Literature summary for 1.14.14.12 extracted from

Dresen, C.; Lin, L.Y.; D'Angelo, I.; Tocheva, E.I.; Strynadka, N.; Eltis, L.D.
A flavin-dependent monooxygenase from Mycobacterium tuberculosis involved in cholesterol catabolism (2010), J. Biol. Chem., 285, 22264-22275.

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Cloned(Commentary)

Cloned (Comment)	Organism
expression in Escherichia coli	Mycobacterium tuberculosis

Crystallization (Commentary)

Crystallization (Comment)	Organism
hanging drop vapor-diffusion method at 18°C, the crystal structure of HsaA to 2.5 A resolution reveales that the enzyme has the same fold, flavin-binding site, and catalytic residues as p-hydroxyphenyl acetate hydroxylase. HsaA prepared aerobically from the Escherichia coli host exists as a mixture of dimers and octamers in solution while HsaA prepared anaerobically from the rhodococcal host exists as a tetramer	Mycobacterium tuberculosis

Crystallization (Comment)

Organism

hanging drop vapor-diffusion method at 18°C, the crystal structure of HsaA to 2.5 A resolution reveales that the enzyme has the same fold, flavin-binding site, and catalytic residues as p-hydroxyphenyl acetate hydroxylase. HsaA prepared aerobically from the Escherichia coli host exists as a mixture of dimers and octamers in solution while HsaA prepared anaerobically from the rhodococcal host exists as a tetramer

Mycobacterium tuberculosis

Inhibitors

Inhibitors	Comment	Organism	Structure
riboflavin	0.004 mM, 50% inhibition	Mycobacterium tuberculosis

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.097	-	3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione	flavin: FADH2, pH 7.4, 25°C	Mycobacterium tuberculosis
0.1	-	O2	flavin: FMNH2, pH 7.4, 25°C	Mycobacterium tuberculosis
0.2	-	3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione	flavin: FMNH2, pH 7.4, 25°C	Mycobacterium tuberculosis
0.35	-	3,17-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9-one	flavin: FMNH2, pH 7.4, 25°C	Mycobacterium tuberculosis
1.44	-	3-hydroxybiphenyl	flavin: FMNH2, pH 7.4, 25°C	Mycobacterium tuberculosis

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione + FADH2 + O2	Mycobacterium tuberculosis	the enzyme is involved in cholesterol catabolic pathway. This pathway contributes to the pathogenicity of Mycobacterium tuberculosis	3,4-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione + FAD + H2O	-	?
3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione + FADH2 + O2	Mycobacterium tuberculosis H37Rv	the enzyme is involved in cholesterol catabolic pathway. This pathway contributes to the pathogenicity of Mycobacterium tuberculosis	3,4-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione + FAD + H2O	-	?

Organism

Organism	UniProt	Comment	Textmining
Mycobacterium tuberculosis	P9WJA1	P96852: oxygenase subunit hsaA. The reductase subunit hsaB of the two-component enzyme utilizes NADH to reduce the flavin, which is then transferred to the oxygenase subunit	-
Mycobacterium tuberculosis H37Rv	P9WJA1	P96852: oxygenase subunit hsaA. The reductase subunit hsaB of the two-component enzyme utilizes NADH to reduce the flavin, which is then transferred to the oxygenase subunit	-

Purification (Commentary)

Purification (Comment)	Organism
-	Mycobacterium tuberculosis

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
3,17-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9-one + FMNH2 + O2	-	Mycobacterium tuberculosis	3,4,17-trihydroxy-9,10-seconandrost-1,3,5(10)-triene-9-one + FMN + H2O	-	?
3,17-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9-one + FMNH2 + O2	-	Mycobacterium tuberculosis H37Rv	3,4,17-trihydroxy-9,10-seconandrost-1,3,5(10)-triene-9-one + FMN + H2O	-	?
3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione + FADH2 + O2	-	Mycobacterium tuberculosis	3,4-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione + FAD + H2O	-	?
3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione + FADH2 + O2	the enzyme is involved in cholesterol catabolic pathway. This pathway contributes to the pathogenicity of Mycobacterium tuberculosis	Mycobacterium tuberculosis	3,4-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione + FAD + H2O	-	?
3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione + FADH2 + O2	-	Mycobacterium tuberculosis H37Rv	3,4-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione + FAD + H2O	-	?
3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione + FADH2 + O2	the enzyme is involved in cholesterol catabolic pathway. This pathway contributes to the pathogenicity of Mycobacterium tuberculosis	Mycobacterium tuberculosis H37Rv	3,4-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione + FAD + H2O	-	?
3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione + FMNH2 + O2	-	Mycobacterium tuberculosis	3,4-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione + FMN + H2O	-	?
3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione + FMNH2 + O2	-	Mycobacterium tuberculosis H37Rv	3,4-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione + FMN + H2O	-	?
3-hydroxybiphenyl + FMNH2 + O2	-	Mycobacterium tuberculosis	2,3-dihydroxybiphenyl + FMN + H2O	-	?
additional information	the enzyme is able to catalyze the o-hydroxylation of a range of p-substituted phenols to generate the corresponding catechols	Mycobacterium tuberculosis	?	-	?
additional information	the enzyme is able to catalyze the o-hydroxylation of a range of p-substituted phenols to generate the corresponding catechols	Mycobacterium tuberculosis H37Rv	?	-	?

Synonyms

Synonyms	Comment	Organism
3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione 4-hydroxylase	the two-component enzyme HsaAB consists of an oxygenase subunit HsaA and a reductase subunit hsaB that utilizes NADH to reduce the flavin, which is then transferred to the oxygenase subunit	Mycobacterium tuberculosis
HsaAB	the two-component enzyme HsaAB consists of an oxygenase subunit HsaA and a reductase subunit hsaB that utilizes NADH to reduce the flavin, which is then transferred to the oxygenase subunit	Mycobacterium tuberculosis

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
25	-	assay at	Mycobacterium tuberculosis

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.031	-	O2	flavin: FMNH2, pH 7.4, 25°C	Mycobacterium tuberculosis
0.075	-	3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione	flavin: FADH2, pH 7.4, 25°C	Mycobacterium tuberculosis
0.11	-	3-hydroxybiphenyl	flavin: FMNH2, pH 7.4, 25°C	Mycobacterium tuberculosis
0.2	-	3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione	flavin: FMNH2, pH 7.4, 25°C	Mycobacterium tuberculosis
0.24	-	3,17-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9-one	flavin: FMNH2, pH 7.4, 25°C	Mycobacterium tuberculosis

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7	-	assay at	Mycobacterium tuberculosis

Cofactor

Cofactor	Comment	Organism	Structure
FADH2	the enzyme can use FADH or FMNH2 as flavin cofactor	Mycobacterium tuberculosis
FMNH2	the enzyme can use FADH or FMNH2 as flavin cofactor	Mycobacterium tuberculosis

General Information

General Information	Comment	Organism
malfunction	hsaA deletion mutant (mutant with a deletion in the oxygenase subunit) does not grow on cholesterol but grows normally on pyruvate. The hsaA deletion mutant (mutant with a deletion in the oxygenase subunit) accumulates 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione	Mycobacterium tuberculosis

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
0.08	-	3-hydroxybiphenyl	flavin: FMNH2, pH 7.4, 25°C	Mycobacterium tuberculosis
0.32	-	O2	flavin: FMNH2, pH 7.4, 25°C	Mycobacterium tuberculosis
0.7	-	3,17-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9-one	flavin: FMNH2, pH 7.4, 25°C	Mycobacterium tuberculosis
0.77	-	3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione	flavin: FADH2, pH 7.4, 25°C	Mycobacterium tuberculosis
1	-	3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione	flavin: FMNH2, pH 7.4, 25°C	Mycobacterium tuberculosis