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Hydrophobic interactions modulate ligand recognition and influence protein function. Here, we interrogated loop\u2010mediated hydrophobic contributions to catalysis in 3\u03b1\u2010hydroxysteroid dehydrogenase\/carbonyl reductase (3\u03b1\u2010HSD\/CR) from\n                    <jats:italic>Comamonas testosteroni<\/jats:italic>\n                    . Residue L193 on the flexible substrate\u2010binding loop was substituted with amino acids spanning a hydrophobicity gradient (W, I, F, M, V, P, A, T, G). Differential scanning fluorimetry showed comparable melting temperatures among variants, indicating minimal effects on global thermal stability. Steady\u2010state kinetics revealed decreased catalytic efficiency with reduced side\u2010chain hydrophobicity and corresponding increases in activation free energy (\n                    <jats:italic>\u0394G<\/jats:italic>\n                    <jats:sup>\n                      <jats:italic>\u2021<\/jats:italic>\n                    <\/jats:sup>\n                    ). With the exception of L193W, L193I, L193F, L193P, and L193T, log(\n                    <jats:italic>k<\/jats:italic>\n                    <jats:sub>\n                      <jats:italic>cat<\/jats:italic>\n                    <\/jats:sub>\n                    <jats:italic>\/K<\/jats:italic>\n                    <jats:sub>\n                      <jats:italic>M<\/jats:italic>\n                    <\/jats:sub>\n                    ) correlated linearly and positively with residue hydrophobicity, supporting a key role of hydrophobic interactions involving L193 in stabilizing the transition state of androsterone in the 3\u03b1\u2010HSD\/CR\u2010catalyzed reaction. Truncated substrates (2\u2010decalol, cyclohexanol) cause a significant decrease in catalytic efficiency, exhibiting similar trends with shallower slopes, consistent with predominant L193 close to the steroid A\u2010ring. Molecular dynamics simulations indicated that NAD\n                    <jats:sup>+<\/jats:sup>\n                    and substrate binding promote loop closure and strengthen hydrophobic interactions involving L193 in the active site. These data establish that the hydrophobic interaction of residue 193 with the transition state of substrate, linked to remote binding interactions of the loop with the unreactive portion of the substrate, significantly contributes to 3\u03b1\u2010HSD\/CR catalysis.\n                  <\/jats:p>","DOI":"10.1002\/pro.70555","type":"journal-article","created":{"date-parts":[[2026,4,6]],"date-time":"2026-04-06T13:01:43Z","timestamp":1775480503000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Hydrophobic interactions of a substrate with\n                    <scp>L193<\/scp>\n                    on the flexible substrate\u2010binding loop contribute to 3\u03b1\u2010hydroxysteroid dehydrogenase\/carbonyl reductase catalytic efficiency"],"prefix":"10.1002","volume":"35","author":[{"given":"Yan\u2010Liang","family":"Chen","sequence":"first","affiliation":[{"name":"Department of Biochemistry, Faculty of Medicine, College of Medicine Kaohsiung Medical University  Kaohsiung Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yun\u2010Hao","family":"Chou","sequence":"additional","affiliation":[{"name":"Graduate Institute of Medicine, College of Medicine Kaohsiung Medical University  Kaohsiung Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hwei\u2010Jen","family":"Lee","sequence":"additional","affiliation":[{"name":"Graduate Institute of Biochemistry, College of Biomedical Science National Defense Medical University  Taipei Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tzu\u2010Pin","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Medicinal and Applied Chemistry Kaohsiung Medical University  Kaohsiung Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chi\u2010Ching","family":"Hwang","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Faculty of Medicine, College of Medicine Kaohsiung Medical University  Kaohsiung Taiwan"},{"name":"Graduate Institute of Medicine, College of Medicine Kaohsiung Medical University  Kaohsiung Taiwan"},{"name":"Department of Medical Research Kaohsiung Medical University Hospital  Kaohsiung Taiwan"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2026,4,6]]},"reference":[{"key":"e_1_2_10_2_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-017-02258-w"},{"key":"e_1_2_10_3_1","doi-asserted-by":"publisher","DOI":"10.1021\/ja016754a"},{"key":"e_1_2_10_4_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi301491r"},{"key":"e_1_2_10_5_1","doi-asserted-by":"publisher","DOI":"10.1021\/ja055493s"},{"key":"e_1_2_10_6_1","doi-asserted-by":"publisher","DOI":"10.1006\/jmbi.1999.2765"},{"key":"e_1_2_10_7_1","doi-asserted-by":"publisher","DOI":"10.1038\/nchembio.232"},{"key":"e_1_2_10_8_1","doi-asserted-by":"publisher","DOI":"10.1021\/jp026686u"},{"key":"e_1_2_10_9_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi0003689"},{"key":"e_1_2_10_10_1","doi-asserted-by":"publisher","DOI":"10.1021\/ar5002928"},{"key":"e_1_2_10_11_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M706336200"},{"key":"e_1_2_10_12_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbapap.2009.06.006"},{"key":"e_1_2_10_13_1","doi-asserted-by":"publisher","DOI":"10.1038\/ncomms5968"},{"key":"e_1_2_10_14_1","doi-asserted-by":"publisher","DOI":"10.1002\/pro.4710"},{"key":"e_1_2_10_15_1","doi-asserted-by":"publisher","DOI":"10.4324\/9780203833575"},{"key":"e_1_2_10_16_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.biochem.3c00290"},{"key":"e_1_2_10_17_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.233.4764.659"},{"key":"e_1_2_10_18_1","first-page":"369","article-title":"Hydrophobic parameters pi of amino\u2010acid side chains from the partitioning of N\u2010acetyl\u2010amino amides","volume":"18","author":"Fauchere J\u2010L","year":"1983","journal-title":"Eur. 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