation. According to x-ray structures of two-component monooxygenases that utilize phenolic compounds as substrates, these two residues are well conserved (24,31,32). Our findings imply that the conserved His/Ser pairs found in other two-component monooxygenases may have similar functional roles.…”

“…Homologues of these residues are found in the oxygenase component (HpaB) of HPAH from T. thermophilus HB8, in which the O atom of Thr-104 and the N ⑀ of His-142 are 2.5 and 2.8 Å from the hydroxyl group of HPA, respectively (24), and in the oxygenase involved in the cholesterol catabolic pathway of Mycobacterium tuberculosis, in which His-92 and Ser-118 are about 2.4 Å from the hydroxyl group of the modeled 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione substrate (32). For the oxygenase (TftD) of chlorophenol 4-monooxygenase from Burkholderia cepacia AC1100, His-289 was proposed to interact with a hydroxyl group of 2,5-dichlorohydroquinone docked in the structure of TftD (31).…”

Activity of 3-Ketosteroid 9α-Hydroxylase (KshAB) Indicates Cholesterol Side Chain and Ring Degradation Occur Simultaneously in Mycobacterium tuberculosis

“…The k cat /K mO 2 in the presence of 1,4-BNC-CoA is an order of magnitude greater than that measured in the presence of ADD (15). Perhaps more significantly, the K mO 2 of 90 Ϯ 10 M is similar to that of other oxygenases in the cholesterol degradation pathway (13,14) as well as in the lung tissues of healthy rabbits (34). This is compared with the value in excess of 1.2 mM previously measured in the presence of ADD (15).…”

“…All previous biochemical experiments on purified Mtb cholesterol ring-degrading enzymes have used compounds representing end products of side chain degradation (13)(14)(15)35). The CoA thioesters examined in this study represent hypothetical intermediates in cholesterol side chain degradation, suggesting that the true physiological substrates for some of these enzymes may also be side chain degradation intermediates, and countering the current paradigm that cholesterol side chain degradation in Mtb is completed before breakdown of the ring structure.…”

“…1) were less than their non-steroidtransforming homologues (13)(14)(15). Genetic knockouts of several of the cholesterol degradation genes have resulted not only in failure to grow on cholesterol in vitro but in attenuated pathogenicity in various infection models (9,11,14,16,17).…”