Comparative Analysis of Bile-Salt Degradation in Sphingobium sp. Strain Chol11 and Pseudomonas stutzeri Strain Chol1 Reveals Functional Diversity of Proteobacterial Steroid Degradation Enzymes and Suggests a Novel Pathway for Side Chain Degradation

Abstract

The reaction sequence for aerobic degradation of bile salts by environmental bacteria resembles degradation of other steroid compounds. Recent findings show that bacteria belonging to the Sphingomonadaceae use a pathway variant for bile-salt degradation. This study addresses this so-called Δ 4,6 -variant by comparative analysis of unknown degradation steps in Sphingobium sp. strain Chol11 with known reactions found in Pseudomonas stutzeri Chol1. Investigations with strain Chol11 revealed an essential function of the acyl-CoA dehydrogenase Scd4AB for growth with bile salts. Growth of the scd4AB deletion mutant was restored with a metabolite containing a double bond within the side chain which was produced by the Δ 22 -acyl-CoA dehydrogenase Scd1AB from P. stutzeri Chol1. Expression of scd1AB in the scd4AB deletion mutant fully restored growth with bile salts while expression of scd4AB only enabled constricted growth in P. stutzeri Chol1 scd1A or scd1B deletion mutants. Strain Chol11 Δ scd4A accumulated hydroxylated steroid metabolites which were degraded and activated with coenzyme A by the wild type. Activities of five Rieske type monooxygenases of strain Chol11 were screened by heterologous expression and compared to the B-ring cleaving KshAB Chol1 from P. stutzeri Chol1. Three of the Chol11 enzymes catalyzed B-ring cleavage of only Δ 4,6 -steroids while KshAB Chol1 was more versatile. Expression of a fourth KshA homolog, Nov2c228 led to production of metabolites with hydroxylations at an unknown position. These results indicate functional diversity of β-proteobacterial enzymes for bile-salt degradation and suggest a novel side-chain degradation pathway involving an essential ACAD reaction and a steroid hydroxylation step. Importance This study highlights the biochemical diversity of bacterial degradation of steroid compounds in different aspects. First, it further elucidates an unexplored variant in the degradation of bile-salt side chains by Sphingomonads, a group of environmental bacteria that is well-known for their broad metabolic capabilities. Moreover, it adds a so-far unknown hydroxylation of steroids to the reactions Rieske monooxygenases can catalyze with steroids. Additionally, it analyzes a proteobacterial ketosteroid-9α-hydroxylase and shows that this enzyme is able to catalyze side reactions with non-native substrates.

Investigations on the Degradation of the Bile Salt Cholate via the 9,10-Seco-Pathway Reveals the Formation of a Novel Recalcitrant Steroid Compound by a Side Reaction in Sphingobium sp. Strain Chol11

“…P. stutzeri Chol1, Sphingobium sp. strain Chol11 wt and ∆nov2c349 were grown with 1 mM cholate as carbon source, P. stutzeri Chol1 pBBR1MCS-5::hsh2 [22] and P. stutzeri Chol1 ∆kstD1 ∆stdA1 pBBR1MCS-5::hsh2 [11] were grown with 12 mM succinate and Sphingobium sp. strain Chol11 ∆sclA [25] was grown with 15 mM glucose.…”

“…Simultaneously, the side chain is degraded via β-oxidation and aldolytic reactions leading to side chain-less androsta-1,4-diene-3,17-diones (ADDs; e.g., 12β-DHADD, IV in Figure 1, in the degradation of cholate). 9α-hydroxylation of ADDs by the monooxygenase complex KshAB leads to cleavage of the B-ring upon A-ring aromatization and yields 9,10-seco-steroids such as 3,7,12-trihydroxy-9,10-seco-androsta-1,3,5 (10)-triene-9,17-dione (THSATD, V) [11][12][13][14]. The A-ring is cleaved by consecutive hydroxylation and meta-cleavage similar to degradation of aromatic compounds [15][16][17][18].…”

“…After A-ring oxidation leading to ∆ 4 -3-keto-cholate (II) during the degradation of cholate [24], a further double bond is inserted into the B-ring upon elimination of the 7OH by the dehydratase Hsh2 [22], leading to 12α-hydroxy-3-oxo-4,6choldienoate (HOCDA, IX) as prominent intermediate. Side-chain degradation via a so far mostly unknown mechanism results in 12-hydroxy-androsta-1,4,6-triene-3,17-dione (HATD, X) [11,25]. Bioinformatic, proteomic, and first physiological analyses indicated that further degradation of HATD most probably proceeds via 9α-hydroxylation and 9,10seco-cleavage [11]: In heterologous complementation experiments, expression of three of five homologs of the oxygenase subunit KshA of strain Chol11 in a kshA deletion mutant of P. stutzeri Chol11 lead to the production of the expected seco-steroid 3,12β-dihydroxy-9,10-seco-androsta-1,3,5 (10),6-tetraene-9,17-dione (DHSATD, XI).…”

“…Side-chain degradation via a so far mostly unknown mechanism results in 12-hydroxy-androsta-1,4,6-triene-3,17-dione (HATD, X) [11,25]. Bioinformatic, proteomic, and first physiological analyses indicated that further degradation of HATD most probably proceeds via 9α-hydroxylation and 9,10seco-cleavage [11]: In heterologous complementation experiments, expression of three of five homologs of the oxygenase subunit KshA of strain Chol11 in a kshA deletion mutant of P. stutzeri Chol11 lead to the production of the expected seco-steroid 3,12β-dihydroxy-9,10-seco-androsta-1,3,5 (10),6-tetraene-9,17-dione (DHSATD, XI). However, this activity seemed to be very low.…”

“…P. stutzeri Chol1 is not able to completely degrade ∆ 4,6intermediates such as HOCDA (IX) and only degrades the side chain, which results in HATD (X) [21]. HATD is then transformed to DHSATD (XI) as well as the dead-end product 1α,2α,12β-trihydroxy-androsta-4,6-triene-3,17-dione (THADD, XII) by two different types of hydroxylation reactions [21] that are both catalyzed by KshAB Chol1 [11]. It is The goals of this study were first to further investigate 9α-hydroxylation in strain Chol11 by providing DHSATD as a substrate.…”