Bacteria degrade nicotine mainly using pyridine and pyrrolidine pathways. Previously, we discovered a hybrid of the pyridine and pyrrolidine pathways (the VPP pathway) in Pseudomonas geniculata N1 and characterized its key enzyme, 6-hydroxypseudooxynicotine amine oxidase (HisD). It catalyzes oxidative deamination of 6-hydroxypseudooxynicotine to 6-hydroxy-3-succinoylsemialdehyde-pyridine, which is the crucial step connecting upstream and downstream portions of the VPP pathway. We determined the crystal structure of wild-type HisD to 2.6 Å. HisD is a monomer that contains a flavin mononucleotide, an iron-sulfur cluster, and ADP. On the basis of sequence alignment and structure comparison, a difference has been found among HisD, closely related trimethylamine dehydrogenase (TMADH), and histamine dehydrogenase (HADH). The flavin mononucleotide (FMN) cofactor is not covalently bound to any residue, and the FMN isoalloxazine ring is planar in HisD compared to TMADH or HADH, which forms a 6-S-cysteinyl flavin mononucleotide cofactor and has an FMN isoalloxazine ring in a “butterfly bend” conformation. Based on the structure, docking study, and site-directed mutagenesis, the residues Glu60, Tyr170, Asp262, and Trp263 may be involved in substrate binding. The expanded understanding of the substrate binding mode from this study may guide rational engineering of such enzymes for biodegradation of potential pollutants or for bioconversion to generate desired products.

中文翻译：

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结构学到的假单胞菌（Pseudomonas geniculata）N1的6-羟基伪拟氧烟碱氧化酶是一种涉及尼古丁降解的关键酶。

细菌主要通过吡啶和吡咯烷途径降解尼古丁。以前，我们在Pseudomonas geniculata中发现了吡啶和吡咯烷途径（VPP途径）的杂种N1并鉴定了其关键酶6-羟基伪氧烟碱胺氧化酶（HisD）。它催化6-羟基伪氧化烟碱氧化成6-羟基-3-琥珀酰基半醛-吡啶的氧化脱氨基，这是连接VPP途径上游和下游部分的关键步骤。我们确定了野生型HisD的晶体结构为2.6Å。HisD是包含黄素单核苷酸，铁硫簇和ADP的单体。基于序列比对和结构比较，在HisD，密切相关的三甲胺脱氢酶（TMADH）和组胺脱氢酶（HADH）之间发现了差异。黄素单核苷酸（FMN）辅因子未与任何残基共价结合，与TMADH或HADH相比，FMN异恶嗪环在HisD中为平面，后者形成6- S-半胱氨酰黄素单核苷酸辅因子，在“蝴蝶弯”构象中具有FMN异恶嗪环。根据结构，对接研究和定点诱变，残基Glu60，Tyr170，Asp262和Trp263可能与底物结合有关。这项研究对底物结合模式的进一步理解可以指导这类酶的合理工程化，以进行潜在污染物的生物降解或进行生物转化以产生所需的产物。